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E}4BRYOGENES IS OF EXPERIHENTALLY INDUCED NEURAL TUBE
DEFECTS IN THE CH I CK EMBRYO
A Thes i s
Presented to the Faculty of Graduate Studies,
Universlty of Manitoba, ln Partîal Fulfiìlment
. of the Requirements for the Degree of
Doctor of Ph i losophy
by
Ra I ph Aì I an l,lann
September '1977

EMBRYOGENESiS OF FXPERII¡ENTALLY INDUCED NEURAL TUBE
DEFECTS iN THE CHTCK EMBRYO
BY
RALPH ALLAN MANN
A dissertation submitted to the Facutty of Graduate Studies of
ttre University of Manitobl in partial fulfillment of the requirements
of the degree of
DOCTOR 'OF PHILOSOPHY
ð rgzg'
Permissio¡¡ h¡s l¡eon grantctl to th(} LIBRARY OF Tllti UNIvUR'
SITY OIr MANITOITA to lcnd or scll copies of tlìis dissert tiolì' to
thc NATIONAL LIBRAIìY OIr CANADA to microlilnr this
dissertation and to lend or sell copies of the film, and UNlvtjRSITY
MICROFILMS to publish at¡ abstract of this dissert tion'
The ¿uthor reserves other pu¡lication ìights, und neither ttre
dissertation nor extensivo extructs tionr it tnay be printed or otlìerwise
reproduced without tho author's writtcn ¡rertltission'
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MY PARENTS
c.J.H.
G. M.

. ACKNOl4lLEDGEMENTS
l',lany peopie have given me lnvaluable help ln carrylng out the
work for this thesls. I would lîke to thank:
Èis. l"larlene Stoddart, 1,1s. Susan pylypas an¿ Mr. David Gray for
technlcal asslstance; lrs. Brenda Bell and Ms. Jean Hay for preparatîon
of the photographs; Mr. Glen Reld, Ms. Lauri Rlchardson and l,ls. Karen
selcho for the llne illustratîons; Mr. üralter Jones for construction of
equipment; Dr. l/.H. Tþurlbeck, 11r. l,layne Gal lagher and l,ls. El izabeth
tJaskîewicz for the use of the Lei tz lmage Analyser; Dr. K,L. Moore,
Dr. F.R. Tucker, Dr. R.K. Greenlaw, Dr. J.B. Hyde, Dr. K. Nagy,
Dr. A.1.1. llaI lbank, Dr. D.V. Cormack, Dr. Helen Battle, Dr. A.F. Holoway,
and Dr. J. Hoogstraten for advlce and assistance; t4r. Tim Ful lerton
for statistical consultatfon; and Hs. Frances Kas,per for typíng the
mênuscr¡pt.
Above all I am grateful to Dr. J.C. Haworth and the Research
Committee of the Chíldrenrs Centre for contÍnued financial supportrand
Dr' T.v.N' Persaud for his unfairing encouragement and enthusrasm through
so many vl ciss i tudes.

lt
Congenital malformêtlons of the ðentral nervous system may be open
or closed. 0pen defects involve the braîn or the spinal cord, or both.
lnvestîgation of the etiology of these defects involves epìdemiological
stud¡es of their distribution in human populaiions and embryological
studies of their development in experimental animals and human abortuses.
For this investigation the chick embryo was ¡nìtíâl ly selected,
because of its accessibil ¡ty to treatment and observation through a
wíndow in the overìyìng shell; the use of an 4n o7)o system al lowed
culture of the embryos to 12 days of incubation. ln addition, early
neurogenesÌs in avian and human embryos is very similar, wÌth development
of the spÌnal cord from neural plate and tai l-bud materials, which
fuse in an overlap zone.
Prel iminary experiments, in which early chîcl< enrb ryos were exposed
to several known teratogenic agents through a window în the shell, revealed
that windowing alone was highly teratogenic. By using a standard
windowing technic at 26 - l0 hours of incubation a range of neurôl ånd
non-neural mal formations were obtained.
The morial ity and malformations produced by windowîng at l4 hours
of incubat¡on were greater than those at 26 hours, but by lB houis the
teratogenic effect was less pronounced. 0bl íterat¡on of the introduced
air space,by the addition of albumen or F 12 medîum or by reexpansion of
the air celì, almost abol ished the teratogenic effect of wíndowing if
performed immediately.
Skeletal staining of 11 - 12 day embryos showed that vertebral
lesions increased in severîty in a cranio-caudal sequence, spina bifida occuì ta

ii¡
occurred in the cerV¡cal and upper thoracic regions¡spina bifida manifesta
(associated wiih opeá cord defects) from the lower thoracic to sacral
regÎons, whlle vertebral deletionswere almost confirmed to the caudal '
r.eg ion ,
Examînation of a closely-spaced series of embryos recovered wíthin
42 hours of windowlng revealed open braîn and cord defects. These
occurred at every Stage after the expected closure of the anterior
neuropore and rhomboid sinus, suggesting a process of non-closure.
Furthermore, lncípient non-closure of the spinal cord could be predicted ..
from the abnormal triangular shape of the rhomboíd sinus.
Serial sectioning of selected early.embryos revealed two types
of open cord defects. HyeloschisÌs arose by eversÌon of the neural
folds at the rhomboid sinus, and formed regular defects w¡th separation
between the neural plate and taÌl-bud sources of neural tissue. The
development of myeloschisis was associated wi th local separation of
the notochord from the open neural tissue, but not with trovergrowthr¡
of neural tíssue.
ln myelodysplasia neural plate material was absent from the êrea
of the open defect, and the spinal cord u¡as derived from tail-bud
material êlone. Myelodysplasia was characterized by a local reduction
in neural volume, and assocîated wîth cystic and hemorrhagic changes
in mesoderm and reductìon in the volume of adjacent somites.

TABLE OF CONTENTS
SECT ION
PAGE
1 INTRODUCT ION 1
1 .1 TERATOLOGY 2
I . 2 CL IN I CAL IHPORTANCE OF B I RTH DEFEETS 2
1.3 INCIDENCE OF BIRTH DEFECTS 3
1.4 coNGENITAL ¡IALFORI'IATIONS OF
THE CENTRAL NERVOUS SYSTEM 3
1.5 SPINA BIFIDA 4
1.5.t Spina Bîfida Occul ta .and
Cystica 5
1 .5 .2 l4en i ngoce I e 5
1 .5 .3 l,lye I omen i ngoce I e 5
1.5.4 l4yelocystocele :
6
1.5,5 Anterlor Spina Bifida 7
1.6 CRANIUI,IBIFIDUM 7
1.6..l Anencephaly, Exencephaly 7
1.6.2 Heningocele,Encephalomenìngocele. . . . I
. 1.6.3 Cranium Bifídum Occul tum. B
1 .7 DYSRAPH IC STATES 8
1.8 PRoGNoSts. 9.
2 REVIEI.I OF LITERATURE 12
2,1 ETIOLOGY OF THE DYSRAPHIC STATES 13
2.2 EPIDEI.1I0LoGICAL STUDIES. 13
2.2.1 lncidence . 13
2.2.2 . Temporal Fl uctuat ions
2.2,3 Seasonal Variat,îon, 15

2.2.4 Sex Ratio 15
2.2.5 Geograph i c Dlstributîon 15
2.2.6 Ethnlc Distrîbution. . 16
2.2t7 Famlly Studles ' . 16
2,2.9 l4aternal Age and Parì ty .. 17
2.2.g soc io-Econornic Status. 17
2.2.10 Urban i zatlon 17
2.2.11 Concluslons 17
t2
EI4BRYOLOGICAL STUDIES 18
2.3.1 Human Specìmens. . . 18
2.3.2 Exper¡mental Oysraphism in Animals 19
2.3.3 Heredi tary Dysraphîsm. 21
2-l+
HYPOTHESES OF THE EMBRYOGENESIS
OF DYSRAPHISM 22
2,\.1 Slmple Non-Closure . . :-. 22
2.\,2 Overgrowth and Non-Closure ' . . 22
2,4.3 Rupture of the Closed.Neural Tube 22
2.1!,\ Abnormal Braîn Growth . , .23
2.,\,5 Abnormal Spínal Flexion . . . 23
2.\.6 Primary Vascular Defects 73
2,4.7 Amniotlc Adhes ions 24
2.4.8 Abnormal Development of the Tail-Bud 2\
2.4.9 Trauma 2\
2.4. t 0 lnfection 25
2.4.11.. Summary 25

},I.ATE R I ALS
3.1 THE CHICK EI4BRYO,
3.2 SoURCE 0F ËûGS AND tNCUBATi0N
3.3 oTHER EqUlPl4ENr
GENERAL ¡4ETHODS
4.I SELECTION OF EGGS
4.f.1
d.1.2
lncubat î on
candl ing
\.2 TECHNIc OF OPENING AND cLoSIIIG EGGS
\,2.1 Prel lminary Exper¡ments
\.2.2 Standard Techn i c
4.2.3 Exami nation of Embryos
\.2.4 Closure of Eggs
4.2.5 Effect of Embryonic Age.
4.3 RE rNcuBATr0N AFTER r,JiNDor,/rNc .
4.+ TERAToGENtc EFFECT 0F opENtNG THE SHELL
4.4.1 Vibration
4,4.2 Parafi lm and Plasticine . . . .
4;4.3 Art if ic ia I Air Space
\.5 BACTERIOLOGIcAL CULTURE
4.6 EXAI'iINATION: OF EARLY EI'IBRYOS
4.6.1 Fixation and Staging
\.6.2 Problems in Examinatîon. . . .
4.7 EXAt.lINATION OF OLDER EMBRYOS
\,7,1 Five Day Emb rYos
\,7,2 Eleven and Twelve DaY EmbrYos
z6
27
27
2B
32
33
33
33
12
33
34
3\
35
35
3B
38
3B
38
\5
\t
45
\5
\6
t+6
\6
46

4.8 H ISTOLOûI CAL EXAMINATION
4.8.
1
Ser ia l Sectloning
4.8.2 Group ing of Embryos
4.8.3 Subd ivi s ion ¡nro Regions
4.8.4 Histo¡ogical Descriptions
\.9 ANALYSIS oF NEURAL CLOSURE
4.lo Rruelysts oF NEURAL voLuMEs
RESULTS OF TERATOLOGICAL PROCEDURES
5.1 TERATOGENIC EFFECT OF t.,INDOWING
5.2 HALFOR}IATIONS PRODUCED BY I,'INDOWING
5.3 INVEST¡GATION OF EFFECT OF WINDOI^'ING
5.3.1 Vibratîon of Unopened Eggs
5.3,2 Parafi lm and Plasticine Alone
5.3.3 0bl iteration of lntroduced Air Space . . .
5.\ BACTERIOLOGICAL CULTURE
RESULTS OF EMBRYOLOGICAL STUDIES
6.1 EI"IBRYoGENESIS oF OPEN NEURAL DEFECTS
L-l
47
47
4B
5\
54
55
56
57
65
69
69
'72
75
92
95
96
6.1.1 Embryoníc Sízes at 26 Hours
6.1.2 Mortal i ty wi th Varying periods
of lncubation after l/ïndowing -102
6.1.3 Neural Closure and Neural Defects lO9
6.1.4 Development of open Neural Defects ll4
6.1.5 Dlstribution of 0pen Cord Defects 144
6.2 SPINAL LEVELS OF oPEN CORD DEFECTS IN 12-DAY E}4BRYos 154
6.3 DESCRIPTIoN oF HISTOI.OGIcAL APPEARANCES 165
6.3.1 Stage l0 controt Embryos 165
96

vt I I
6.3.2 Stage 10 Experímentel Embryos 166
6.3.3 Stage Il-12 Control Embryos 167
63.\ Stage 1l-12 Experimental Embryos 168
6.3.5 Stage 1l-16 Control Embryos 171
6.3.6 Stege f3-16 Experìmental. Embryos 172
6.3.7 Stage 1/-20 Contro¡ Embryos 175
6.3.8 Stage t7-20 Experimental Embryos 176
. 6.3.9 Review of Hîstologïcal Changes 180
6.3.10 Sequentîal ll lustratÎons 199
6.4 DETAILED REVIE}¡ OF HISTOLOGIcAL FINDINGS. . 212
6.5 col'lpARtsoN oF HtsroloctcAL FtÑDtNGs
!úlTH APPEARANCE 0F I,,HOLE E}4BRY0S 253
6.6 DEVELOPMENT OF THE RHOMBIC RoOF 262
6.7 HtSToLoGIcAL CHANGES AssoctATED
WITH NEURAL DEFECTS 276
6.8 EXTENT OF THE OVERLAP ZONE . 302
6.9 ANALYSIS OF NEURAL VOLUMES 318
Dtscuss toN 340
Animal |lodels 341
lJindowing of Eggs 342
open Neural Defects in Chick Embryos , 3\2
Spinal Defects at 11-12 Days. . 3\3
. Somíte and Vertebral Levels . 3\4
Process ing Art i facts 3\6
No rrna I Neurulatîon. 3\6
l,lyeloschisis 347
l,lye I odyspl as ia 3\B

tx
Rhombic Roof 3\9
Notochord 349
Som i rês 351
Cystic Changes ...
35tt
Ectoderm 355
Ta i l -B ud
Overlap Zone 356
Neura I Vol ume 357
Neural l4itosis ... 355
Hypotheses of Human Dysraphîsm 359
Slmple Non-C l os ure
. Overgrowth and Non-Closure 361
Closure and Rupture 362
0ther Hypotheses 363
Spina Bifida occul ta .....-¡. 363
' Chíck Embryo as a Hodel 365
I'lechanisms of Neurulation . . . 365
Neural lnductîon .. 367
Ce.l I Death 369
Regulative Ab¡ ¡ í ry 3'69
'370
. . Principles of Teratogenesis .
Phys ical Teratogenic Agents 371
Chenlcal Teratogen i c Agents 372
Windowi¡g as a Teratogenlc Agent 373
SUMHARY AND C0NCLUSI0NS . 375

9 APPENDTCES 378
APPENDIX A .
379
1 Preparatlon of Earìy Chick Embryos for
APPENDIX B
Ser ia I Sectioning
Staining of Carti laginous Skeleton at ll-12 Days
l0 B |BLI0GRAPHY 382

I NTRODUCT I ON

1.1 TERATOLOGY
Birth defects have attracted popular curîosity from the earlìest
tlmes, and scientífic attentìon more recently. Thei r study constitutes
the specÌal ity of Teratology, whîch combines many dîscipl¡nes with¡n the
areas of Developmental Bíology and Cl inical l4edicine. Teratology originally
implled the study of rmonstersr, but has now expanded to embrace the
whole fleld of structurâl and functional defects present at birth.
Three degrees of structural abnormal í tíes may be defîned:
a) \,ariations are slight deviations from the range of normal, such as
the delayed appearance of an ossificat¡on centre.
b) Anomalies are minor structural defects which may remain undetected
and do not produce marked functionaì disability, Examples in the vertebral
column are sacral izatîon of a lumbar vertebra or a symptomless spina bifida
occu I ta .
c) Malformations are more extensive defects present at birth. They may
be major, as in anencephaly (which ís uniformly fatal) or more mînor, as in
the congenital fusíon of two vertebral bodies.
1.2 CLtNICAL il'1PoRTANCE q!: BrRTH pEFECTS
ln recent years congenital malformations have become increasingly
important in cl inical prêctice. Appl ication of the prînciples oi publ ic
health and preventive medicîne, followed by the introductlon of antimicroblal
êgents, have produced a steady decl ine in nrortality from acute
infections. Thus a relatlvely hîgher proportion of deaths in înfancy and
childhood are now attributable to congenital nalformations. Horeover,

improvements in the quality of antenatal carg and in the treatment
of certâ1n deformîtles after birth have produced an absolute increase
ln the population of affected children.
1.3 tNctpENcE oF B!¡I!_!!!!!I!
There is an extensivc Iiterature on the'incidence of bi ¡-ih defects
but the statistics are subject to mâny sources of error, such as:
a) the unknovln rate of prenatal loss
b) incomplete d i agnos is
c) under-reporting of defects
d) vâriations în recordíng methods
è) the preponderãnce of dâta from hospital series,
ln addition, the figures are greatly êltered by the inclusîon or
exclusìon of stillbirths (Keonedy, l!6/; Persaud , 1g7il. ln an international
survey of two huridred and thirty-eight reports,covering twenty
million b¡rths, congenital defects occurred ¡n one to five per cent of
líve births according to the cr¡teria used (Kennedy, 1!6/).
one of the most meticulous investigations of the incidence of
bîrth defects by the World Heal th Organization has achieved a high degree
of comparabil'ity between twenty-four centres in sixteen countries, by
uniform recording methods and standardization of frequencies for,maternal
age (Stevenson et al ., 1966).
t.4 coNer¡!!rA!_lALroRr{nloHs or rú¡ cr¡lrRnl NeRvous sysreil
Abnormal development of the central nervous system may produce a
wide range of malformations of the braln and spinal cord. A simple
class¡fication of these defects, however, ís difficult to achieve because

4
of the complexity of normal development (t/arkany, 1971). Thus the
splnal coid may be asymmetrical, double (d¡plonyelìa), or even ôbsent
(arnyeìia). These cord defects are col lectively known as myelodysplasias.
The brain nay be enlarged (macrencephaly) or reduced în size
(microcephaly) , or show more locallzed enclosed defects of the cerebel lum,
corpus caìlosum, cerebral cortex (porencephaly) or the whole forebrain
(arhinencephal.les). Dilation of the central canal of the cord or of the
braln ventrì cles consti tute hydromyel ia and hydrocepha ly respective'ly.
Hydrocephaly, however, is not a single disease, but the end resul t of many.
dlfferent and often unreìated processes.
ln a rather separate group of malformations neural tlssue is eîther
exposed or herníated. lnvolvement of the spine produces spína bifida
(of several distinct types), and involvement of the skull c¡-anium blfidum.
ln some câses ân extensíve open lesion called craniorachischisis (fig. 2)
involves both skull and spine, suggesting a close relationship between
the tv'ro defects.
As a further compl ication spina bifida is very frequently accompanied
by hydrocephalus (Fol tz and Shurtleff, 1972), usually in the presence
of an Arnold ChíarÍ malformation (Russell and Donald, 1935) Emery and
llacken z ie, 1971 ) .
1.5 SPINA BIFIDA
The tern spina bifida rvas ¡ntroduced by Nicolas Tulp (1652), and
lrnplles a nidllne defect of the vertebral column.

5
1.5.1 SÞlri¿i B_!ll i dê Occúl rå ând Cysr¡ca
ln spina blfîda occulta there ls no open neural lesion,but ro_
en tgenog rans revda I a defect of or¡e or more spinous processes or raminae.
The site of this bony defect may occasionar ry be marked by abrrormar ities
of overlying skin, such as pigmented or. hairy patches. Though often
undetected, spína bifida occulta is sometimes accompanied by symptoms
suggesting ínvolvement of the spinal cord or cauda equina. This could be
caused by ê tight filum terminale, fibrous bands, ¡ntrathecal I ipomas,
or frank myelodysplasia (¡ames and Lassman, ,|967). The symptomless and
symptomatic forms of spina bífida occulta may represent two distinct
leslons, the former being prima,r.iry a skeretar defect and the ratter
secondary to cord or cauda equ¡na defects,
By contrast, ân external ly visible defect is called a spina bifida
manífesta, or apertâ, or cystica (if cystíc). Several types may be distínguished.
I.5.2 Men!!rgoce I e
A meningocele invorves defects of severar neurar arches wrth herníation
of meninges but not of neural tissue, though the underlying cord may be
dysplastíc. For this reason a meningocete cannot always be diagnosed
w¡th certa¡nty, and may prove on exploration to be myelocele (Laurence,
1964',).
1,5.3. Myelomeningocele
An open lesion consisting of neural tissue, accumulated fluld,
abnormal vasular tíssue, and a variable amount of epithel íumrwith the
loss of several neural arches is tradítionar ry calred a myeromeningocere
(fig.3 ¡. However, it is now bel ieved that the tesion originates as a

6
flat, exposed plaque of neural tissue (a m'¡,e¡6t"¡¡rts or neuroschlsls).
ln most èases thls open plaque undergoes secondary changes. Accumulation
of fluld (which elevates and disrupts the plaque) leads to formation of a
cyst which ís progressívely covered by squamous epithel îum and scar
tissue, suggesting ên ¡ncorrect diagnosis of ulcerating rmyelomeningocelet
(Cameron, 1956). ln a few cases the exposed neural plaque is still
evident at the tíme of birth.. Some authors, therefore, call any lesion
with cord involvement a myelocele (Cameron, 1!!6; Laurence, 1964).
Roentgenograms of a typical mye I omen i ngoce I e at birth show absence
of spinous processes and reduction of the laminaeuextending from the
upper end of the lesíôn ¡nto the sacrum. The pedicles of affected
vertebrae are splayed out i¡n an oval shape, with the distances between
art¡culãr processes increasíng to a maximum at the centre of the lesion.
The intervertebral disc spaces are reduced, and there may be abnorr,ral
verticaì bars between the lateral masses of involved vertebrae (FiS.. 4).
ln many myelomeningoceles the vertebrôl bodies are well formed,
but some cases may show associated skeletal defects such as:
a) hemivertebrae with congenital scol ìosis
b) anteríor. wedging with congenital kyphosîs
c) partial or complete sacral agenesîs (Sharrard, l97l).
1.5.4 Myeloqlllqqele
A much rarer form of spina bifída cystica (representing another
form of myelocele) is the myelocystocele, ín which the leston contêlns
both meninges and dilated spinal cord, Thís is associated with local
enlargement of the centrai canal of the ¡ntact spìnal cord (hydromyelia),
so thêt the sac is not traversed by spínal nerves.

7
1.5.5 Anter ìor Spina _Bif idg
Flnally, defects of the vertebral bodies rather than the neural
arches may occur. These anterior spina blfidas appear to be of two
distlnct types ;
a) lsolated anter¡or meningoceles in the thgracic cr lumbar region
are very often associated w¡th cutaneous neurofibromatosis, and may be
a complicatîon of von Reckr inghausenrs disease (ta viene and campber r,
1958; Sammons and Thomas, '|959).
b) ln another group varying degrees of connection may occur between
the gastro¡ntestinal tract or an enteric cyst,ând the spina.l cord or
even overlying skin. These connect.ions pass through the anterior spina
blfida, which may be accompanied by dupl icatíon óf the notochord or even
of the spinal cord (Bremer, 1952; Fal lon et el., 1954).
ln some cases the neuro-ente¡¡c connectícn and.anterior spina
bífida are combîned with an open posterior spina bíf¡da (Saunders, 1943).
1.6 CRANIUM BIFIDUM
The term cranium bifidum may be used to embrace a comparable group of
open defects of the skul l.
1.6,I Anencepha,b¡, Exenqeph¡¡ Ly
The commonest of these malformations involving both skult Ur"in
ls "n¿
known as anencephaly, though the term is misleading as the brain is
rarely completely absent. riost fut term fetuses show ross of a variabre
amount of braín tissue and replacement by abnormal neuro-vascular materiar,
sometlmes called pseudencephaly (
Geoffroy_St. Hílaire, t836).(Fis. l).

8
However, several early embryos in the I iterature show wellpreserved
brain tlssue protruding through the cranial defect, formîng
an exencephaly rather thên the anencephaly of later stages (Huntet, 1|g34-?¡51 .
1 ,6 .2 t4en î njc!:e I e , Enrephq I gmed rìgoce I e
ln another group of lesions cranium bifidum is accompanied by
hernîation of cranial contents,without direct exposure of the brain tissue.
A cranial meningocele involves. herniation of meninges through the skull
defect, and may be compared to a meningocele of the spine.
Protrusion of brain tlssue as wel I as meninges through a cranium
blfidum constîtutes an encepha I omen i ngoce I e. This is perhaps nÌo re comparable
to â myelocystocele than to a mye I omen i ngoce I e în the spine,
as the herniated brain is invariably enclosed by meninges and sometimes
covered by normal skin (Bal lantyne, l!04).
1.6.3 Cran ium Bifidum Occultum
Finally, examples of cranium bifîdum occultum,without -hern¡ation of
the underlying brain or meninges,are occasional ly seen (Caffe)r, 1972)..
1.7 DYSRAPHIC STATES
The dîversity of neural malformations reflects the complexity of
neural development. However, ê fundamental áistinction may be made between
open and closed defects of the central nervous system. .Spina biîida and
cranium bifidum constitote the open defects (or dysraphic conditîons),
and are of considerable clinical împortance.
Open defects have been described in both human and experimental
embryos at stages of development as early as the perîod of normal neural
closure. Any experimental study of the pathogenesis of spina bifida and

cranlum bifidum thus involves the establ ishment of a regular serles of
speclmens between pre-neurulation and Iate fetal stages.
1.8 PROGNOS tS
The current emphasis on early surgical correct¡on, facilitated by
the development of antibiotics and the control led drainage of hydrocephal
ic fluîd, l^:s greatly increased the survivar of dysraphic infants
(Sharrard et al., 1967) . However where neural tissue ís involved the
damage is irreversible, so that anencephaly is always fatal vrhí le myelcmenlngocele
invariably produces some degree of neurological impairment,
The survival of an increasing number of affected infants poses serrous
social and economic problems (Tizard, l968; Lîghtowler, 1971). These
wîll only be avoíded when the dysraphic states themselves are preventable
through a fuller understanding of their etio¡ogy.

.1-
5

REV IEW OF L ITERATURE

l2
2.1 ErI0L0rL!! fïE qYs34t!1!!jrArEs
lnvestlgatîon of the etîology of neural defects falls into the two
separate fîelds of Epídemiology and Embryology. Ëp l clem i o log i ca l studies
descrìbe the distribution of defects within a defined population,and
attempt to analyse the factors producing this distrîbution. Embryologîcal
studies involve both human material and experimental lesions in animal
models.
The lack of unÍform termínology to describe the dysraphic conditions._
complicates any review of the I iterature, Host authors exclude the occult
I es ions and refer to:
a) anencephaly, pseuclen cepha I y, cranioschisis, meningocele or menîngoencepha
locele cran ial ly;
b) spîna bifida, rachischisis, meningocele, mye I omen i ngoce I e , myelocystocele,
or myeloceIe caudal ly.
ln addition, the lesions seen in experimental animals may bê called:
a) exencephaly, brain hernia, cleiencephaly, or acleiencephaly cranially;
b) myeloschisis or neuroschisis caudal ly.
2.2 E!_LDExlqror!!êL_gM t Es
2.2.1 lnc idencq
L'arge variations in the estimated incidence of neural malformations
are found in different publ ications. A comparative review of fifteen
hospital seríes (Alter, 19621 showed a frequency per l,OOO.births varying
between :
0.5 and 5.9 for anencephaly
0.2 and ).2 for spina bifida manifesta
0.J and 4.2 for hydrocephalus

t3
The value of such col lected series is limited by heterogeneìty of the
data,gnd correlation wìth naternal age and parlty. Stevenson et al.
(1966) therefore collected data from twenty-four centres simultaneously
and appl ied a correction for maternal age per thousand births. They
found that anencephaly, spîna bifida, encephalocele and hydrocephalus
occu!- th!'eughout the vlorld, though at very dî'fferent frequenc?es. The
hÌghest values were shown by Belfast and Alexanclría,with high levels
ln Helbourne, Bombay and Mexico City. Some towns in eastern lJales have
recently been shown to have an incidence as high as 12 per thousand
births of anencephaly and spina bîfida cystica combined (Laurence, 1976) .
2.2.2 Temporal Fluctuat¡ons ln lncidence
ln areas where records are available for a long periodr,gradual
changes in incídence mây be detectable. Rogers and Morris (197.|) found
that mortal íty from spina bifida in England and kales showed a steady
lncrease between 1848 and 1920, wirh a sharper r.ise between lgZO and ,l942,
followed by a declíne until the present (apart from rr"l i". peak in
"
1954 ).The recent fall in mortal ity might partly result from the improved
prognosis due to early closure, but the peaks must have some sepa!.ate
significancå. A dramatîc epidemic of anencephaly and spina bifida
occurred in Birl in between 1946 and 1950 (Gesenius, 1952). These postwar
European peaks, however, were not seen in New England.where a r+ther
uníform incidence of anencephaly and spìna bif¡da between 1890 and 1920,
and sharp increase between 1920 and 1932,have been followed by a ãteady
decl ine (Macl'lahon and Yen, 1!/t).
Edwards (1958) found that the overall reductîon in anencephaly and
spina biflda ln Bl rmingham and in Scotland since 1939 has not been
accompanîed by a similar fall in the íncÍdence of congenital hydrocephalus,

4
which has rema¡ned fairly constant.
2,2,3 Seasgna I Variation
ln Br'ltain durÌn9 the 1940ts and 1950rs thè rate of anencephalic
births was higher in winter than in summer (McKeown and Record, l95t;
Edwards, l95B; Record, 196t), Allowi.ng for prematurlty (commonly found
In anencephaly) the highest conception ,."r", to occur between
"OOu".ud
l4arch and July. This seasonal variation, however, was not apparent in
New England (HacMahon, Pugh and lngalfs, 1953) and has subsequently disappeared
în Britain (Leck and Record, 1!66).
2.2,4 Sex Rat io
ln both anencephaly and spina bïfida females predominate. The sex
rat¡o for spina bifida is around 1.2 (MacMahon, pugh and lngal.ls, 1953),
but ratios quoted for anencephaly vary between .I.1 (Sea.rle, .l959) and
4.2 (Coffey and Jessop, 1957). The reason for this disproportion is
not clear, though a hígher loss of male fetuses is often suggested.
Correlation of sex chromatin with sex. features shows concordance
in âlmost every case (Benirschke, 1966).
¡n contrast with dysraphism,the sex ratío for congenital hydrocephâlus
shows a slight excess of maìes (Record and McKeown, 1!4!;
Alter, 1!62), probably due to a group of sex-l inked cases (Shannon
and Nadler, 1968).
2.2.5 GeograÞh ic Di str î but ion
The ì,/.H.0. survey (Stevenson et al, 1966) allowing direct comparison
of hospital del iveries in different centres,shows wide variàtions between
different countries. Hany other studies of individual populations have
also shown local variations wlthin a country or region.

t5
ln general the lncldence of dysraphlsm shows an east-west cllne,
whlch decreases across Nôrth Amer¡ca (Hewitt, 1963) and increases across
the British lsles (Elwood, 1970). Th¡s pattern could be due to genetic
or envlronmental factors, or both.
2.2.6 Ethnìc DistributîoL
There are deÍlonstrable differences in incidence between different
ethnic ArouPs. living within the same region (Naggan and t4acHahon, 1967) '
ln the southern U.S'A. and in South Africa, Negroes show a much
I ot¡er incidence of dysraphism than whites (Alter, 1962; Penrose, 1957) '
Thls may partly be due to under-rePort¡ng.r ðs the ínc¡dence among
Negroes in Kenya is comparable to that in the white Population (Khan,
1965). A similar paradox is seen in lndia where reported dysraphism
ls uncommon except êrnong Sikhs, who show one of the h¡ghest rates in
the vrorld that persists after emigration (Searle, 1959) '
These ethnic differences within a community might also reflect
genet¡c or envîronmental variables, though some insìght into their
relatlve roles is offered by studies of imrnigration. l-ect (t969)
found that immigrant groups in Birmingham showed a change toward the
nBlformation .rates
of the host commun î ty, though marked ethnlc differences
were st¡ll âpparent. Th¡s suggests an envi rorimental modiflcatlon
of underlying genetic d i fferences.
2.2.7 Fami ly Studïes
Neural defects tend to be repeated in a slbship,but the recurrence
pâtterns of dysraphism and congenital hydrocephalus differ from each
other (!4acHahon, Pugh and lngal ls, 1950). The recurrence risk for
dysraphism has been calculated as about 53 after oi¡e affected slbllng

6
and about 112 after two (Laure,nce, 1969). These rates fall well short
of the 257. level suggesti,,,e of recessive inheiîtance (Pen'rose, l!å$) .
Twîn studies show a lower concordance than might be expected, with
a risk to the co-twin of about lt% (Record and l"lcKeown, 195]). When a
woman remarries the recurrence risk for maternal half-síbl ings is at
¡east ês great as for fuii sibiings (Yen and l"iact4ahon, 1968).
2.2.8 Maternal Age and Parity
Maternal age and parÍty are difficult to anâlyse separately. Dysraphic
pregnancies are commoner ín primipara and grand multipara than at inter- ..
medìate parities (Record and McKeown, ,|949). Some (though not all)
¡nvest¡gators êlso report a higher frequency in older mothers as an
independent effect (Edwards, t95B; Record, l96l).
2.2,9 Soc io- Ecojgdqlta tus
Poorer fami I íes show a higher incidence of dysraphism (Ceffey and
Jessop, 1957; Edwards, l95B; Pleydell, 1960) though nor of -other
lethal
bîrth defects (Anderson, BaÍrd and Thomson., I95B). This social gradient
however is not shown by Jewish famil ìes (Naggan and HacMahon, i967) and
does not apply to the Negro, populatíon of the southern U.S.A. (Alter,
1962) .
2.2.10 Urbanîzation
u".,1* ,JJreport
ê hígher incidence of dysraphísm in industriat
and urban communities, especially among poor familíes (Anderson, Baird
and Thomson, 1958; Edwards, .t958; Pleydelt, f960).
2.2.11 0o0c Llq íons_
Epidemiological studíes have thus revealed many assocîations but
no clear etîology for neural tube defects. lmportant environmental

t7
factors êre suggested by the correlation w¡th maternal age, parity,
economic status and urbanization, as well as by temporal fluctuatîons
and the discordance of most twins.
Genetic factors are suggested by the marked dìfferences between
ethnîc groups (only slov¡ly modified by ìmmigration), and perhaps by the
famîl ial trend and high proportion of affected females. However, if
a genet¡c component is ínvolved it is likely to be polygenic (Penrose,
l)jf; Carter, 1969).
2.3 EMBRYOLOGrcAl STUDIE!
The I iterature contains many descriptions of dysraphic human
înfants or abortuses, and many hypotheses to explain their development.
Unfortunately, the dearth of very early human mater¡al.limits the
extent of embryological studies. Experirnental teratology however can
partly compensate for this deficiency by producing simi lar,.malformations
in many animal nrodels, though the lesions.induced may not be strictly
comparable to the human defects.
2.3.1 Human Spec i mens
Anencephalic human fetuses are either stiìlborn or die soon after
birth. Most specimens show an open cranial vault with abnormal vascular
tissue- replacing the cerebral and cerebel tar hemispheres, but tÈe midbrain
and pons are usually present. The eyes, olfactory bulbs and cranial
nerves are often well developed, showing that different¡atîon of the
forebrain preceded the loss of cerebral and cerebel lar tissue. However,
this degree of differentiatíon is not ¡n itself evidence that the brain
had developed normally until the onset of these secondary changes.

l8
ln some very early human embryos wlth cranioschisís an exposed
mass of well-preserved brain tissue is seen, forming an exencephaly
rather than an anencephaly (Hunter, 1934-,35; van der Zrrran,..195l). Thts
suggests that exencephaly gives rise to anencephaly by necrosis and
sloughing of the exposed mass of brain tîssue, followed by vascularization
of the open area.
ln spina bifida cystica a comparable process wâs demonstrated
by Cameron (1956) who found that the basic dêfect was probably an open
neural plaque. Secondary overgrowth by squamous ep¡thelium and scar
tissue with accumulation of fluid suggested an încorrect diagnosis of
ul cerat i ng meningocele or rrmyelomeningocele't.
Several early human embryos with establ ished spina bif¡da cystica
do indeed show an exposed neural plaque and open ependymal cana! wíth
no covering of epithel ial or vascular tissue (lngalls, 1932i patten,
1953; Lemire et al,, 1965).
2,3.2 Productíon of Experimental Dysraphis¡n in AnÍmal Molþls
The belief that external factors may influence embryonic development
is an ancîent concept common to many cul tures. ln the nineteenth
century congenital malformations were frequently produced in lower
animals, but the mammalian embryo was thought to be protected by its
uterine envlronment. During the present century, however, experimental
terêtology has produced many defects in mammalian embryos, and demonstrated
many malformation syndromes in man due to environmental êgents.
Agents whích have been reported to produce experîmental dysraphism
in mammalian embryos by maternal treatment are shown in Table l.

9
TABLE l._
EXPERTHENTAL pySRApH_t SM tN MAMi"1ALt4N E}4BRYO_S
Agent Species Refe rences
X-rays rêt t{arkany a Schraffenberger, l!4/; Hicks, f954
. mouse Rugh ê Grupp, 1959
Hypoxla
mouse lngal ls et al., f953
Trypan blue rat Gillman et al,, l94B¡ 1951; ütarkany et al., l!!B
. mouse Hamburgh, 1952; l9I\
. hamster Ferm, l !!B
Hypervi taminosis A rêt Cohlan, 1954: Giroud Ê l,lartin et, 1957
hamster Marin-padilla o Ferm, l!6!
Dimethyl sulfoxide harnster Ferm, .l966
Sal icylates îìouse tJarkany ê Takacs, 1.959
Urethane mouse Sinclair, l9!0
Morphine mouse Harpel ê Gautieri, 1!68
Sodium arsenate hamster Ferm 6 Carpenter, 1968
Avian embryos are usuaìly treated dírectly, ei ther in ouo or .ín uitro,
and may be observed directly. Agents whích have produced experímental
dysraphism in chick embryos are shown ín Table 2.
TABI-E 2 . -
EXPERIHENTAL DYSRAPHISI,I IN THE CHICK EMBRYO .
Agent
References
X-rays Reyss-Bríon, 1956
Ultrasound Lutz et al., 1955
Ul traviolet-l ight Davis, 1!44
Vl ruses lJi I I iamson et al . , 1953
Robertson et al., 1960
Hypoxia Gàl lera, 1951
Hypercarbîa Gallera, 1951 , Lutz s Lepy, 1958

2l
and kinky-ta¡l as disorders of segmentation
c) congenital hydrocephalus and screw-têil as dlsorders of the membrenous
ske leton
d) diminutive, blebs, and disorganization as more generêl dîsorders.
This classificatìcn emphasizes the. diversity of processes causing
heredÎtary neural defects .in
a single experimental animal. The relatïonship
of hereditary lesions to experimental dysraphism in the mouse is
not clear.
2.\ HYP0.rHEgEs_!E JIE !t4!|RYoGENEslg 0F qlqRAPHIs¡,t
Many attempts have been made to explain the embryological mechanisms
producíng dysraphism, though some of these hypotheses are of only
h i stor ica I ¡nterest.
2.4.1 Sìmple Non-elosu[e
Von Recklinghausen (l886) suggested that â primary defect of neurulation
led to non-closure of the anterior poster¡or neuropore, followed
_or
by the invasion of epithelial and vascular tissuE,to produce anencephaly
or spína b îf ida manifesta.
2.4,2 OvergrowtLand Ncn-Closure
Patten (1952; 1953) described a marked infolding or excess of neural
tissue in several embryos, some of whîch had no external defect.. He
suggested that local rrovergrowthrr of the neural plate might prevent closure
and lead to dysraphism. More extensive overgrowth might be responsíble
for development of an associated hydrocephatus and the Arnòld-Chiari malformation
(Barry, Pêtten and Stewart, 1957).
2,4,3 Rupture of the Closgd_lleural Tubg
More recently Gardner (l9Sg; t964; 1972) and padger (1968; 1970)
have revived an alternative hypothesis, first proposed by I'lorgagn i (176Ð,

22
suggest¡ng that dysreph¡sm resul ts from di iation and rupture of a
previous ly closed neura l tube.
Gardner maintains that dilation of the closed neural tube could
êccount for hydrocepha I us, encepha locel e, hydromye I i a, syr ì ngomye loce I e
and the Arnold-chiarl malformatîon. Dilation and rupture of the neural
tube,followed by varying degrees of heal ing could account for exencephaly"
anencephaly, mye I omen i ngoce I e , meningocele, spína bifìda occulta, distematomyelia,
anterior spina bifida and various neuro-enteric connections.
2.4.4 AbnormAL-:Lrain G rowth
Because of the íntact hindbrain and well developed eyes and craníal
nerves' several authors have argued that anencephaly results from degeneratÎon
with¡n a fully-formed braïn.
Frazer (1921-22) suggested that ¡nadequate flexion of the enlarging
braîn shears off the major arteries at the base of the brain, producing
ischemic necrosîs and sloughing of brain tissue and the ovérlyíng cranial
vault.
2.\.5 Ab¡orEl *spinal Fl ex ion
Browne (1934; 1967) maintained that a variety of malformatíons,
including spina bifida, were produced by embryoníc compress-ion in uteto-
He suggested that rtundue spatial pressurerr might cause hyperflexion
of the primitive trunk and trinterfere w¡th the fusing oi the ridge
which should form the spinal canalr'.
2.\.6 Primary Vqscular Defects_
Anencephalîcs typically show a dÍsorganîzed network of sinusoîds
and anomalous pêttern of larger vessels ãt the exposed brain surface,
combined with relatively normal development of brainstem and midbraín.

23
Vogel and McClenahan (1952) tnus s.uggested thðt a prîmary defect of the
cerebral vessels produces local hypoxia and degeneration of the involved
area of the bra i n.
2,4,7 Amn i ot ic- Adhesions
During the nineteenth century fetal constriction by amniotic bands or
adhesîons was held to be responsîble for' many'congenital malformations
(Dareste, 1877). Certainl¡i amnÌotic adhesion to open.brain defects does
occur ín man (Torpin, 1968) as well as in experimental animals. However
this is now usually regarded ês the resul t rãther than the cause of
anencepha I y.
2.4.8 Abnormal Development of the Tai l-Bud
The origîn of the most caudal part of the spinal cord must differ
from that of the rest of the neuraxis, as the posterîor neuropore closes
before the definitive length of the embryonic axis has been achieved. The
terminal section of the cord is apparently developed by growth of the undífferentiated
taíl bud, at least in the chick (Romanoff, 1960; Hami ìton,
1952), the rat (Benrl iff and Gordon, .l965) and perhaps in man (Lemire, 1969).
Crlley (1969) demonstrated an area of overlap and fr,lsìon between these two
sources of neural material in the chick embryo.
tenire (1969) suggests that some neuraì malformations în the lower
lumbar and sacral regíons (especial ly those covered by skin) mig[t arise
by abnormal development of the tail bud material.
2.4.9 Trauma
Reviewing the co¡ lection of embryos studied by Sternberg fi929),
Pol ltzer (1954) note¿ that some narrow mídline braîn or cord defect'showed
a break in continuity between neural tissue and the skìn. He suggested that
these particular lesíons m¡ght be trãumatìc in origin.

24
2,4.10 lnfect ion
FInally, some of thè earlier authors such as Brouwer (1916) conãidereC
that the marked disturbance of neural tissue in anencephaly might be
due to embryonic infectionrcausing an extensive encephalomyel itis.
2.lt.11 Surlma rL
Many of these conflicting hypotheses are based on the study of
establ ished Iesions in human specímens collected at random. Assessment
of their validity requíres further stud¡es of early human embryos, and
experimental studies of the development of dysraphism in a range of
animal ¡nodels.

I'IATE R IALS
25

26
3.1 THE CHTCK EMBRYo
The chlck embryo was se¡ected as the expêrimental model in the
present study for the fol lowîng reasons (Hatthews et al., 1974):
a) fertile eggs êre cheap and avaìlable throughout the yeâr
b) avian embryology has a long history añd.an extensive I iterature,
with a well defined system of Staging based on simple morphological
criteria (Hamburger and Hamilton, 1951)
c) the developing chick embryo is more readily accessible than the
mammalîan embryo to dìrect observation and manipulatîon th.rough
a window in the shell (though thìs has certaîn concomitant disadvantages)
d) whole embryos as well as ísolated fragments can be cul tured in oityo
or 4n uiuo, with direct observation of the embryo by either technic.
Despîte the advantages offered by the varÍous in oitro techirics, an
in oi¡to (in ouo) method was finally chosen because:
a) it provided a method of producing open neural defects by the use of
a simple physical procedure
b) it allowed prolonged culture of embryos with various congenîtal malformations
to advanced stages of developmeni.
3.2 souRcE oF Ëqq ¿N!__!xlrjBiqll0N
The eggs used for this work were obtained from a second generation
hybrid tfhite Leghorn flock c.ontainíng one colored and three pure Leghorn
línes, maintained by the Department of Animal Science of the Uníversity
of I'lanítoba. The incidence of spontaneous malfornrations in this flock
cannot be establ îshed because of the reluctance of the oríginal commerc!at
suppl iers of the stock to supply appropriate information.

27
lf necessary, eggs were stored at 10oC for up to four days before use.
The two incubators used were both suppl ìed by the Blue M Electric
company (Blue lsland, ll l.). The larger convection model (2004) measured
48 cm. x f6 cn. x 4! cm.,and contained a 15 cm. x lB cm. x 5 cm. dísh of
water for regulation of humidîty. lt was used maînly for reincubatlon
of eggs after some form of treatment, especially in experiments involving
prolonged culture. Satisfactory regulation of temperature (37.50 t loC)
and humidity(60?. t 4%) was achieved.
The smal ler forced-draught model (VP-1004T-1) consigted of a 42 cm.'
x \2 cn. x 30 cm. plexiglass contaìner above a humidifier, wlth a fan
provídîng a continuous airflow at wel l-control led tenperature (37.50 t
0.5oc) and humidity (60Z ! 1Zl. lt was used mainly for short term
exper¡ments and for the ¡nitial incubat¡on of eggs before treatment
(Fis. 5).
The work descrîbed in thîs thesîs was performed ín a single room
facing south, with large windows and no air-condítioning. Regulation
of heating and ventilation gave partial control of the ambient env¡ronmentrand
the two incubators used provided stable incubation conditions.
Exper¡ments were performed throughout the yeâr.
J.3 OTHER EQUIPI4ENT
All operations were performed under steríle or semi-steriÌá
conditions in a dust-free plexiglass cabinet cleaned with 70% alcohol
before each exper iment.
stainless steel instruments, such as forceps, were steril ized
before use and passed through the flame of an alcohol burner several
tlmes during an operat ¡ on.

2B
To expose each embryo a wlndow was cut in the shell rvith a 1.5 cm.
dental separatíng dlsc on a 5 cm. mandrel,mounted in a hand_held
electrlc drîll (B¡ãck and Decker, model /010).
Albumen was removed from opened eggs wíth disposable sterile 5 ml .
syi'inges artd #16 gauge urrpointed needles (Bec.ton Dickinson e Co.).
After treatment, those eggs cultured with an artificial air_space
above the embryo were sealed with a J cm. circle of ster¡le parafilm
(American Can Co., Neenah, t^/is. ),attached to the shell by a ring of
plasticîne-l ike materiar (caurking cord, gtop Hardware products, ttontrear,
Ouá.¡. These eggs were not turned during subsequent íncubatîon (Fîg. 6).
Other eggs, from whích the introduced aír was removed (either by
re-expansion of the punctured air cell or by filiing the eggs rvith
albumen or Fl2 medium) were closed with â I cm. circle of sterile para_
film, sealed to the shet with a square of frexibre Erastoprast(product
1211; Smith E Nephew Ltd., Hull, England)

FÍg. 5.
lncubator allowing precise controi of temperâture
and humidity. Chamber contains a batch of
windowed eggs.
Fís. 6.
Windowed eggs seen frorn above,

GENERAL I.,IETHODS

3l
4. r sELEcr I oN !F EGGq
4.1,1 . lricub¿it i on
Experlmental and controì .eggs were rout¡nely cul tured from the
beglnning of lncubation at 37.54C and 60? humidlty, lying on thelr
sldes wlth the long axls horizontal.
At 26 hours of incubatlon (al lowlng one extra hour for rewarmìng
after remova l from the refrígerator) candl ing "ras perfo¡:med în the dark,
by layîng each egg horizontal ly over a 4 cm. x 3 cm. lîght-source and
marking the shell with a penci I at the site of the embryo
\ .1 .2 Cand I ïng
Ëmbryos were graded (accordlng to an êrbitrêry scale determined
by previous experience) ¡nto three sizes -small, medium and largewhîle
infertile eggs were rejected. ln this study only the eggs with
medlum-sized embryos were used. Eggs with large-sized embryos were
rejected, while those wîth smal l-sized embryos wåre al lowed to deveìop
for a further two to four hours and then used only if they had reached
medium size.
\.2 TECHNtq 0F_Q!!¡r!NG ANp cl0stNq EGGS
4.2,1 PreliminqllExperiments
ln ê ser¡es of prel iminary experiments, attempts were,made to produce
open defects of the central nervous system by the use of several proven
teratogenic agents introduced through a shall window. These experiments"
however that the well-known technic of openíng a wíndow in the
"showed
shel I above the embryo and removing 1 - 2ml . of albumen (to prevent adheslon
of the embryo to the cut edges of shell),was in itself highly
teratogenic at early stages of avian development. lllndowed but otherwise

7.2
untreated embryos showed almost as hÌgh an incidence of deaths and malformations
as windowed and treated embryos. l,loreover the defects produced
lnvolved predomlnantly the central nervous system.
4.2.2 Standard Techn îc
From thîs observation a standard experimental method was developed
which produced a high incídence of open neural defects. Each egg wíth
an embryo of medlum-size on candl ing at 26 to 30 hours of incubation
was wiped wíth a gauze square of 702 alcohol and cuts made in the shell.
During the operation the egg was held obliquely to avoid damaging the
embryo, and the shell membrane careful ly preserved. A short cut was
made over the air cell and a 1.0 cm. x 1.5 cm. hexagonal or rectangular
window made over the embryo. The egg was cleaned with 7Oy" alcohol and
transferred to a holder in tlre experimental cabinet, with the shell
window uppermost. The shelI membrane was then punctured gently with
sterile forceps, first over the air cell and then over the-embryo, allowíng
air to be drawn into the egg with col lapse of the air cell. 2 ml . of
albumen were then carefulìy withdrawn with a sterile syringe and widebore
unpoînted needle. E99s in which the vitelline membrane was damaged or
those where the embryo was not îmmediately beneath the shell window were
rej ected .
By this means embryos of quite a narrow range of developmenial stages
were exposed to the act¡on of an artíficial. air space, with no protect¡on
from an overlying layer of albumen but Iittle distortion from excessive
f latteníng or stretching.
\.2.3 Examinatíon of Embryo.s
As each blastoderm was fully exposed by this technic of windowing,
It was lnspected and its diameter measured 1n nillimeters with a plast¡c

uler. Accurate Staging (Hamburger and Hamllton, t95l) at the tlme of
wlndowlng was not attempted,as thls necessltêtes vltal stalnlng, whlch
has several disadvantages:
a) the tlme cluring whîch the egg rs out of the incubator rs increased
b) dlrect manipulation of the embryo ls unavoídable
c) teratogeníc effects bave been demonstrèted for several of the vltal
stains commonly used such as Nl le blue sulfate and neutrat red (Menkes,
et al., 1964).
For...lhese reâsons embryonic age êt the time of treatment was assessed in ":
terns of sîze rather than Stage.
\.2.\ Closure of Eggs
After measurement of the blastoderm, each e99 was sealed with a 3 cm.
clrcle of sterile parafilm applied to the shell with a 2 cm. ring of
plastícine, leaving ên artîfícial air space above the embryo (FÌS.7 ).
The eggs were reíncubated in a horizontar positíon without being turned.
' Thls technîc of wíndowing eggs with medium-sized embryos at 26 to 30
hours of incubation was found to produce open neurar defects ín about 502
of the treated ernb ryos .
4.2.5 Effec! of gmbryolíc Age
The relationshíp between embryonìc êge at the time of wrndowlng and
the development of neural defects rvas investigated by perforring'thu ,"r"
procedure on eggs at f4 and J8 hours of incubation.
l{here appl icabie, the significance of differences between experímentar
and control values in windowing experîments was determined Uy the Cni
Square test.

Ëigs.
t4. Human neurcr*sp ina I d)¡sraphism:
Fis. t.
Anencephal ic înfarit with typîcal facies and
a cap of neurovascular tìssue (pseudencephaly)
Fig. 2.
Craniorachischisis involving the bra¡n and
spinal cord. No external defect in the sacral
regîon.
Fis. 3"
Child with a healcd myclomenìngoccle lesion
and paralysis of the lower ìimbs.
Fis. 4.
Rad iograph of myelr:meningocele extending
from thoracic to sacral regions. Shows
l.ateral dìsplacement of pedicles, a lateral
bar, wedging of the body of L. 5, and
reduct i on of the sacrum

.\lz
'?.
t

JO
,\.3
REIN!UBATION AFTER. li,INDOh,Illc
A large humber of experimental and control embryos were fixed at
the tlme of opening (O hours) or 6, lB, 30, or 42 hours after wíndowing.
These embryos (designated Oc,6E,6c, lgE, lBc, 30E, 3oc, \28, and 42C)
were examlned, drawn by camera iucida, and selected for serial sect¡oning.
' other embryos were curtured for severar days to estabrish the fu
range of external malformations produce! by this techníc. Some were
fixed after five days, when the externar embryonic form was fuly estabr ished.
0thers were cultt¡red for ereven or twerve days to assess the rerationship
of neural de fects to skeletal development in the spine.
\.4 FURTHER tNVEsflçAIr0N 0J THE TERAToGENtc EFFECT 0F 0pENtNG THE SHELL
The teratogenic effect of the standard windowing technic might be due to3
a) vibration of the embryo by the dentð¡ separat¡ng disc during windowing
b) toxicíty of the plasticine or parafîlm used to crose the window
c) the ¡ntroduction of infectíon
d) a direct effect of the art¡f ¡ål air ipace above the embryo.
These possible factors vúere systematícal ry investigated by further experîments.
4.4. t V ibrat ion Alone
One group of eggs was subjected to vibrêtíon of the shelr above the
embryo for thîrty seconds(without opening a window) at 0,26 and-33 hours of
incubation.
4,4.2 Parafilm and plasticÍne Alone
ln another group the plasticine ring and parafîlm clrcle were appl ied to
the areas of shell overrying each embryo at 1,26, and 33 hours of incubation,
wi thout openíng the shell.

Fis. I AsB,
Windowìng followed by obl iterâtìon of the
ïntroduced air space by adding albumen or
F 12 med i um.

IÆ
6

Fis. 9 Aã8.
t/indow?ng followed by oblìteration of rhe
introduced air space by reexpansion of the
air-cel ¡.

4ì
,'ä:
1
Æ(o)
\/u
I

42
( \--l /)
^
)
t0

43
\,\.3 Effect of Artìficial Aìr S
The role played by the artificîal air space was thus further examined
by either restoratîon of the originâl stête of the eggs, or by modifications
of the external envíronment of unwindowed eggs during incubatîon.
Experiments designed to avoid exposure of the developing embryo
to the air space took two forms. After closure of the window with ê 3 cm,
circle of parafilm and a squaqe of elastoplast, a second 5 mm. x 5 mm,
window was made near the pointed end of the egg. ln some cases the egg vJas
fll ted with F, culture medíum (Ham, 1!6!),
cr with ålbumen from '
an egg of the same developmental age (fig, Bn ¿ s) .
Alternatîvely the introduced air was removed by re-expansion of the
collapsed air cell (with air from a rubber bal loon) before seal ing the
second window. (Pig. 9n a g) .
4.5 BACTERIOLoGIcAL CULTURE
The sterility of the operative techn¡c was assessed by making
bacteriological cul tures of albumen on bldod-agar plates at the time of
wîndowîng and at fixation of the embryos.
\.6 ExAMtNAT|oN oF EARLY EMBRyQ!
4.6.1 Fi xat ïon and Stagíng
,^oñto-;ãr,
embryos recovered 0 tq 4z.hours afrer windowins
were washed in Howardrs salÍne, fixed in Bouints fluid, and bleached in 702
alcohol contaíning 22 ammonia solution. Curl ing during fixatlon was prevented
by includîng a square of filter paper in the dish above the embryo.
For full details of the method see Appendíx A.
After bìeaching, all the embryos were examined for defects and Staged
(Hamburger and Hanilton, t95t)'. To Preserve a permênent record. before

44
serîal sectlonlng a camera lucida drawi.ng was made of every embryo.
4.6.2 Problems in Examlnation
Examlnation.of these early embryos presented severê'l problems:
a) Staging by somîte counts was not always easy because of necrosis or
cyst-formatlon in the somite regíons, and torsôon or opacity of older
embryos
b) the same processes often involved the neural folds, making assessment
of neural closure diffîcult
even death was not always obvious because some embryos showed
")
excessîve. necrosís when still alive, whereas other embryos without a beating
heart mîght be well-preserved though technically dead.
\.7 EXAMtNATIoN oF oLDER EMBRYoS
defects.
0lder embryos were fixed in Carnoyrs fluid and exdmlned for external
\.7.1 F ive Day Embryos
'
Some embryos were recovered at a totâl of fíve days incubation, with
the externâl configuration establ ished but well before the second peak of
embryonic mortal ity (Hamilton, 19!2).
\.7.2 Eleven - Twelve Day Embryo:
To correlate skeletal defects of the spine with spinal cord lesions,
however, a further period of development was necessary, "f,i"t'
resulted in
further mortal ity. As windowing produced some growth retardêtion, experîmenta¡
embryos were recovered at twelve days and control embryos at èleven
days of total incubation. After examination and measurement of all open
neural defects, the cartîlaginous skeleton was stained with alcian blue
(O;eda et al, 1970) and the embryos cleâred ln xylol and benzyl benzoate

4S
or 22 KOH. Three types of skeleta¡ defects were recc¡rded în the spine:
a) part¡al or conplete deletions of vertebrae
b) splna biflda manifesta in the region of an open neural defect
c) and spina bifida occulta.
Detalls of skeletal stainÌng and clearance of these older embryos are given
in Appendix B .
4.8 HISTOtOG¡cAt FEATURES OF. NEURAL cLosURE AND NEURAL DEFEcTS
u.r.,|
A representôtlve series of experirnental and control embryos (from
Sectlon 4.6), in good condition at fixation 0 to 42 hours after windowing,
were selected for serial sectioning. 0nly those control embryos showing
normal development and little necrosís were included.
After light staining with eosin in 702 alcohol, embryos were dehydrated
ln 802, 902 and 952 alcohols, processed wíth amyl acetate, and embedded in
pa raff í n wax.
Some !0,000 seríal sect¡ons were cut, gach at a thicknèss of 10 microns,
and stained with hematoxyl ìn and eosin.
4.8,2 Grouplnlqf E¡Þq¿os
Because of the rapid progress of the early part of neurulation (after
Stage B), exper¡nental and control embryos could only be compared ât identical
Stages. ln later neurulation several Stages were combinäd.
As no experimental embryos were available at Stage 9, the four groups
cons ¡ sted of:
Group 1 Stage 10
Group 'l 1 Stages 1 1- l2
Qroup 111 Stases tr3-16
Group 1V Stages I /-20

46
Thus the embryos selected for serial sectioning were rearranged
and examined by developnentar stages rather than hours of incubation,
4.8.3 súbdivislón lrirö Rédións
Examlnation of the serial sectrons reveared marked differences in
the development of the neural tube, notqchord., and somites at dìfferent
levels of al I embryos.
Each group of embryos was thus subdivîded into regions of neurar
development on the basls of non-neural morphological markers. These
markers were - found to correspond quite closely to developmental changes
along the length of the neural tube.
Because of the striking changes between Stage 10 and Stage 20 the
regîons werê not identîcal in each group, but were ¿rranged to encomp¿rss
the same developmental areas. For this reason the rela.tive size of each
region varied in the embryos of different groups.
The most important non-neural marker was somite mesoderm, divided by
reg lons in to:
a) presomite regîon: grouped w¡th the brain region,because after rotatÌon
of the head fotd the two areas courd not be separated rrn obr ique sections
b) upper and lower somite regions: grouped together for lack of çlear
morphol og i ca I separatîon
c) protosom¡te regîon: with club-shaped protosomites showing líttle
separation into dermatome, myotome, and sclerotome
d) unsegmented mesoderm region: with loosely arranged mesoderm not con_
densed into protosot¡î te s
e) posterlor region: with developing notochord ( p rotonotocho rd) still
continuous wlth developing mesoderm and neural t¡ssue.

47
I'leurulation showed a comparable series of changes characterîstìc
of each region în each group of control embryos. These changes Ìn the crosssectîonal
shape of neural tissue were described as:
a) closed neural tube O
b) closíng neural rube C)
c) inverted neural folds (-)
d) elevated neural folds tJ
e) everted neural folds --.\ñ
f) flattened neural plate
-vln
thís way the normal progress of neurulation could be closely
deflned for each group of embryos in terms of six regions,
(Tabtes 3 -6 ).

48
TABLE 3. srAGE t0 El,tBRyos (cnoue | ¡
Reg.i ons
l4a rke rs
A) forebrain (short)
opt ic veslcle J
midbraln
I
hlndbraln
t
presomlte area (short) f
1 no notochord
J
I
neural tube closed or closìng,
notochord, pharynx, heart, head
mesenchyme, anterior intestinal
Porta I '
B) upper somite area I neural folds closing or inverted,
lower somite area
Þ
J
notochord, somites.
C) protosomî te a reê neuraI folds înverted, notochord,
protósom i tes .
D) anteríor rhomboid sinus neural .folds elevated, notochord, unsegmented
mesoderm.
E) poster¡or rhomboid sinus neural folds elevated or flattened,
protonotochord, fused mesoderm.
F) Hensenrs node deep primîtive pit, developinþ neural
t i ssue.
prlmitive s t reak
shal low primitive groove, no neural
t l ssue,

49
TABLE 4.
. .STAGE. t 1:l2. .EtrtBRYoS. . (GR0UP. il)
Reg l onsr ...... . . . . . . . l-4arkers .
A) forebra in
optic ves¡cle
mldbrain
hlndbrain
p resom i te area
]
l
no notochord
neurql tube closed, notochord, pharynx,
dorsal aortae.
neural tube closed, notochord, pharynx,
heart, dorsal êortâe, anterior intesti¡al
portá I .
B) upper somi te area
Iower somi te a rea
C) protosomi te a reê
neural tube closed, notochord, somites,
heart, dorsal aortae.
neural folds closed or closîng, notochord,
p rotosom I tes.
D) anterior rhomboid sinus neural folds ¡nverted or elevated, notochord,
.upper overlap zone without accessory
cana I s, unsegmented mesoderm.
E) posterior rhomboid sinus neural folds elevated, protonotochord,
'
overlap zone with accessory canals, fused
mesoderm.
F) Hensen¡s node
prim¡t¡ve s t reak
deep primitive pit, developing neural tissue.
shallow primitive groove, no neural tissue.

50
TABLE 5 ,
Reg i ons
srAGE 13:16' EMBRYOS (enour ttt¡
l'larkers
A) forebra í n
mldbra in
hlndbrain
presomîte a rea
(åbsent by st. 16)
B) upper somite area
lower somi te a rea
c) protosomi te a rea
-t
)
no notochord
overlappîng mî dbra i n and hînclbrain,
otocysts, notochord , pharynx, heart,
dorsa I aor Èae.
notochord, pharynx, heart, dorsal aortae
neural tube closed, notochord, somîtes,
anterior intestinal porta I .
neural tube closed, notochord, protosomites,
upper overlap zone.
D) unsegmented mesoderm area closed neural tube dorsal to accessory
canals, notochord, unsegmented mesoderm.
E) caudal a rea
closed or closing neural tube dorsal to
accessory cana I s, protonotochord, fused
mesoderm.
f) anterior tai t-bud
(absent by St. 16)
posterior tai I -bud
short primitive streak
sha I low surface pit,
no notochord.
no surface pit.
prîrnitive groove,
(absent by st. 16)

5l
TABLE 6,
%
STAGE lT-20 EHBRyos lcRo p tvl ... ... . ..
Reglons Markei.s.. .......
B) prebrachial cord -l overlapping brain and cord, notochord,
forebrain
brachial cord
forebraln
postbrachla¡ cord
J
1
J
It
somltes, foregut, heart, dorsal aortðe.
overlapplng braln and cord, notochord,
somltes, wîng b,uds, single dorsal aorta.
cord, notochord, somites, midgut, dorsal
aortae, mesonephroi , Wolffian ridges.
C) crural cord cord, notochord, somites, leg buds,
mesonephroi , mesonephric- ducts, cloaca.
D) postcrural cord cord, notochord, somites or unsegmented
rnesoderm, cloaca, developing tail .
F) tail tip no cord, no somites,
E) caudal cord cord, taÍl-bud, caudal somites or unsegmented
mesoderm, protonotochord, caudal gut.

52
4.8.4 HistologÌcal_De:plþtÍons
To test the hypothesis that delãyed onset of the passage of cerebrosplnal
fluíd across the rhombic roof might produce rupture of the closed
neural tube, the progressive thinning of the rhombìc roof was tabulated
for each group of embryos (Tables 113-46
).
The possìble contributicn of excessive cãvitation of the tail-bud to
abnormal neurulation was assessed by tabulating the nunbers of accessor¡.
canals in the overlap zone and tai l-bud of each group (Tables 5l - 54).
However, a detal led .ieview
of the histological changes during neurulation
revealed varlous dífferences between experimentâl and control embryos
(Section 6.4.). These differences were thus analysed ín terms of the
reprèsentative ãppearance of Regions A-E in each group of embryos, to
determine whether they were significant (Tables 35-38).
4,9 ANALYStS oF NEURAL cLosuRE
From the results of the histological studieè (section-6,4)
several aspects of neurulation were analysed quantÌtatively, as percentages
of each regilon and percentages of each embryo (Tables. 47-50),
Histological features analysed ín this manner were:
a) progress. of neural closure
b) extents of myeloschisis
c) extents of myelodysp I as ia
d) length of the overlap zone
e) cover of neural tissue by ectoderm
f) contact of neural tissue with notochord
S) contâct of neural tlssue wîth somîtes
h) possible reduction of somìte volume with cystic changes.

53
Because of the difficulty in separatíng the brain from the upper
cord this analysis was confined to the spinal cord and tail-bud (Regions B,
C, D and E).
4. to ANALYS ts !r ],IEURAL voLUt4Es
Flnally, an attempt was made to compäre the volumes of neural tíssue
în embryos with and without neural lesions over regions C and D. To
el iminate variations due to dífferent sizes of indivídual embryos,and
dîffering lengths of regions C and D, values were expressed in the form
of ratios of mean neural tissue to mean notochord (which showed little
fluctuation), and analysed by multiple T-tests.

5 RESULTS OF TERAÎOLOGICAL PROCEDURES
54

55
5.1 TERAToGEN!! !FFECT 0F,WtNp0I{1!Nq
Tt¡e effect of windowîng 999s in the early developmental period
ls closely related to embryonic age. To învestigate this effect, the
standard wlndowing. technîc was performed on eggs lncubated for l\, 26
and 38 hours. After windowi.ng, enbryos were reincubated to a tot¡l of
/2 hours.
Eggs ln the control group were not opened, but were removed from
the lncubator for a simîlar length of time to the experimentâl groups.
None of the eggs were turned.
4t.72 hours all the embryos were fixed and examined under a
dissectlng mlcroscope. Table 7 and Fîg. l0 show the mortal lty and
overall malformation rates for experrmentar embryos windowed at r4, 26,
and l8 hours, together wlth the control embryos.
Hany experlmental embryos, especially those windowed at l4 hours,
showed early death and such complete degeneratìon that embryonic
structures were.unrecognizab,le within the_smal I nodule of necrotic tissue.
For this reason, individual malformation rates are not given as percentages
of the number of embryos whìch continued to develop after windowing.
survivî.ng embryos deveroped to stages 13-20 and were recovered dead
or al ive, w¡th or without malformations, at 72 hours. Fig.l0 ¿s¡q¡strâtes
a decrease in early deaths betv,reen the four groups, and.an increãse in
survival wlthout defects âmong the embryos which contínued to develop.
Statlstical analysls reveals slgnificant dlfferences in:
(a) early deaths and survivrng embryos between the combrned experimenta¡
groups and the control group (p < O.Ol)
(b) early deaths and survlving embryos in each experimentêl group
(p < o.ot)

(c) deaths and defects in the surviving embryos of each experimental group
(p < o.o5) .
56

Numbers of Eggs 31 73 31 135 47
Eârly Deaths 28 (90.32) zo (27.40) 2 (6,4Ð 50 .2 (\.26)
Developing Ernbryos 3 53 29 gS
45
38 (80.85)
1 ( 2.13')
r ( 2.13)
5 (10.64)
TABLE 7. MORTALITY AFTER l,/lNpOwtNG AT 14, 26.and 38 HoURS
. llindow ì./indow l,/îndow Ëxperimentêl No VJindow
at l4 Hrs. (%) at 26 Hrs.(?) at !8 Hrs. (%) 'iot"ilcontràrr
tzl
Al ive wíth No Defects
Al ive wi th Defects
Dead wi th No Defects
Dead wi th Defects
0 (0)
1 (3.26)
0 (b)
2 (6.\5)
21 (28.77)
25ß\.25)
1 ( r.37)
6(8.22)
17 ß4.84)
I (25.81)
2 (6.4r)
2 (6.45)
38
34
?
10
Stages at Fixat ion 13-17
15-20
13-'t9
13-20
14-20

Fi g.
Percentagcs of early deaths and later deaths
and deformitîes after windowing aL 14, 26 and
JB hours. Embryos recovered at 72 hours.

14 HRS" 2ó HRS. 38 HRS. CONTROLS
:
Nl Eorly Deoths
N=182
N Al¡ve, No Defecfs
Nl Alive, Defects
Nl Deod, No Defects
N Deod, Defecis
tn
o
cô
t, ''
tu
u-
o
Èe
MORTALITY AFTER WINDOWING AT .14,26 & 38 HOURS

6o
MoRTALtTY, AFTER ì,'tNpowtNq,AT t¡;26; AND 38 H0uRS
. Analysís of early deaths and developing embryos in the combîned
experimental groups and the control group:
Observed Va I ues 50 2
85 .4s
D.eg rees of Freedom
t
Chi Square (Yates Correction)' 16.7g
P < 0.01
Analysls of early deaths and developlng embryos ¡n the experlmental
I roups :
0bserved Val ues
Degrees of Freedom
Chl Square
P
28202
35329
2
53.09
< 0.01
Analysls of later deaths and defects in developing embryos of each
expêr lmenta I group:
Observed Va I ues
Degrees of Freedom
Ch i Sq ua re
P
021 17
1258
012
262
6
14.46
< 0.05

6t
0n examlning. the survivi.ng embryos (table I ), the commonest malformations
were found to lnvolve the central nervous system and the eyes.
The braln was often reduced în slze Ìn the experlmental embryos, as well
as ¡n some controls. Two embryos windowed at 26 hours, and one control
enbryo showed an open anterlor neuropore. As. the bráin normally closes by
Stage 12 (Haml I ton, 1965),. these embryos were regarded as showìng open
braln defects after Stage 12. Closure of the rhomboid sinus, which is
normal ly complete by Stage 15 (Haml t ton, t965), was êssessed in the four
groups. ln many embryos wlndowed at 26 hours the neural folds proximal
Éo the rhomboîd slnus were st¡ll open. At Stãges 13 and 14 these open
areas were adjacent to the rhombold slnus, but after Stage 1! they
extended up lnto the somlte reglon. As these defects were ín continuity
wlth the rhombold sinus they appeared to aríse through non-closure of the
neural folds. ln embryos older than stage l6 they are therefore recorded
as open defects of the neural tube.
Many embryos, especially those dead by /2 hours, showed hemorrhages
and cysts of the trunk, sometimes causîng great distorsion, and obscuring
the structures in this region. There were few non-neural defects apparent
by 72 hours.

TABLE B.'DEVEIOPI{ENT'AFTER'WINDOIiIING AT 14;'26:IAND' 38'HOURS
Wlndowing Windowing l,/î ndow Î ng No
at..14 hr;. .. . at 26 hr.s, . at..38 hrs. \Jindowing
Developing Emb ryos
53
45
open Anterior Neuropore
tJ
2
¡
0pen Rhomboìd S inus
?
5
2
\
Open Neural Tube Defects
I
25
3
0
Mlcrocephal y
3
t4
5
4
Eye Defects
1
4
3
2
Facia I Defects
1
0
2
0
Cardiac Deiects
2
3
0
0
Trunk Cys ts
0
6
3
0
Limb Bud Defects
0
0
0
0

63
5.2 MALFoRMAT I 0NS .P¡OpUCEp BY \ir l ND0Vill NG .
Analysîs of the nalformatlons produced by the windowing technic
required culture of treated embryos to the perìod when the external embryonic
form had been establ lshed. By this time, however, the mortal ity among
developing embryos was substantial, and many of the dead embryos were so
necrotlc that full examìnatlon was impossible.
For thîs reason, embryos wlndowed at 26 hours and developing to
5 days or 12 days were compared to the 72 hour (3 days) group in Section
5.1.
-
Rs ihis was an analysis of the external malformatîons produced by
the wlndowing technlc, it includetl only well-preserved experimental embryos
showing con'tinued dèvelopment after windowìng. Table 9
and Fi9.11
show the external defects recorded at 3,5 and 12 days of incubation,
expressed âs percentages of those developlng embryos which could be examined
fully. These values do not represent the ou..ull malformadion rates in
treated embryos but, rather, reflect the changing pattern of malformations
wi th prolonged g rowth .
ln Fi9. Il the most striking finding is the uniformly high incidence
of open cord defects in all three groups.
0f the non-neural ma I fo rma t,îions , def ects învolving the face-and beak,
trunk, rump and ta¡1, anterior body wall,and lower I imbs appear with íncreasing
frequency at progressively later stages of development.

TABLE 9. ¡4ALFORMATIONS PRODUCED'BY [TINDO\iING'AT'26'HOURS
?:?:v: {ll::: ¡ ?ly: {Tì
l2 Days (Z)
Developlng Emb ryos
53
109
69
open Cord Defects
25&7.12')
ç3 (57 .8)
41(59.42)
0pen Bra ln Defects
2(3,77)
4 (3.671
6 (8.70)
Ml crocepha I y
14(26.\z',)
1o (9. r 7)
6 (8.70)
Eye Defects
\0.55)
\2(38.53)
19Q7.5\)
Face E Beak Defects
0 (0)
1l (10.09)
21(30.43',)
Trunk Defects
6(11.32)
8 (7.34)
lo(14.49)
' Rump e Tail Defects
0 (0)
30(27.521
31(44.93)
Ectopia V I sce rum
0 (0)
0 (0)
27 ß9.13)
Lowe r Llmb Defects
0 (0)
\ß.67',)
17 Q\.6\)
Upper Llrnb Defects
0 (0)
1(0.92)
0 (0)

Fis.
Percentages of neural arrd non-neural defects
in survîvìng experimental embryos windowed
êt 26 hours. Embryos recovered at 3, 5 ancl
12 days.

N=231 ffil e ooyi
ffi s oays
[il t2 ooys
OPEN CORD ÞETECTS
OPEN BRA¡N DEFECTS
MICROCEPHALY
EYE DEFECTS
FACE & BE.AK DEFECTS
TRUNK DEFECTS
RUMP & T,AIL DEFECTS
ECTOPIA VISCERUM
LOWER LIMB DEFECTS
0r020304050ó0
EXTERNAT MATFORMATTONS (%) AFTER WtNDOW|NG
(2ó HOURSI

67
5.3 INVFSïIGATtON 0F THE TERAToGENTC EFFECT:0F t;/lNpor,ilNG
ln an attempt to define some of the factors leading to abnorrnal
development after windowing, several experiments were performed to
investigate varlous aspects of the windowi.ng technic, ln one group of
experiments eggs were trear:ed bv vîbration, of by the applicatíon of
parafî lm and a plasticine iing, wÌthout windowîng. ln other experiments
the ¡ntroduced air space was obl iterated at the time of windowing.
5.3.1 Vibf¡itiori óf UnoÞened Eggs,
,.m;ratÎonProducedbythedentalsandIngdiscusedto
cut the shell wlndows, unopened eggs were vlbrated for J0 seconds wlth a
carborundum ball mounted on the dr¡ll. Thls procedure was performed ât
0, 26 and 33 hours of încubatlon, and the eggs then relncubated to a total
of / days without belng turned.
Tables 10 and tt show the mortal ìty and malformation rates for the
three experîmental groups and the controls. Bêcause of the relatively
small numbers ín each group, values are not converted to percentages.
Desplte the small numbers ît is clear thðt V¡brat¡on by itself is not
responsîble for the teratogenic effect of the windowíng techníc. There
werè no open èord defects in any of the experimentêl or control groups.

6B
TABLEt loj lloRTALtTy AFTER VtBRAT|0N ALoNË AT 0; 26 AND 33 H0URS
Vi bra t ìon Vl brat ion Vibrat i on No
at 0 Hrs. at 26 Hrs. at 33 Hrs. Vibration
Number of Eggs
12
22
10
22
Early Deaths
0
1
0
1
Developlng Emb ryos
Al ive wlth No Defc.cts
12
II
21
r5
10
9
21
18
Al lve wîth Defects
1
3
1
2
Dead with No Defects
0
0
0
I
Dead wîth Defects
0
3
0
0

69
ÏABLE 1 I. MALFORMATIONS AFTER' V I BRATI ON'AIONE'AT O ; . 26' ärid' 33 HOURS
Vi brat ion Vìbration Vibration No
at 0..Hrs. . .. at.26..Hrs,......at..33..Hrs.... Vibration
Numbers of Emb ryos
12
22
10
22
0pen Cord Defects
0
0
0
.0
Open Brain Defects
1
I
0
0
Ml crocepha ly
0
2
0
1
Eye Defects
0
3
0
t
Face E Beak Defects
I
0
0
0
Trunk Defects
1
3
0
0
Rump e Tai I Defects
2
0
0
0
Ectop la Vlscerum
0
0
I
0
Limb Defects
0
0
0
0

70
5,3.2 Pa-rôf i Idì ¿irid PIèsticIrie l,Jìthoút 1^1î ridoli,î n9
Chemical agents ¡n the plasticÌne or parafi lm used to seal rv i ndowed
eggs, rather than vîbratîon by the dental disc, might alternatîvely be
responsible for the teratogenîc effect of windowlng. This possibil ity
was tested by applylng a parafílm circle and.plasticìne ring to the
intact shell overtylng three groups of embryos.
Eggs were first candled to locête the postion of the embryos,
and the plasticîne,/parafl lm covers applìed at 0,26, and 33 hours of
incubation. Embryos were recovered after a total of / days.
Tables 12 and 13' show that the mortal ity and overall malformation
rates are lowest ln the control group and hlghest in embryos treated
from the begînning of incubation. Because of the smaìl numbers in each
group values are not converted to percentages. The results, however,
show that there ls no obvious teratogenic factor emanatîng from the parafilm/
plasticlne covers alone. ln the control group there was onu ,pont"nou,
open cord defect.

71
TABLE I2;},IORTALITY AFTE-R PLASTICINE,/PARAFILH ALONE AT O; 26 AND.33 IIOURS
' Cover Cover Cover No
at 0 Hrs. at 26 Hrs. at 33 Hrs. Cover
Number of Eggs
Early Deaths
Developlng Emb ryos
Al lve wlth No Defects
lt
2
?
r
2
18
1
17
12
12
0
12
t0
32
0
32
26
Al ive with Defects
3
I
4
Dead wi th No Defects
1
0
1
I
Dead wl th Defects
1
2
0
t

TABLE 13; MALFORI4ATIONS AFTER PLASTICINE/PARAFILl:I'AtOi{E
Cover Cover Cover
at 0 Hrs. at 26 Hrs. at 33 Hrs.
No
Cover
Numbers of Emb ryos
0pen Cord Defects
0pen Brain Defects
Mi c rocepha I y
Eye Defects
Face ê Beak Defects
Trunk Defects
Rump € Ta 1l Defects
Ectopla Visceru¡h
Limb Defects
11
0
0
0
0
0
0
2
1
0
12

73
5.3.3 .o¡literation,lof
lt¡tro¿u¿¿¿
eir space
There fs llttle evidence that vibration of the eggs or materials
ln the p!astîcine/parafilm covers are responslble for the malformations
produced by wÌndowÌng. Thîs suggesb that the presence of an introduced
alr space above the developing embryos may be the causa¡ agent. To
test this posslbilîty the lntroduced alr space was oblíterated in three
dlfferent ways at varying ìntèrva¡s after windowing at 26 hours of
lncubatlon. ln each experlment eggs were sealed wîth parafilm and relncubated
to 72 hours wlthout turnlng but w¡th the sealed lirindow facing
downwards (figs. 8n and B and 9A and B),
' ln the first experiment the lntroduced aîr space was filled with
albumen from unwindowed eggs of the same batch, at 26,32,38, 44 and 50
hours of încubation (or 0, 6, 12, l8 and 24 hours after wîndowing).
Tables 14 and 15 and Fig.12, show that mortal ity and the overall malformation
rates decl ined with earlier obl iteratíon of the air space.
ln the second experiment F 12 culture medium (ttam, I965).
was used to obl îterate the introduced air space êt the same intervals
after windowing. Tables l6 and 17 and Fi9. l3 also reveal a
reduction in.mortality and overal I malformation rates with earlier
obliterat¡on.
Finally, reexpansion of the air cell with a rubber ¡al loon,to
dlsplace the introduced air space, was performed at 26, 38 and 50 hours
of íncubation (or 0, 12 and 24 hours after windowing). Tables 18 and
19 and FiS. l¡r show that aîr cell reexpansion also reverses the terâtogenic
effect of wîndowing, especlal ly when performed Ímmediately.

41
13
0
23
No l^l i ndow
Control s (Z)
l1
l1
o (o)
9 (81 .82)
2(18.18)
0 (0)
0 (0)
TABLE 14. I4ORTALITY FOLLO}'ING INTRODUCTION OF ALBUMEN AT,VARIOUS INTERVALS AFÎER WINDOT,.IING
Al bumen
at
50 Hrs. (?)
Al bumen
at
44 Hrs. (Z)
Al bumen Al bumen
at at
JB Hrs. (l) 32 Hrs. (%l
Albumen Tota I s
at with
26 Hrs. (?) Albumen
Number of Eggs 17 27
Earfy Deaths 4(23.53) 2(7.41')
Developing Embryos 13 25
17 15
,+(23,531 2(13,33)
13 13
13 89
o(o) 12
13 77
Al ive w¡th No Defects
Al Íve wi th Defects
5Q9 .\1) 1 1 (40. 74)
2(11.76) 4(14.81)
9$2.94') 5ß3.33)
2(11.76) \(26.67)
1 1 (84.62)
1(7 .69)
Dead wl th No Defects
0 (0) 0 (0)
0 (0) 0 (0)
0 (0)
Dead wî th Defects
6(35.29 10(37.04)
2(11.76',) \(26.67)
1(7 .69)

No t'i i ndow
Controls
0
0
0
1
0
0
0
0
TABLE I5. I'{ALFORHATIONS FOLLOI"'ING INTRODUCTION OF ALBUMEN AT VARIOUS INTERVALS AFTER I,JINDOl^'ING
Albumen Albumen Albumen Albumen Albumen
êt 50 Hrs. at 44 Hrs. at 38 Hrs. êt 32 Hrs. at 26 Hrs.
Open tord Defects
'5
0
1
0
Open Brain Defects
I
2
I
I
0
Microcephaly
6
5
1
4
0
Eye Defects
5
1
2
6
1
Facl al Defects
1
2
2
0
0
Trunk Defects
0
0
0
0
n
Trunk Cys ts
t
?
0
2
t
Limb Bud Defects
0
0
0
2
0

Fíg.
Percentages of early deaths and later deaths
and deformities fol lowing ìntroduction of albumen
at varying perïods after windowing at 26 hours.
Embryos recovered ðt 72 hours.

N Eorly Deoths
N=100
.n
9óo
d
co
=!r¡
lJo40
ñ
N
N
N
N
Alive, No Defects
Alive, Defects
Deod, No Defects
Deod, Defects
5O HRS.
44 HRS. 3B HRS. 32 HRS. 2ó HRS. CONTROLS
MORTATITY AFTER INTRODUCTION OF ALBUMEN
AT VARYTNG PERIODS AFTER WINDOWING {2ó HOURS}

78
ALBUMEN INTRODUCTION
Analysís of early deaths and developing embryos ín the combined
experîmental groups and the control group:
Observed Val ues 12 0
77 lt
Degrees of Freedom 1
Chî Square (Yates Correction) 0.65
P
groups:
N.S.
Analysís of early deaths and developing embryos ¡n the experimental
observed Values 4 2 4 2 0
13 25 13 13 13
Degrees of Freedom 4
Chi Square 5,82
P
N.S.
Analysis of ìater deaths and defects' în developlng embryos of
each experimental group cannot be performed because of 0 vˡlues for
embryos classified as Dead w¡th No Defects.

F 12 I'IEDIUH AT VARIOUS INTERVALS AFTER WINDOI,/ING
97
8
89
33
33
4
r9
9
0 (0)
9
7 07 .78)
1(11.11)
1(11.1f)
0(0)
\l
\o
. F12 F12 F12
ât at at
50 Hrs. (t) 44 Hrs. (Z) 38 Hrs. (?)
F12 F12
at at
32 Hrs . (Z) 26 H rs . (%)
Totals wî th No Window
F12 Control s (Z)
Numbers of Eggs
Êarly Deaths
Deve lop ing Emb ryos
16
2(12.501
l4
32 ?o
4(r2.50) 1(5)
28 19
14
13
r(7.r4)
15
15
o(o)
Al ive wlth ¡¡o Defects
2(12.501
11(34.38) 6(30)
5ß5.71)
e (60)
Al îve wîth Defects
Dead wÌth No Defects
3fi9.75)
0 (0)
9þ8.13, 10(50)
4(12.50) 0(0)
6 (42.86)
0 (0)
5ß3.331
0 (0)
Dead wìth Defect s
9G6.25')
4(12.50) 9(ì5)
2(14.29)
t (6.67)
.,,.

0
1
0
0
a
o
TABLE 17. I'lALF0RÌ1ATi0NS F0LL0l/lNG INTRODUCTI0N 0F F 12 MEDIUM AT VARI0US INTERVALS AFTER !/INDO!/ING
F12 F12 F12 F12 F12 No \^l indow
at 50 Hrs. at 44 Hrs. at 38 Hrs, at 32 Hrs. at 26 l1rs. Controls
open Cord Defects
Open Brain Defects
6
0
I
I
0
1
2
1
1
1
0
0
Microcephaìy
10
5
E
2
3
Eye Defec t s
B
3
2
1
2
FacÌal Defects
2
0
1
0
1
Trunk Defects
I
I
0
4
0
Trunk Cys ts
L imb Bud Defects

Fis.
13.
Percentages of early deaths and lâter deaths
and deformÌties following ìntroduction of
F 12 medium at varying periods after windowing
at 26 hours. Ernbryos recovered at 7Z hours.

N Eorly Deothi
N=10ó
N
a
ú,
o
d,
cô
=¡t¡
u-
o
Èe
ó0
N Al¡re, No Defects
N Al¡ve, Defects
Nl Deod, No Defects'
Nl Deod, Defects
50 HRS.
44 HRS. 3B HRS. 32 HRS. 2ó HRS. CONTROLS
MORTATITY AFTER INTRODUCTION OF FI2 AT VARY¡NG PERIODS
AFTER WINDOWING (2ó HOURS)

B3
Fl2 INTR0DUCTtqN
Analysis of early deaths and developing embryos in the combined
experimental groups and the control group:
Observed Va I ues 8 0
899
Degrees of Freedom
I
Chì Square (Yates Correction) 0.06
P
N.S.
i
group:
Analysls of eorly deaths and developing embryos în experimental
0bserved Values 2 4 1 1 0
Degrees of Freedom
14 28 19 13 15
lr
Chi Square 2.80
P
N.S.
Analysls of later deaths and defects in developing embryos of
each experimental g roup:
Observed Va I ues
Degrees of Freedom
Chi Square
P
211 659
3glo65
04000
94321
12
30. 44
< 0.01

103
51
52
28
14
4
6
No W î ndow
Control s (2)
11
tt
0 (0)
r0(90.91)
1 (9 .09)
0 (0)
0 (0)
CELL REEXPANS iON AT
Not Reexpand. Reexpand. Reexpand.
Reexpand.(8) at êt ar
50 Hrs. (*) 38 Hrs. (B) 26 Hrs. (%)
l/lNDol.,lNG
Tota l s
Reexpanded
I'lumber of Eggs \7
23
23
10
Early Deaths 29ß1 .7ol
1 1 (47.83)
1 1 (47.83)
0 (0)
Developing Embryos lB
12
12
t0
Al Íve with No Defects 8(17.02)
5þ1.741
5Q1.74)
1o(1oo)
Al îve with Defects 5(10.64)
501.7\)
4(17.39)
0 (0)
Dead with No Defects 1(2,13)
1 (4.35)
2 (8. 70)
0 (0)
Dead wi th Defecrs 4(8.51)
I (4 .35)
1 (4.35)
0 (0)

No Wi ndow
Control s
0
0
0
0
0
1
0
0
co
Not Reexpand.
Reexpand. at 50 Hrs.
ANS ION AT VAR I
Reexpand.
ãt 38 Hrs.
Reexpan d .
at 26 Hrs.
Open Cord Defects
7
2
1
ll
open Bra in Defects
2
0
2
0
Mi c rocepha I y
2
0
I
0
Eye Defects
5
1
2
0
Facial Defects
4
0
2
0
Trunk Defects
0
1
0
0
Trunk Cys ts
0
3
1
0
Lîmb Bud Defects
0
0
0
0

Fi g. 14. Percentages of early deaths and ìater deaths
and deformÍtìes fol lowÌng reexpansion of the
air-ceìl at varying periods after windowing at
26 hours. Embryos recovered êt 72 hours.

NOT REEXP. 50 HRS. 38 ¡-IRS. 2ó HRS. CONTROTS
¡.\
oo
N Eorly Deothi
Nl Aliue, No Defects
Nl Alive, Defects
N beod, Nlo Defects
N Deod, Defects
MORTATITY AFTER REEXPANSION OF AIR CEIL AT..VARYING
pERroDs AFTER WTNDOW¡NG 12ó HOURS)

88
AtR qELL, REEXPANSI0N
Analysls of early deaths and developing ernbryos in the combined
experímental groups and the control group:
Observed Val ues Sl 0
Degrees of Freedom
52 tl
Chi Square (Yates Correction) 7.g5
p
. O.OI
l
groups:
Analysis of early deaths and deveroping embryos in the experimentar
Observed Values 29 lt ll 0
18 12 12 rO
Degrees of Freedom 3
Ch i Square n.65
P < 0..01
Analysis of later deaths and defects in developing embryos of each
experimental group:
Observed Val ues
Degrees of Freedom
Chi Sq ua re
P
8 5 5 ro
5540
r120
41 o
9
14.05
N.S.
\

Ro
Comparison of Figs. 12, lJ and 14 shows thät obliteration of the
introduced air space reduces its teratogenic effect, part¡cularly when
performed immediately after windowíng. comparlson of the statisticêl
analyses, however, shows some incons¡stencies between the three
expe r¡ ments 3
(") early deaths and deveroping embryos in the combined experimental
groups and the control group:
albumen ¡ n troduct ¡on
F12 înt roduct i on
air cell reexpanslon
N.S.
N.S.
P < 0.01
(b) early deêths and developing embryos in each experimental group:
albumen introduction
F12 i nt roduct i on
aír cell reexpansion
N.S.
N.S.
P < 0.01
(") deaths and defects in developíng embryos of each developing group
of experimental emb ryos
albumen ¡ntroduction
F12 introduction
air cel.l reexpansion
P < 0.01
N.S.
0bl iteration of the introduced air space thus reduces the early
deãth rates within and between the experímental groups when albumen or
F12 (but not air cell reexpansion) are emproyed. A more subtre reduction
ln later deaths and malformatíons is achieved by air cerr reexpansion
(but not by albumen or Fl2), Despite these difference the experiments
confirm that the presencè of an artìf ical. air space above.
chick embryos at early developmental stages is hlghly teratogenic.

90
5.4 BACTERt4L CUTTURE 0F UlNpohrEp EGGS
One of the hazards of the windowing technic is the possibîlity of
acquired infection, which might play a role Ín the teratogen¡c action
of ân introduced air space. Furthermore, one of the hypotheses to
account for the pathogenes¡s of anencephaly s.uggests thât it may result
from an extensive întra-uterine encephalomyel îtis (Brouwer, t9l6).
For both these reasons the sterility of the standard wîndowing
techníc was assessed by making aerobic cultures of albumen on bloodagar
ât the times of windowing (26 hours) and of fixation (72 hours).
Bacterial cultures were taken from experimentêl eggs wíth embryos
shovring early death and necrosis, as wel I as those developing to later
stages' The control eggs were only cultured at the time of fixation.
Examínation of the plates after A days growth at 3go0 reveôled
some colonies on the track smeared by the wire loop,and some on background
areas of the medium (which were regarded "a
contaminants).
"¡a-uoan.
Tables 20 e 2_1: show that the rnortal ity and malformations rêtes in
experîmental (windowed) and control embryos at 72 hours correspond to
those in other exper¡ments. ln Table 22 a number of blood-agar plates
show Oorynebacterium colonies as background contam¡nants. There is no
evídence of significant ínfection in wíndowed eggs.

9l
TABLE 2P" MORTALITY AFTER Ii'INDOl,,'ING AT 26 HOURS..(FOR BACTERI{L CULTURE)
Exper ¡ men ta l s
Control s
Number of Egg s
Early Deaths
Developing Emb ryos
34
7
27
l0
0
10
Al ive wîth No Defecrs
Al ive wi th Defecrs
Deâd wi th No Defects
Dead wi th Defects
9
t0
1
7
9
1
0
0
TABLE 21. l"lALFoRI4ATtl
I,,IALFORI4ATI ONS AFTER Ì,'I NDOhII NG AT
(FOR BACTERIAL
Exper í menta I s
Control s
Numbers of Emb ryos
0pen Cord Defects
open Brain Defects
llî crocepha I y
Eye Defects
Facial Defects
Trunk Defects
Trunk Cys ts
Limb Bud Defects
27
l0
0
tl
t0
3
1
4
D
10
0
0
I
1
i
0
0
0

92
TABLE 22. ¡¡UI'ISELTS OF PLATES {ITH COLONIES AFTER,,¡[,DAYS GROWTH
Bacl I I us
Staph. albus Corynebâct.
Exp erlmenta I s 0n Track
0
1
f
(2.6 Hrs. ) Backg round
t
2
2
Experimentaìs 0n Track
(72 Hrs. ), Backg round
Controls
(72 Hrs. )
0n Track
Background

6 RESULTS OF EMBRYOLOG I cAL STUD I Es

94
6.1 EMBRY0ûIIES I s 0L 0!!\- NEUR4L lErElrs
To study the development of open neural defects, embryos were
recovered at various intervals after windowing at 26-30 hours. Large
groups of experímental and control embryos were fixed êt thê tíme
of windowing, or 6, 18, _32
and 42 hours lateÉ (groups 0C,6E, 6C,!BE,l8C,
30E, 30C, \2E \ZC). Excluding embryos showing early death and necrosis,
each embryo was Staged, examîned for defects, and drawn with a camera
I ucida attachment.
'
Embryos in good condîtion at fixation were selected for serial
sectîoning. Their histological changes were later compared to thelr
appearance as whole embryosoas recorded in the camera lucída drawings
(see Section 6.5 ) .
6.1.1 Embryoníc- 9izes at 26 Houri.
Because of diffîculty în Staging early emb,ryos without vîtal
stalning, development at the t¡me of windowing *", "r."r."1
in terms of
blastoderm diameter (measured with a miliimeter ruler). ln embryos
recovered at the time of windowing (OC), tneir sízes could be compared
to the range of Stages, to índicate the Stages of all the other exper¡-
mental and cdntrol groups at 26 hours. The embryos recovered at the t¡me
of wîndowín9 (OC) provided a base line for all the experimental ,groups
(6E, tBE, 30E,428) and conrrol groups (6C, tBc,30c,42c).
The sizes and Stages of group 0C embryos are given in Table 2l ,
ênd the sizes of all other windowed groups are shown in Table 24.
The range of sizes in group 0C is compared with sizes of group 6E, lBE,
JOE, and 42E embryos ln Fig.l5A bB,and with the range of Stages ín Fig. 16A e B.

95
TABLE 2?, SI,ZES AND STAGES OF DEVqIOIINC GLOUP OC EMBRYOS
Slzes (mm)
Stages (H¿H)
1i
1Z
13
5 6 7 8 9 t0 Totals (2)
I
2
3
3
4
7 ¡3.\6'
7 (13.46)
I (15.38)
14
4
5
I r (21.15)
15
l6
1
1
2
3
2
I
7 u3.\6,
7 (3.46)
17
1
1
2 (3.85)
18
1
2
3 ß.77)
rotårs 3ß.77) 2(3.85)lo(19.23)13QÐ 18(34.62) 6(11.54152
TABLE 24. SIZES OF DEVELOPING GROUP 6E. 18E. 3OE Ê 42E EMBRYOS
S izes (mm)
Groups
r8Ê Tota I s (?)
il
3
0
1
I
5 Q'1t+)
12
13
2
,7
3
11
2
7
7
3
14 (5.98)
28(11.97)
14
15
1'
17
15
62(26 50)
15
16
13
4
14
t4
r5
2\
14
I
56(23.93)
50(21,37)
17
t
3
5
5
14( 5.98)
r8
t
2
2
0
5( 2.14)
Tota I s
\6
73
53
23,9.

Fis. I5 A¿8, Range of s izes (mm.)
68, tBE, 3cE and 42E
of embryos in g roups
conpared to group 0C.

97
N=234
ttt
o
d,
ao
ã
t¡¡
llo
bq
Ir t2 13 14 15 16 17 l8
SIZES (mm) lN GROUPS óE,l8E,308,42E
N=52
il1213 14 15 ló17 18
SIZES (mm) tN GROUP OC
stzEs tN GRouPs oc,óE,l8E,3oE,42E

Figs. 16 AaB.. Range of sizes (mm. ) and Stages (UaH) ¡n
group 0C.

N=52
56789r0
STAGES (H.&H.) rN GROUP rOC
an
o
d
co
ñro
IL
o
bq
It 12 t3 t4 t5 ló t7 t8
SIZES (mm) !N GROUP OC
SIZES AND STAGES IN GROUP OC

100
6.1.2 f4ortal í!y with Vsrying Periods of lncubation _Af
ter"}jindowin-9.
The experimental embryos recovered 0, 6, 18, 30 and 42 hours after
wlndowing showed an increasing mortal íty with longer periods of incubatìon
(Table 25A and Fis. 17 ). The controls, by contrast, showed very
few early deaths. (ta¡le Z5S and Fig. l/i.
Statistical analysis revealed a signÌficant dìfference in early
deaths between the combined experímental groups and the combined controls
(P . 0.01). There were also signîficant dífferences in deaths ênd defects
betwêen (P

\28
73
20(27 .40)
53
21 (28.77')
25ß4.25)
1(1 .37)
6(B,zz)
15.20
TABLE 2
I'tO RTAL I TY
URS AFTER I,J I N
(EXPERIMENTAL GROUPS)
qc
Expe !'i men ta I
6E
Groups (%)
188 308
Number of Eggs
iz
h7
66
77
Early Deaths
0 (0)
1 (2.13)
4 (6.06)
4(5.19)
Developlng Emb ryos
52
46
62
73
Al ive with No Defects
48(92.31)
350\.47)
19Q8.79)
20 (25.97)
Al ive with Defects
3ß.77)
6(12.77)
17 Q5.76)
26(33.77)
Dead with No Defects
1(1.92)
3 (6.38)
16 (z\.2\)
8 ( 10.39)
Dead wi th Defects
0 (0)
z(4,26)
10(15.15)
19 (24 .68)
Stages at F Í xat ìon
5 -10
5.- 12
9-15
11 -17

421..
26
25
I (3.85)
20 (76 .92)
1 (3.85)
0 (0)
4(15.38)
r4-20
25 S. t'loRTALtrY' O. 6. 18. 30; e 42 HOURS AFTER lllND0WlNG (CoNTRoL ûRoUPS)
0c
cont rol
6c
eroups (E)
18C 30c
N¡rmbe rs of Eggs
52
22
27
25
Early Deaths
0 (0)
0 (Ò)
1 (3.70)
0 (0)
Developing Embryos
52
22
26
25
Al ive with No Defects
48(92.31)
20 (90. 91 )
20 (74.07)
22 (88)
Al ive with Defects
3ß.77)
0 (0)
2(7.41)
2 (8)
Dead wl th No Defects
1(r.92)
1(4.55)
0 (0)
0 (0)
Dead with Defects
0 (0)
1(4.55)
4(14.81)
1 (4)
Stages at Fl xat ion
5 - l0
I - 11
1t 13+
13 - 1'Ì

Fig. 17.
Percenteges of early deaths and later deaths
and deformities 0, 6, 18, 30 and 42 hours after
windowing at 26 hours.

00
N Eorly Deoths
,n
-¡
F-
ô-
xu.t
Èa
b\
N
N
N
N
Alive, No Defects
Alive, Defects
Deod, No Defects
Deod, Defects
0c
óE
r8E
30E
428
0c
óc
18C
30c
42C
al,
J
L.'
d.
t-
z
o
(J
Ès
100
MORTALITY O,ó,18,30&42 HOURS AFTER WINDOWING (2ó HOURS)

05
I,IORTALITY O; 6; IB: 30 Ê.42'IIOURS AFTER WINDOI,/ING
Analysls of deaths and developing embryos in the combined experimentâl
groups and the combined control groups:
Observed Val ues 29 z
286 l5o
Degrees of Freedom 1
Chí Square (Yates Correction). 9.07
P < 0.01
Analysis of early deaths and developing embryos in the experimental
groups:
. observed Values o t 4 4 20
52 46 62 73 53
Degrees of Freedom '4
Chl Square 39.25
P < 0.01
Analysîs of early deaths and developing embryos ¡n the control groups:
Observed Values O O I 0 l
,2 22 26 25 25
Degrees of Freedom 4
Chi Square 3.79
P
N.s.
Analysís of later deaths and defects in developing embryos of each
experlmental g roup :
0bserved Vâ I ues 48 35 19 20 21
36172625
1 3 11 8 1
0210196

ì06
Degrees of Freedom 12
Chi Square 102.\7
P < 0.01
Analysls of later deaths and defects in developîng embryos of each control
g roup:
observed Values 48 Zo 20 Zz zO
30221
11000
0l4tt1
Degrees of Freedom 12
Chi Square 15.15
P
¡1.s.

107
6.1.3 l:legral Cþ;ure and Neglq I qe!e!:ts
By examlnlng and drawing such large numbers of embryos, normal and
abnormal neural closure could be followed ín detall. The anteríor neuro,
pore, normally closed by Stage 12 (Hami lton, 1965), hras still open in some
experìmental and control ernbryos at Stailes 13 - ZO. Such a contînuous
series of embryos showing an open anterior neuropore at Stâges immediately
after normal closure suggests that the establ ished open brain defects seen
În groups 3OE, l+28, € 42C ar¡se by non-closure (Table 26; Fi9s. tg €. 20)..
During normal development, the rhomboid sinus assumes an oval shape
and closes by Stage 15 .(Hami lton, 1965), though fÌnal closure cannot be
fully confirmed until Stage 16 in whole embryos. ln sonie embryos with
ên open rhomboid sínus, the neural folds formed an inverted triangular
outl íne rather than the normal oval shape (F¡gs. 24 ê 29. ln groups lgE,
t8C, 30E 6 JOC open neural defects were present just craniaì to the
rhomboîd sinus, sometimes showing contînuity with the neu.al fold, of a
triangular rhomboid sinus. This suggests that establ ished open cord
defects aríse by non-closure of the rhomboid sinus, whose neural folds
form a trìangular rather than an oval contour during non-closure
(Flss. 19 È 2A.
Ar
.
slightly later Stages (30E, 3OC, \zE, 42c) the additíon- of more
somític mesodern in the caudal region resulted in open cord defects beíng
located in the somite regîon. lJhen these lesíons were examîned careful ly
some formed a regular open area while others showed an irregular contour
(F¡s . 20).
The histological appearances of regular and irregular
open defects were later found to be quite d¡stinct (see Section 6.3 ).

hzE
53
42C
z5
51(96.4)2\(96)
2(3.77) r (4)
14(26.\2) 3(2)
\0.55) 4(16)
1(1.89) o(o)
48 (90. 57) z r (84)
r3(24.53) 0(o)
12(22.64) 1(\l
15-20 1\-20
co
TABLE 26. NEURAL CLOSURE O, 6, 18" 30 E 42 HOURS AFTER I,/INDOI,,IING (BY GROUPS) (Z)
0c 6E
18E 18C
308
30c
Numbers of Eggs 49
45
22
62
26
73
25
Open Ánter¡or Neuropore 40(81.63)
25 (55. 56) 1 1 (50)
7 (1.29)
3 (1r .54)
Closed Anterior Neuropore 9(1r8.37')
20 (44.44) il (50)
55 (88. 71 )
23 (88.46)
68(93.15) 25(1oo)
open Brain Defects
Hiçrocephaly 0 (0)
2 (4.44) o(o)
2(3.23)
0 (0)
5 (6.85) 0 (0)
23ß1.5Jt 2 (8)
Oval Rhomboid Sinus 49(100)
4\(97 .78)22(1oo)
42(67 .7\l
26(roo)
\2(57.531 19Q6)
Triangul ar Rhombold Sînus 0(0)
Closed Rhomboid Sinus 0(0)
Regular Cord Defects 0 (0)
1(2.22) 0(0)
o(o) o(o)
o(o) o(o)
19 (30. 65)
1(1.61)
r (1 .61)
0 (0)
o (o)
1 (3.85)
18(24.66) o(o)
13(17.81) 6(2\l
26(35.62',) 1 (4)
lrregular Cord Defects 0 (0)
0 (0) 0 (0)
1 (r .61)
0 (0)
5 (6.85) o (o)
Stages at Fìxatlon 7-10
7-12 8-11
9-15
t1-t3'
11- -17 13-17
)

Fig'
18.
Percentages of experimental ancl conirol embryos
showing closure of the anterior neuropore 0, 6,
18, J0 and 42 hours after windowing at 26 hours.

ffil Op"n Anterior Neuropore
I I0
N=38o
f] Closed Anteiior Neuropore
i-i Opun Broin Defecl
,n
F
o-
xt¡¡
ñ
óc
r8E
GROUPS
lBc
30E
30c
42Ê
V'
o üFz
o()
bq
CLOSURE OF ANTERIOR NEUROPORE BY GROUPS

Fi g'
19.
Percentages of experimental and control embryos
showing closure of the rhomboid sínus 0, 6, 18,
30 and 42 hours after wìndowîng at 26 hours.

N=380
El Ovol Rhonnboid 'Sinus ì
looun
ffi Triongulor Rhomboid SinusJ
I Closed Rhomboid Sinus
lD
t-
o-
xu¡
ÈE
óc
r8E
GROUPS
t8c
30E
30c
428
tn
o
ü,
F.
z
o
U
Èq
CLOSURE OF RHOMBOID S¡NUS BY GROUPS

lig. 20. Development of open brain and cord defects
0, 6, 18, 30 and 42 hours after wíndowing at
26 hours.

Øl Open Broin Defecls
N=384
ffi F"sulor Cord oefects I
Iopen
El lrregulor Cord DefectsJ
t,
t-
È
x¡¿¡
Èe
30E
428
30c
42C
at,
o
æ,
z.
o
u
Èq
OPEN NEURAT DEFECTS BY GROUPS

I l5
l'/hen neural closure was assessed, only embryos with neural tissue
(after St¿ge 6) could be incìuded (Hamilton, 1952). ln Table 26 and
Flgs, 18 ê 19, closure of the anteríor neuropore and rhomboîd sìnus can be
followed from 26 to 72 hours (Stages 7 - 2O'). The open braîn defects,:.regarded
as arÌsing by non-closure of the anterior neuropore, were only clearly
establ ¡shed in groups 30E g 42E, with one ,pont"n.ou, defect ln group 4ZC
(nig. zo)
Regular cord defects, regarded as arising from a tr¡angular rhomboid
slnus, coexist with an open rhomboid sînus as the position of the rhombold .l
slnus changes wlth lncreasing growth of the embryo. Open cord defects were
recognlsable in groups 18E, 30E, ê q2E, with spontaneous exarñples in groups
18C, 30C, E 42Ci in Fig. 20 they are subdivided inro regular and irregular
les lons.
Figures 21-J0 show camera lucida drawíngs of the typical changes in
embryos of the collected series:
St. 9 - at the time of windowing (0C 15)
St. 11 - open anterior neuropore and cysts (l8E 9)
St. 1B - open brain defect and anophthalmia (42E'20)
St. 13 i normal oval rhomboid sinus (l8c zo)
St. l3 - abnormal triangular rhomboîd sinus (l8E 6l)
st. 15 - early regular cord defect (30E 25)
St. 19 - later regular cord defect (4iE l)
St. 16 - early lrregular cord defect (lOt 73¡
St. 17 - later irregular cord defect (4ze 52¡
St. f5 - large cyst of caudal resion (3Og 3Z).

ligr. 21 - 30. Camera lucida drawings of a series of whole
embryos (1 mm scaìe indicated) :
FiS. 21 ,
Normal St, 9 - embryo at the time of
windowing at 26 hou rs .

117
lmm
AT WINDOWING
2ó-30 HOURS
STAGE 9'
octS

Fi s.
22.
lrregular open anteríor neuropore and trunk
cysts in St. 11 embryo 1B hours after windowing.
Fi g.
23.
Establ ished open braîn defect and anophthalmia
in St. 1B embryo 42 hours after windowing.

lmm
OPEN ANTERIOR NEUROPORE
TRUNK CYSTS
48 HOURS
STAGE II
l8E9

lmm
OPEN BRAIN DEFECT
Á,NOPHTHALMIA
72 HOURS
STAGE I8
42820

Fis.
2l+ .
Normal oval shape of the rhomboid sinus in
St. 13 control embryo lB hours after the
t i me of w i ndowi rrg.
Fis.
tE,
Abnormal tr¡angular shape of the rhonboid
sÍnus in St" 1l embryo 1B hours after windowing,

lmm
OVAL RHOMBOID S¡NUS
48 HOURS
STAGE I3
r8c20

123
t1
lmm
TRIANGUTAR RHOMBOID SINUS
48 HOURS
STAGE 13
18EóI

FiS. 26,
Open neural folds extending from the rhomboid
. sinus into the somite region, forming an early
egulirr open cord defect, in St. 15 embryo
J0 hours after wÌnciowìng.
FiS, 27.
Establ ished regular open cord defect in
St. 19 embryo 42 hours after wíndowing.

t--J
lmm
EARLY OPEN CORD DEFECT
REGUTAR TYPE
óO HOURS
STAGE I5
30 E2s

t-l
lmm
IATER OPEN CORD DEFECT
.
REGULÁ,R TYPE
72 HOURS
STAGE 19
42Et

Fìs. 28.
lrregular neural folds without an open rhomboìd
sinus, forming an early irregular open cord
defect, ¡n St, 16 embryo 30 hours after windowîng.
FiS. 29.
Estãbt ¡shed i rregular op.n "o.d
defect in
St. l7 embryo 42 hours after windowing.

l2B
t--l
lmm
EARLY OPEN CORD DEFECT
IRREGULAR TYPE
óO HOURS
STAGE Ió
30E73

129
lmm
LA,TEROPEN CORDDEFECT
¡RREGULAR TYPE
72 HOURS
STAGE 17
42E 52

Fis.
30.
Large caudal cyst Ìn an otherwise normal
St. 15 enbryo 30 hours after wÍndowing.

3ì
lmm
CAUDAL CYST
óO HOURS
STAGE 15,
30E32

132
6.1 .4 DeveloÞñent of oÞen Neúr¿il Deþslq
l,lhen embryos are analysed in experlmental and control groups
(Section 6.1.3) the exact tlmîng of neural closure cannot be assessed
because each group incorporates a range of Stages. Analysîs of the
comblned experímental groups and the coribined control groups ¡n terms
of indlvÌdual Stages. (including group 0C ín both categories), provides
more Information about neural closure after Stage 6.
ln Table 2l and Fis. 3t , closure of the ânterîor neuropore is ._ "i
complete in the control embryos by Stage .l3, so that an open neuropore
after thís Stêge cên be regarded as an open brain defect.
Closure of the rhomboid sinus (taUle Z8 e Fig,32 ) occurs at
Stages l!:.l6 ¡n both experimental ênd control groups. A triangular
rhomboid sînus is seen only in experimental embryos between Stages 11
and 16. Open cord defects first appear at Stage i3 (Table 29 and FiS. 33 ),
and occur at all Stages in the experímental group, as well as in three
control embryos. The first appearance of open cord defects at Stage 13
ls consístent w¡th the suggest¡on that they are preceded by a triangular
rhomboid s i nus.

133
TABLE 27, ANTERIOR NEUROPORE CLOSURE BY STAGES
S tages Expe r I men ta I s (?)
Open
C I osed
controts(%)
Open e I osed
7
I
12(4.26)
15ß.32)
0 (0)
0 (0)
¡2(8.16) o(o)
16(1o.BB) o(o)
9
24 (8.5 r)
0 (0)
21(1t+.2-9) 0 (0)
10
tl
12
13
l3 (4. 61 )
6(2.13)
2(o .71,
2(0.71)
r I (3.90)
23 (8. 16)
10 (3. 55)
21(7 .45)
2(1.36') l1(7.48)
2(1.36) 12(8.16)
1(0.68) 8(5.44)
o(o) r3(8.84)
r4
15
16
1 (0.35)
2(0.71)
0 (0)
40(r4.18)
23ß.16')
20(7.09)
o(o) 2(1.36)
0(0) 7$.76)
o(o) r3(8.84)
17
18
19
1 (0. 35)
r (0. 35)
o (o)
23 (8. 16)
21(7.45)
8 (2. 84)
I (0.68) 8(5.44)
0(0) 5(3.40)
0(0) 10(6.80)
20
0 (0)
3(r.06)
0 (0) 3 (2.04)
Totals Numbers
of Embryos

Fig.
31 .
Percentêges of experinental and control embryos
with an open or cìosed anterior neuropore at
each Stage. Open anterior neuropore after
Stage 12 regarded as an open braìn defect,

ffi Opàn Anterior Neuropore
135
I Closed Anlerior Neuropore
lrl = 3Bo
[-Ì Op"n B¡'oin Defects
EXPERIMENTATS
gi ro
À
xtf,¡
Àe5
0
0
sT.7 I 9 r0 il 12 t3 14 15 tó 17 18 19 20
.n
.J.
oa'
e,
z
o(,
E
Èe l0
coNTROTS
CLOSURE OF ANTERIOR NEUROPORE BY STAGES

36
TABLE ?8. RH0I4B0lD stNUs ctôs RF Ry ç-rarìF(
Stages Experìmentals (?) Controls (%)
Ova I Trìangu I ar Cl.osed Ova I Tr Iangul ar C I osed
7
I
9
10
t1
12
13
14
15
16
17
18
19
12(4.26)
15ß32)
29 ( 1 0.28)
24(8.5t)
22(7.8o)
15$32')
12(4.26)
27 ß.57)
23ß.16)
2(0.7r)
o (o)
0 (0)
o (o)
0 (0)
0 (0)
0 (0)
0 (0)
2 (o.71')
t (0.35)
g(r. rg)
14 (4.96)
5(.77¡
I (2. 84)
0.(0)
0 (0)
0 (0)
0 (0)
o (o)
0 (0)
o (o)
0 (0)
0 (0)
o (o)
0 (0)
1 (0.35)
4(1 .t+2)
24(8.5t)
22(7.80
8 (2. 84)
r2(8.16)
,16 ( 10.88)
25fi7)
l3 (8. B4)
1o (6.80)
9ß.12)
13 (8.84)
2(1.361
1 3 (8. 84)
7 G.76)
0 (0)
0 (0)
o (o)
o(o) o(o)
0 (0) 0 (0)
o(o) o(o)
o(o) o(o)
o(o) o(o)
0(0) o(0)
o(o) o(o)
0(0) 0(0)
0(0) 5(3.40)
o(o) 3(2.04)
o(o) 6(4.08)
-o(o)
5(3.40)
o(o) 5(3.40)
20
0 (0)
0 (0)
3(1.06)
0 (0)
o (o) 32.o\l
Total! Numbers
of Emb ryos

Fì s.
32.
Percentages of experImental and control embryos
with an open or closed rhomboid sinus at each
Stage. Open rhomboid sìnus divìded into oval
and tríangular types.

N=38o
H ovol Rhomboid sinus l^ r38
ffi Triongrlor Rhomboid Sinusl
fl Closed Rhomboid Sinus
l(Jpen
:i to
t-
o-
xu¡
ñ5
sT.7 I
9 l0 rr t2 '¡3 t4 15 ló 17 18 19 20
CLOSURE OF RHOMBOID SINUS BY STAGES

t39
DEFECTS BY STAGES
Experimentals .(Z)
Regular lrregular
Control s (Z)
Regular lrregular
13
r4
15
7 Q.48',)
5(.77)
B (2. 84)
0 (0)
I (0. 35)
r (0. 35)
I (0.68) o(o)
0(0) 0(0)
0(0) 0(0)
16
17
IB
19
20
I (2. 84)
4(1 . \z)
\(1.\2)
2(o .71')
2(0.711
5u.77)
6(2.13)
56.tt',t
0 (0)
0 (0)
I (0.68) o(o)
0(0) 1(0.68)
0(0) o(o)
0(0) o(0)
0(0) 0(0)
Total Numbers
Emb ryos

F¡ S. 33. Percentages Õf open cord defects at each Stage
after St. 13. Defects divíded into regular
and i rregul a r types.

4l
N=38o
ffi! Regulcr Cord
ffil lrregulcr Cord
O"f".t, l
Iop*n
DefecrsJ
.J'
o-
xl¡.¡
Èe
.n
o
É
t-
z
o
L'
Be
OPEN CORD DEFECTS BY STAGES

142
6.1.5 Distríbution of 0pen CorrJ Defects
Thé condtant addition of somltes during embryonic growth results
ln open corcl defects being found at a greater distance from Hensenrs node i¡
older embryos. This is demonstrated by dividing the trunk into somite
and post-soml te reglons,
Table 30 and Fig.J4 show the numbers of embryos with regular and
irregular cord defects ín each of these two regions, ln the experímental
embryos, lesíons occur at the post-somite region in group l8E, at both
regíons in group 30E, and at the somite region in group 42E,
When the experimental and control groups êre rearranged in terms of
Stêges, experimental embryos show lesions in both regions êt Stages
t3-16, but only in the som¡te region by Stages 1/-20 (taUle 3l and Fi9.35 ).
lf the mid-point of each lesion în experinental embryos of Stages
17-20 is determined in the camera lucida drawings, the distribution of
open cord defects can be expressed in terms of theír somite levels.
Table 32 and Fig.36 show that the mid-poínts ofmost lesions ¡ie between
somítes 21 and ll, with little difference between the regular and i rregular
defects.

143
TR IBUTION OF
D DEFECT5
N umbe rs
of
Embryos
Regular
Somî te
level
Q)
Below
somitgs
lrregular (?)
Somite
level
Below
som i tes
18E
62
0 (0)
1(0.53)
o(o) r (0.53)
3oE
4zE
73
53
eþ.7e)
13,(6.91)
19(10.1r)
0 (0)
3 (l .60) 2 (1 .06)
r2(6.38) o(0)
r8c
30c
42c
26
25
25
0 (0)
1(1 .32)
0 (0)
1(r.32)
0 (0)
0 (0)
0(0) o(o)
0(0) o(o)
1(132) o (o)

Fi s.
"l!
Percentage distrìbution of open cord defects
at somÌte or post-somite levels 18, 30 and
42 hours after windowing. Defects divided
into regular and irregular types.

145
ffit Regulor Cord Defects
ñ=2ó4
El lrregulor Cord Defects
SOMITE
ar)
o
æ
cô
ã
It¡
Dq
BELOW SOMITES
t8E 30E 428 ,t8C 30C 42C
DISTRIBUTION OF OPEN CORD DEFECTS AT SOMITE
oR POST-SOMTTE LEVELS (BY GROUPS)

lrregul ar (Z)
somi te be low
level somites
0(0) 0(0)
0 (0) 0 (0)
o(o) o(o)
o(o) o(o)
l(1.32) o(o)
o (o) o (o)
0 (0) 0 (0)
0(0) 0(0)
o\
TABLE 31 . DISTRIBUTION OF OPEN CORD DEFECTS BY STAGES
Stage
Regu l.a r
somi te
level
Exper i men ta I s
&) lrregular (?)
below som ï te below
somites level somi tes
Controls
Regular (%)
somi te below
level somites
13
14
15
16
17
18
19
20
3(r.60) 5Q.66) o(o)
2(r.06) \(2.13) o(o)
2(1.06) 52.66]. 1 (0.53)
2(t.06) 6(3.19) 3(r.60)
5Q.66) o (o) 6(3.19)
4(2.13) o(o) 5Q.661
2(1.06) o(o) o(o)
2(r.06) 0(0) 0(o)
o (o) o (o)
1(0.53) 0(0)
0 (0) 0 (o)
2(1.06) 1(1.32)
o(o) o(o)
0(0) 0(0)
o(o) o(o)
0(0) 0(0)
1(1.32)
o (o)
0 (0)
0 (0)
o (o)
0 (0)
0 (0)
0 (o)
Numbers of Emb ryos
188
76

Fis.
35.
Percentage d¡stribution of open cord defects
at somlte or pcst-somite ìeveìs at each
Stage after St. 13. Defects divíded into
regular and irr:egular types"

ì48
N=2ó4
ffi
m
Regulor Cord Defects
trregulor Cord Defects
,t;
l--
A-
X
¡¡l
ñ
5
5
tn
o
&L
'20
o
I
ñ.
sr. t3
SOMITE LEVEL
BELOW SOMITES
t8 19j 20
SOMITE IEVEt
BELOW SOM¡TES
D¡STRIBUTION OF
OR POST.SOMITE
OPEN CORD DEFECTS AT SOMITE
TEVELS (BY STAGES)

149
TABLE 32. SOI4ITE LEVELS OF l4ID;POINTS.OF OPEN NEURAL DEFETTS
Som i tes Resular (%) I rr"eg u I ar (Z)
17
18
19
20
21
22
23
2l+
25
26
27
28
29
30
3l
1(0.53)
0 (0)
o (o)
0 (0)
0 (0)
I (0.53)
2(1.06)
2(f.06)
0 (0)
0 (0)
0 (0)
6(3.19)
I (0.53)
2(1.06)
0 (0)
0 (0)
0 (0)
0 (0)
0 (0)
r (0.53)
r (0.53)
0 (0)
0 (0)
2Il .06)
7 ß.72')
3(r.60)
1(0.53)
r (0.53)
0 (0)
r (0.53)
_.
Total Number of
Experimental Embryos
188

Fis.
36.
Percentage distrîbution of the somite level.s
of mid points of open neural defects ¡n
experimental embryos of Stages 1/-20,

SOMITE LEVELS OF MID-POINTS OF OPEN NEURAL DEFECTS
ffil Regulor Cord Defects
N =188
I lrregulor Cord Defects
at,
F
Â-
X
l¡J
ÈR
soM.r7.
t8
19
20 2'l 22 ¿3 24 25 26 27 28 29 30 3r
¡N EXPTAI. EMBRYOS AT STAGES 17.20

152
6.2 sPtIA! LFVELS 0F oPEN CORp DEF.ECTS rN 12 pAY EMBRYoS
The range of malformations produced by windowing at 26 hours încreases
wlth prolonged embryonic growth. Embryos recovered at 72 hóurs show neural
defects and trunk cysts. At 5 days non-neural defects are apparent, and these
êre more widespread by f2 days (Sect.ion j.2)..
The group of experimental embryos that survîved to 12 days were
examîned for external .malformations, and then :ubjected to cartìlage
staining with alcian blue .(Ojeda et aì., 1970) to display any skeletal .. .:
defects. This revealed the level and extent of the vertebral abnormal ities
associated with each open neural defect.
Because the distinction between myeloschisis and myelodysplasia was
not absolutely clear at 12 days, the two are combined în Table 33 and Figs.3?a38.
Control embryos (with no growth retârdation) were recovered at 11 days,
to provide a comparable range of Stages.
Figs. 39 and 40 demonstrate the difference in grosa appeârance between
the two types of open cord defects in their extreme forms. Myelosch,î.sis
consists of an exposed, regular neural plaque, while myelodysplasia
involves a more irregular defect, part¡ally covered by skin.
A coìlection of four embryos with open neural defects (Fig. 41)
demonstrates the fairly uniform level of the defects, with reduc.t¡on of the
rump and tail in two embryos.
Non-neural malformations (Fig. À2 ) consist of:
a) ectopia v i s cerum
b) uni lateral anophthalmia (wlth a crossed beak)
c) bilateral anophthalmia (wíth a short but central upper beak)
d) reduction of the rump and tail

t53
TABLE- 33,.VER,TE-BRAL qEFqcTs AT 11' 12 DAYS FOLLqvJING.l¡/INDOT.¡ING-
4T 26 tIOUBs
Numbers of Emb ryos
Spina
.Bifida Occul ta
Spîna Bifida Manífesta
Vertebra I Del et i ons
Lengths of S. B. 0cculta
Lengths of S.B. l4anifesta
69
t8
42
30
1-11 verteb rae
3-15 vertebrae
62
0
1
l1
0 verteb rae
6 verteb rae
Lengths of vertebrar Deretions 1-1g vertebrae 6-t5 vertebrãe
Range of S tages 35-39 35-39

Fìgs. 37'38,
Vertebral defecrs in Ìndividual experimenral
and control embryos at 1Z.and ll days. Each
bar represents one embryo.

CERVICAL
THORACIC
LUMBAR
FUSED
CAUDAL
FREE
CAUDAL
PYGOSTYTE
N=ó9
ø Spino Bifido Occulto
n Vertebrql Deletions
tr Spino Bifidq Monifesto
I Open Cord Defect
.n
l¡¡
lt¡
J
d.
cô
t¡l
t-
æ,
1¡¡
w
VÅ øt
wm
w
!ND|VIDUAL EXPER|MENTAI EMBRYOS (12 DAYS)
VERTEBRAL DEFECTS IN EXPERIMENTAT EMBRYOS

CERVICAT
THORACIC
LUMBAR
FUSED
CAUDAL
FREE
CAUDAT
PYGOSTYTE
\o
N=ó2
@
@
Spino Bifidq Oèculto
Verîebrol Deletions
tr
I!
Spino Bifido Monifesto
Open Cord Defects
.u,
t¡¡
u¡
d.
co
t¡¡
l-
É,
t¡¡
INDIVIDUAL CONTROT
,VERTEBRAT DEFECTS IN
EMBRYOS {il DAYS}
CONÏROL EMBRYOS

Figs. 39 - 42. Malformations ¡n exper¡rneñtal embryos êt i2 ciays:
Fis. 39. Oþen cord defect (probably regular type) .
FîS. 40,
Unilateral anophthalmia, crossecl beak, rumplessness,
and open cord defect (probabîy írregular type) .
FiS. 41.
0pen cord defects and varying degrees of rumplessness
ìn four emb ryos .
Fig. 42,
Ectopìê vìscerum, open brain ¿efect and short upper
beakrbilateral anophthalmîa and short upper beak,
un¡laterâl anophthalmiã and crossed beak.

---l
r
r'I
*
40

ì58
One embryo shows ðn open brain (with a short upper beak), which can be
compa red to rhe same defect at 72 hours (f¡g. Z3 ).
Skeletal stainlng of the experimental and control embryos revealed
three types of vertebral defects:
a) splna biflda occulta (with no external neural defect)
b) splna bifida mênîfesta (associatecl with myeloschisis and myelodysplasia)
c) reduction oi irregularíty.of the lumbar, sacral and caudal
vertebrae. F.igs.43-46 demonstràte spîna bifida occul ta, spina blflda
manifesta, rumplessness, and lumbo-sacral irregularity in four cleared
emb ryos .
There is not full agreement on the number of vertebrae in each
region of the chick spine, because of the difficulty in assigning
transitional vertebrêe to a particulãr region. The control embryos in
this study general ly possessed:
l4
cervi ca I vertebrae
7 thorac i c vertebrae
4 I umbar verteb rae
2 sacral verteb räe
6 (5-7) fused caudal vertebrae
6 (5-7) free caudal vertebrae
4 (3-5) pygostyle segmenrs.
Table ll
and Figs. 37 and JB show the numbers of experimental
and control embryos with three recognisable vertebral defects -spina
bifida occulta, spina bifida manifesta (enclosing the Open neural defects),
and deletîons of whole vertebrae. From Fí9.J/ ît is apparent that:

t59
a) spîna bif¡da occulta is seen mainly in the cervical region
b) splna b.ifida manifesta occurs from the rower thoracic to the upper
caudal reg ions
c) vertebral deletions are almost conflned to the caudal region.
The control embryos show a simílar pattern of spontâneous vertebral
deletions, and one spontêneous spîna bifida manlfesta(Fig; 38 ).

FÌgs. 43 - 46, Vertebral defects in the lumbo*sacral region
of 12 day embryos I
Fis.. tú.
Spina bifìda manìfesra.
FiS. 144.
Spina bifída manifestâ and rumplessness.
Fig. 45.
Spina bifida occul ta (only rarely seen in lumbosacral
region).
Fig. 46,
Spina bÌfida manifesta, rumplessness, and extensîve
vertebral i rregulari ty of cauilal region.

:al .
'**Ç- 4
ff,
À(,)
ftø 9-' o-
,.¡å ,S,
À
o

t6l
6.3 DESCRIPTION OF HISTOLOGICAL APPEARANCES
Embryos recovered 0 to lt2 hours after wíndowing (Section 6.1)
were examlned hîstological ly in serlal sections. Because the neural
defects observed after dífferent perìods of incubatìon showed a progresslon
of changes, the embryos were examined ln four groups of Stages
(see Sect ion 4.8.2) .
The general description of the histology typical of Group I - lV
embryos ís based on the appeêrance of each embryonic system.
ln some embryos there was dorsal splitting of the closed neural tube
(at later s tages ) ,...or- sepa ra t î on of notochord and somites from the neural
tissue (at earl ier stages). Both were caused by shrinkage of the embryos
during processing and were quíte different from the appearance of open
neural defects (figs. 89 - 94).
6.3.1 Stage !0 Control Embryos (Group l)
The neural plate was closed or closing cver the brain-and presomite
areas, closing or inverted in the somite area, inverted ln the protosomlte
area, elevated at the anter¡or rhomboid slnus, and elevated or
flattened at the posteiior rhomboid sinus,
The neural folds about to close showed swell ing and rounding of
the free edges, which were often inverted into the future neural- canal.
At thê rhomboid sinus differentîat¡on of the neural plate was revealed
by marginal foldîn9 and elevation above the level of the adjacent
ectoderm. This region of neural plate showed a regular êrrangement of
cells perpendicular to the exposed dorsal surface. After neural closure
(at more cranial levels), these cells retaîned the same orîentation to
each other and to the surface (which then enclosed the lumen of the neural
tube).

162
lmnediately after closure, separation of neural crest tissue was
apparent: Cránially the. rhombîc roof was seen to be closing at Stage lO,
and thìckened by Stage 10. Caudal ly there were no âccessory neural canals,
and no other signs of an overlap zone. Differentiation of neural tissue
from ectoderm was alreâdy apparent, though the ectoderm was cont¡nuous
w¡th the neural folds at unclosed areas and ¡n contact v,r¡th the neural
tube at closed areas.
The notochord was ln close contact with neural tissue from the future
midbraln down to the protonobchord. Somitic mesoderm was general ly in .. ":
contact with neural t¡ssue at the level of the somites, but not in the areas
of unsegmented and fused mesoderm at the rhomboid sinus. The somìtes were
all well formed, with no evidence of reduced volume and no cyst¡c or
hemorrhag i c changes .
The prîmitive streak and Hensenrs node were prominent, with no formation
of a ta¡l-bud at thîs Stage.
63,2 Stage l0 Experimental Embryos (Gtoup l)
The histologícal appearances of these embryos were similar to those of
Stage 10 control s for:
a) neural cJosure
b) neural foldins
c) formatîon of neural crest
d) development of the rhombic roof
e) absence of an overlap zone
f) continulty of neural folds with ectoderm
S) close contact of notochord with neural tissue
h) contact of somites with neural tïssue
î) no cyst¡c or hemorrhagic changes

t63
j) ênd prom¡nence of Hensenrs node and the primltive streak.
Howéver in two embryos (68 4l , 6E 45) there was a defînite appeêr_
ance of sllght eversion of the neurar fords at the posteríor rhomboid sinus,
rather than the wlde erevatron or frattenrng seen in the contro¡ and other
experlmental embryos. These two embryos still showed an orderly arrange_
ment of cells perpendicular to the well-preserved dorsal surface of the
open neural plate (Fi9. 47 and 48).'
6.3.3 Stage l1-.|2 Control Embryos (Grouo ll)
Neural closure extended from the brain down to the antèrior rhomboid"
slnus, with closing or elevated neural folds at the posterior rhomboid
sinus. The rhombic roof was thinner by Stage ll+ than at Stage tO
(rls. lo3).
The first signs of accessory neural canars were seen at the rhomboid
sinus. The overlap zone could in fact be traced up as far as the proto_
somite area when asymmetry of the neurar tube rather than the presence of
accessory canals was taken as a criterion..
As în Stage lO the areas of unclosed neura.l plate were continuous
wlth, but sharply distinguishable from, adjacent ectoderm, .ln the braln
regign, ectoderm was separated from the underlying neural tube by neural
crest cells,but not at this stage by migrating somit¡c mesoderm,
The notochord was in close contact with the neurêl tube or neural
plate over the somite, protosomíte, and anterior rhomboid sinus areês, but
was general ly not in contact with the midbrain and hindbrain. somites and
protosom¡tes were also in contact with the neurar tube,but this contact was
not maintained with unsegmented and fused mesoderm of the rhomboid sinus.
The somltes showed a normal sequence of changes, wlth no cyst¡c or hemo_
rrhagic areas and no reduction .in volume.

164
Hensenrs node and the primitive streak were stil I prominent, w¡th
no eví de¡ice of ta I I -bud format Ìon.
6.1.t+ Stagé 1l-12 Exöéf irhérital Embivos (Groun I l)
All experimental embryos showed retardation of neural closure.
ln general, the braÌn region was closed but the neural plate wss still
closlng over the somite and protosomlte areas, inverted or elevated at
the anter¡or rhomboid slnus, and elevated or flattened at the posterior
rhombold s inus .
One embryo (6E 34) showed definite eversion of the neural folds at
the posterîor rhomboid slnus, similar to the appearance of the two stage to
experimental embryos. Together with the retarded closure of the neural
folds was an appêrent delay ín appearânce of the overlap zone, as no
accessory canals were visible (f¡gs. 49 and 50)
ln other respects the appearances of these experîmental embryos resembled
those of the contro¡s, in that:
a) the rhombic roof was thîckened
b) ectoderm was in continuity with the neural folds
c) therê were no somitic mesoderm and few neural crest cells between
areas of cìosed neural tube and the overlyîng ectoderm
d) notochord was in close contact with the neurar tube over the somite,
protosomite, and anterior rhomboid sìnus areas, but not generally in the
brain regîon
e) somitic mesoderm was usually in contact ur¡th the neural tube in the
somite and protosomite areas, but not at the levels of unsegmented and
fused mesoderm
f) the somîtes were well-developed, wíth no cysts or hemorrhages and no
reduct ion in vo I ume.

Figs. l+7 ^ 5A, Ëversic¡n of the neuraT foÏds as the first sígn oi'
non-closure of the rhomboîd sinus. Developing protonotqchord
(l ¿ r; x4o) :
Fíg. l+7.
Control St. 10 embryo, 6 hours af ter the tîr'le of
windor,ring, with clevaied neuraì folds at the posterior
rhomboi d s i nus (6C Z1) .
Fig. 48.
Experimental St. t0 embryo, 6 hours after windowing,
with everted neural folds êt the posterior rhomboid
sinus (61 45) .
FiS. 49.
ContrÕl St, l1+ embryo, 18 hours after the. time of
windowing, wi th further differentiation of elevated
neural folds ar the posrerior rhomboid sinus (18C 4).
FiS. 50.
Experìmental St. f l-F embryo, 6 hours after wîndowing,
w¡th further differentiatîon of everted neural folds
at the posrerior rhomboid sinus (6E 34).

-l
::
l
48
I
49
i
:.
t:
.lì
50 ì:

166
6.3.5 staEe 13-16 córitról Embryos (Qroup lll)
All stage l6 control embryos showed complete neural closure, whereas
the stage l3 cont!'ol embryos were stlr crosing ât the posterior rhomboid
sinus. The overlap zone was fuly deveroped, with êccessory canals at the
unsegmented mesoderm and caudal areas, and recognizabre overrapping w¡thout
canals in the protosomite area. rhe rhombic roof was th¡n at stage rJ and
very thin at Stage f6.(Figs. 104 and 105).
The neural tube wâs separated from overlying ectoderm by neural
crest ceìls in the braln region at stage rJ, and by somitîc mesoderm and
neural crest cells over the braìn and somite areas by stage 16. The notochoid
was in contact with neurar tube în the regions of the somites,
protosomites and unsegmented mesoderm but not in the brain or caudal
âreas.
Somite development was normal,
.
with no reductlon ín volume and no
cys ts or hemorrhages.
Posteríorly, Hensenrs node had given way to a recognizable taìl_
bud. This was associated with the formation of the protonotochord,
fused mesoderm, and the tail-bud contribution to the neural tissue of
the caudal region. The primitive streak was much shorter than în
Stage .11-f2.
. In Stage 1l embryos, with an open posterior neuropore, an" iup".-
ficíal neural folds (deríved from neural plate) could be traced down
from above,othrough continuity of their future lumen with the lumen of
the closed neural tube. Deep to this was canaljzed neural tissue
(derived from the tail-bud) with no singre rumen. There was however no
clear llne of demarcation between these two sources of neurar materiar.
Neural tissue derived from the taîr-bud could be traced up to the proto*

t67
somite area (through asymmetry of the neural tube) but lts fusion to
the neural plåte materîa.l was so gradual that the upper limit of the
overlap zone was difficult to determîne (Fîgs. !1-54 änd 55-66).
t/here neurâl closure was st¡ll occurfing în the caudal region of
Stage 13 embryos, the exposed surface of the neural plate was wellpreserved,
with normal or¡entation of cells perpendicular to thÍs surface.
6.3.6 Staqe I3-16 Experîmental Embrvos. (Grouo I I t)
ln this group of experimental embryos two types of establ ished
defects were ev i den t.
The majoríty of Stage ll-1! embryos showed elevatlon or eversîon
of the neural folds in the protosom¡te, unsegmented mesoderm and caudal
êreas, constitutlng neural defects whích could be followed into Group lV
embryos. The defects consisted of unclosed neural folds, showîng marked
necrosis of the exposed surface and loss of cell orientation, lying dorsal
to more normal neural tissue derived from the ta.i l-bud. ln many cases
there was some distlnction between the two sources of neural tissue at
some part of the lesions,though no clear line of separation. As similar
lesions were present ln a more advanced foim.in many Group lV experimental
embryos, these were regarded as the f¡rst stage of estêbl ished myeloschisis.
ln experîmental embryos wìth and without myeloschísís the notochord
was in contact with both closed and open sections of the neural .tube over
the somite, protosom¡ìe, and unsegmented mesoderm areas, but not ¡n the
braîn or caudal areas. Areas of myeloschisis showed continuity with adjacent
ectoderm, implying that the neural folds at these sítas had never
c I osed. (r¡gs. 6S-Zo).
Somltic mesoderm was generally ín contact with neural tube at the
somite level , but separated from neural tube at the unsegmented mesoderm
and caudal areas. ln the protosomite area the degree of cóntact varied,

t68
with lack of contact in severar embryos showing myeroschisis. somite
developmènt aþpeared to be normal, with no cysts or hemorrhages.
0f the Stage 16 experlmental embryos several showed normal neural
closure. Several others showed rnyeloschisis at the lower somlte, protosom¡te,
êrid unsegmented mesoderm areas, giving way to a closed neural tube
(showing accessory canars and so derived from tair-bud materia¡) in the
caudal reg ion.
Two of the Stage t6 embryos however (lOf 35, 3OE 76) showed a different
type of neural defect in the lower somîte, protosomite, unsegmented meso-.
derm, and caudal areas. ln these embryos the neural tissue formed a
V-shaped or U-shaped mass, open on the dorsal aspect. The total volume
of neural tissue at the site of each lesion (but not at more cranîal levels)
was reduced when compared to Stage 16 contror or normar experimentar embryos.
0n fol lowing the canal of the neural tube down from the somite region
în these two embryos' it courd not be trêced con.tinuous¡y into the dorsar
half of the lesions. The neurar prate materiar was progressivery reduced
at the cran¡al end of each lesion, wh¡ch thus appeared to be composed
entlrely of ta¡l-bud marerial.(F¡gs. 77-82).
An absence of accessory canals, in contrast to the multiple canals of
Stage 16 control and normal experímental embryos, suggested an early
maturat¡on of the tail-bud material in the two resions. The defects were
covered by ectoderm above end berow and open in the mîddre section, though
not so smoothly contínuous with ectoderm as in the examples of myeloschisîs.
ln the caudal region, each les,ic¡n gave wây to a ctosed neural tube covered
by ectoderm and closely resembr in9 the caudar tube of Stage 16 control and
normal experimental embryos, though slightly reduced ín size.

t69
ln both embryos the volume of neural tÌssue was so reduced that
somitic ñesoderm encroached on the midlìne, dorsal to the les¡ons where
they were covered by ectoderm. This again suggested that the dorsal
contrlbution to the neural tube (derived from neural plate materîal)
was considerably reduced in the area of the lesions (Flg,. 77) .
Examlnation of somitic mesoderm în one embryo (SOf lü revealed some
reductlon in volume at the protosomíte and unsegmented mesoderm areas
(though not elsewhere) with a diffuse arrangement of cells and some
cystic spaces (f ígs. 77-8Zl ... "r
Unsegmented mesoderm was in contact with neural tissue in one
embryo (30E l!) because of the encroachment of somitic mesoderm across
the midline dorsal to the lesîon
The.lesions in these two embryos thus appeared to show:
a) reduction of total neural volume
b) marked reduction in the neural plate contiibution, but faîrly normal
ta I I -bud contribution
c) early maturatîon of the tail-bud material
d) formation of normal cord from tail-bud material in the caudal
reg i on
e) exposure of the central part of the lesion, but ectodermal cover
above and below this
f) encroachment by somitic mesoderm ôcross the dorsal aspect of the
lesion where ectodermal cover was preserved
9) some reduction ín the local volume of postsomitic mesoderm
h) the occurrence of cyst¡c areas within the local somitic mesoderm.

170
These two lesions were clearly separable from myeloschisis and so
were called myelodysplasias. Because of the U-shaped contour of the
defects and the apparent reduction of the neural plate contrîbutìon,
this form of myelodysplasla was called a hemÌmyel îa.
ln all the experlmental embryos of.this group (Stages 1l-16)
histologîcal appearances away from the a!'eas of neural defects closely
resembled the findings ¡n Stages tJ-16 control embryos for:
a): thlnning of the rhombic roof
b migration of neural crest and somitic mesoderm cells
c) reduction of the primîtive streak .
6.3.7 Staqe 17-20 Control Embrvos (Group lV)
As well as full neural closure these control embryos showed complete
fusion between the two sources of neural tissue in the overlap zone.
There were no accessory canals,and it was imposslble to dîstinguish neural
plate material from taíl-bud material"by any criterion (inCluding asymmetry
of the closed tube). H¡toses were restr¡cted to cells lining the lumen.
Llmb buds were cleaily distinguishable and provided boundarîes for
subdivision of the embryonic spinal cord. ln the"caudal region the taílbud
showed progressive reduction, with disappearance of the primitive
streêk. By Stage l9 a differentîated notochord was replacing the protonoiochord
and caudal somîtes were replacing unsegmented mesoderm in the
caudal regíon (figs. 6t - 64).
The caudsl notochord preserved its close contact wíth .the spinal
cord, whereas the caudal somites were not ¡n contact with the cord at
the lower postcrural and caudal areas. At the upper somite region,
however, the neural tube was separated from notochord by mesenchyme cells
in one Stage 20 Embryo (42C 21),

171
The rhomblc roof became membranous by Stage 18, w¡th a choroid
plexus developing in the fourth ventricle (F¡g.. 109 ).
Between the.spìnal cord and overlying ectoderm somitic mesoderm
cells were present in the brain and somite ateas, whereas neural crest
cells were observed from the sonite region down to the caudal areâ by
Stage 1!. Somîte dlspersal was well advancèd down to the postcrural
region, whíle fully developed somltes (with no cystÍc areas) extended
to the tip of the tail by Stage 20.
6.3.8 Staqe 17-20 Experîmental Embrvos. (Grouo lV)
These experîmentâl embryos could be divided înto three types -
those wíth-no defects, those wíth myeloschisis, and those with myelodysplasia.
A sinþle embryo (42E 21) showed both myeloschisis and
myelodysplas í a.
The embryos wlth no defects close¡y resembled Stage 1/-20 control
embryos (Sectîon 6,3,71 . By Stage 20 the closed neural tube was sêparôted
from notochord by mesenchyme cells at the somîte region in one case (t+ZE 73).
The embryos with myeloschísis showed a progression of the Iesions
seen in experimental embryos of Group llt (Secrîon 6.3.6r. llyeloschisis
occurred in the êreas of postbrachîal, crural, and postcrural cord, giving
way to an âpparently normal cord in the caudal region (Fi9s. 71-76).
The cranial part of each defect consisted of a widely euurtåd pl"te
of neural tissue, with the cells perpendîcular to the well-preserved dorsal
surface. ln most cases the notochord was widely separated from neural
t¡ssue at this level by mesenchymal cells (Fig. 72).
l,{ í toses were
restrlcted to cells ofthe exposed surface of the everted neural plate.

172
The centraì part of each defect showed separatìon of neural material
lnto an open plaque (lyln9 at the same level as adjacent ectoderm) dorsal
to a closed tube (whlch was deficlent ln dorsal materlal). ln almost all
cases there was a clear ì lne of demarcation between the superficlal plaque
(apparently derived from unclosed neural plate material) and the deeper
tube (apparently derlved from taìl-bud materlal). The cells of the plaque
were arranged perpendicular to the exposed dorsal surfacerwhi le those of
the tube were perpendiculâr to the lumlnal surface. llithín a few sections
of the first appearance of the tail-bud material the notochord was ìn close
contêct wlth neural t¡ssue (Fi9. 73) . H¡toses were seen ín cells on
the exposed surface of the dorsal plaque and ín cells ìîning the lumen of
the closed tube.
At the caudal end of each defect the neural plate material dísappeared,
leavîng a narrowly everted mass (whose cells were perpendicular to the
exposed surface) continous with the caudal cord. The notochord remained
ln closè contaét with the neural tube (r¡g. 75).
ln the lower postcrural and caudal regíons an apparently normal neural
.
tube reformed from tail-bud materíal (Fig, 76).
Because of the clear
separatlon between neural plate and tail-bud materials the development of
myeloschîsîs thus revealed the true extent of the overlap zone at Stages
17-20.
ln myeloschisis the neural tlssue uras in contact, but no longer in
continuity, with adjacent ectoderm by Stage 17. (F¡gs. 72-74). The rhombïc
roof in embryos wlth myeloschisis was membranous and indistinguishable from
the
âppearance în both control and normal experimental embryos, w"ith
a chorold plexus developing after Stage 17. (Fi9 111)'

173
At areas of myeloschisîs contact between somitïc mesoderm and
neuraì tissue was general ly lost, whereas contact was maintalned at the
same levels in control and normal experimental embryos. . Somîte develop-
'ment was general ly well maintalned, wlth normal somite volume and no
cysts or hemorrhages.
The development of myeloschisis dîd not prevent the local formation
of neural crest tissue (riss. 73-75 ). Migratlon of neuraì crest and
somitic mesoderm cells between neural tube and ectoderm in areas away
from the defects however showed sl ight delay when compared to control
embryos. Regression of the tail bud was almost complete by Stage 20.
Embryos with myelodysplasia also showed progression of the lesions
seen at Stage 16 (Section 6.3.6). Myelodysplasia occurred at a slîghtly
more caudal level than myeloschisis, extending from thè postbrachial
area înto the crural ênd posÈcrural regions and somet¡mes down to the
caudal reg i on
llith only two exceptions myelodysplasia took the form of hemimyelía,
One embryo however (428 561 showed two smal I and irregular masses of
neural tíssue wîth residual accessory canals in the caudal region
(diplomyel ia).. The other exception was an embryo (\28 69) showing
marked local necrosis associated hrith complete absence of neural tîssue
in the postcrural and caudal regions (amyel ia).
Examlnation of the hemîmyálias revealed no obvious demarcation
between neural plate and tal l-bud materials. The craníal end of each
leslon was covered by ectoderm and marked by reduction in size of the
neural tube, w¡th the wide gap between ectoderm and neural tube filled
by migrating mesenchyme cells. The reduction in neural volume affected
malnly the dorsal part of the closed tube, producing a rather triangular

t74
contour and an Irreguìar central canaì (F¡g ' 83)'
ln the central part of each hemimyel îa neural tissue formed a
V-shaped or U-shaped plaque in cont¡nuîty with the tall-bud material
of the caudal region. The volume of neural tissue was consíderably
reduced and lay at a deoper level than the adjacent 6ctoderm' givìng the
lmpresslon of reduced neural plate material (Figs' 84-85) '
The mîd-zone
of each plaque was exposed for a short distance but just cranial to thîs the
plaque was covered by ectoderm and often by mesoderm encroaching on the
mldl ine from the adjacent somites (fig 5. 84 , 85,88).Mìtoses were largely
restrlcted to cells of the dorsal surface of the plaque but were not as
numerous as in myeloschisls.
ln the caudal part of each hemimyel ia a closed neural tube was reformed,
though reduced in size when comparec to control and normal experimental
embryos, and usualty covered by somìtic mesoderm across the midline
( Frs. 88)
The notochord was uniformly în contact with neural tÎssue at all
levels of the myelodysplasias, in contrast to the wîde separation of notochord
from the cranial part of most myeloschisis lesions at stages l7-20.
Accessory canals were present in the caudal regïon of three embryos
wlth myelodysplasia, suggesting some deìay ln maturatîon of the tail-bud
¡n these embryos. The rhombic roof in dysplastic embryos had a membranous
appearance and an early choroîd pìexus, similar to thât in all other
Stage 17-20 emb ryos .
Somitic mesoderm adjacent to myelodysplasia was often'reduced În
volume and loosely arranged, wlth cystic spaces in some areas and occasional
hemorrhages from ìocal vessels (Figs' 83-88) '
Contact of

175
neurêl tîssue wíth mesoderm however was maintained at the sites of
myelodysplasias because of the encroachment of somitic mesoderm dorsaj
to the I es ions .
The most marked cystlc and hemorrhagic. changes extended from the
postbrachlal area to the caudal region and were a:íociated with defective
mâturatîon of the tail-bud. The embryo wìth ãmyel ìa (4Zf 6!) provided
the most extreme example of this process, with hemimyelia of the crural
regîon, amyel ia of the postcrural region, and loss of all recognîzable
structures în the cauda! region.
The embryo with diplomyel ia (42E 56) simllarly showed hemimyelia in
the postbrachiaì, crural and postcrural regions, giving way to d¡plonryel
ia in the caudal region assoclated ùrith the persistence of accessory
cana I s.
ln the slngle embryo showing both myeloschlsis and myelodysplasia
(428 21'r, ê rather smal I cord în the postbrachial region gave bray to an
everted myetoschisis (showing wide separation from the notochord) and
then ên exposed dysplastîc plaque (in contact with notochord) at the crural
region. There was a smal I irregular mass of uncanalized neural tlssue
(covered by ectoderm and somitic mesoderm across the midline) ín the postcrural
regfonl and amyelia in the caudal region. Neural. pìate and taÌlbud
materials were not clearly separated at the êrea of myeloschisis.
6.3.9 BgyjeU pt Histologîcal Changes in Experimenral Embrvos
A review of the histological features descríbed in Sectíon 6.3
revealed certain dlfferences between experimental and control embryos ln
each g roup.
ln the regions of myeloschisîs or myelodyspìasia experimental embryos
showed varlous changes which appeared to be assoclated consistently with
\

t76
the lesions. The interpretation of these cha.nges was complicated by
the artifactual distorsion of some embryos, which was assessed by a
detaÌled tabulatîon of the appearance of each region in every embryo
(see Sect ion 6.4) .
Group I {Stajç ll0 Embryós)
a) the neural plate în two experimena"l .rb.yo, showed slight eversion
of the neural fo¡ds at the pos.terior rhomboid sinus, compared to the
elevatîon or flattening seen in control embryos.
Group ll (Staqe 11-12 Embrvos)
a) the neural plate in all experimental embryos showed some delay in
closui'e, and in one case showed defînlte eversion of neural folds at
the posteríor rhomboid sinus
b) the appearance of the overlap zone was retêrded in all experimental
emb ryos .
Group lll
(Stagg t3-16 Embryos)
a) early myeloschlsis (caudar to the somite region) consisted of wrdery
everted neural folds in continuity wittr adjacent ectoderm, lying dorsal
to more normal taîl-bud materîal
b) several embryos wìth myeloschisis when compared to their controls
showed loss of contact between protosomite mesoderm and neural tissue
c) early myelodysplasla showed narrowly everted neural tissue 'in
cont¡nuity with tail-bud material, but apparently deficient in neural
plate mater i a I
d) ectoderm wâs present over the cranial and caudal sections of myelodysplasia,
but in the exposed middle sectíon was not in such smooth
cont¡nuity wlth adjacent neural tissue as in myeloschisis

t77
e) encroachment of somitîc mesoderm towards the mîdline dorsal to
areas of myelodysplasiã was assocîated wíth reduced neural volume
f) below the level of myelodysplasia rhere was slight reduction ln
the volume of protosomite and unsegmented mesoderm, wìth some local
cystic changes but no loss of contact with neural tissue.
Groúp lV (Staqe l7-20 Embrvos)
a) myeloschisis showed clear demarcation between neurar prate materíal
and tâ î I -bud material
b) ln most cases the notochord was wldely separated from the upper
thlrd of an area of myelosihisls (derived from neural plate material)
c) somite mesoderm showed loss of contact *lth nuur"l tissue at areas
of myeloschisis when compa red to the contro.l s
d) myelodysplasia occurred at a slightly more caudal .level than
myeloschîsis, and consisted of hemimyel ia, diplomyel ia, or amyel ía
e) in myelodysplasla there hras no separêtÍon between neural plête ênd
ta i I -bud mater¡âls
f) the myelodysplasias showed reduction in neural volume, and were
pêrtly covered by ectoderm
S) three embryos with myelodysplasia showed prolonged retention of
accessory canalsh)
somitic mesoderm often encroached on the midrine dorsar to ãreas
of myelodysplasîa and the caudêl cord
¡) somitic mesoderm adjacent to myerodysprasia was in contact with
neural tube, but often cyst¡c and reduced in volume.
These histological findings were tabulated by regions for all control
and experimental embyos (See Section 6.4).

Figs. 51 - 54, Normai de'¡elopment Ìn a St. l3 control enrb ryo shovring
the overlap zone from above down (lBC 27) (H â Ë; x40):
Fig. 51 .
Protosomîtesi notochord; slightly. âsymmetrical neural
canal marking upper end of overlap zone.
FiS. 5?. Protosomí tes; notochord; one accessory canal .
FiS. 53.
.Unsegmented
sornì.tic mesoderm; notochordl one accessory
canel opening lnto neural canal.
[¡s. 54, Unsegmented sonitic mesoderm; protonotochord; several
accessory cânals.

ôt
rf)
, ,,. ì _ ^iiir¡:ù\. I r i.

Fí9s" 55 - 60. llcrmaI development in a St. I6 control embryo showing
the overlap zone from above do¡in (30c Zz). Mitotic
fîgures adjacent to lumina of neural canal and
----^^ory canals. Ectodermal cöver. but no neuraì
crest material (H.a E; x4O) :
FÎS. 55,
Protosomites i notochord; asymmetrical neural canal
reveals upper end of the o.¡erlap zone.
FiS. 56.
Llnsegnented somitic mesoderml notochord; asymmetrical
neura I tube.
FiS. 57.- 60. Unsegmented somitic mesoderm; protonotochord; neural
canal extending dor¡rn to caudal region; accessory
canals extending up from caudál regîon.

l
55
56
57
58
59
ó0

F;gs 51 - 64. Normal devcìopn¿¡ii il a St. 1B controì etrìbiyu s¡rüia¡i¡,g
the caudal regîon from above down (hZC 7) " NeuraÏ
. canal symmetrical . Notôchord in close contact with
neural tube. Large somitesshowing differentÌation
Neural crest present, Cl

Fiss. 65
Early myeloschisis in a St. 14+ experimental embryo,
1B hours after wincion,î¡g (tBe 36) . Open neural folds
extending ciovr¡r to the cauciaì region anc{ overìying
eccessory canals in the tail-bud material. Extensive
necrosis of cells on exposed surface of open neural
plate. Normal protosomîtes. Notoch

:
ó5
66
67
69
70

fiSr. 71 - 76. Later myeloschisis in a St. i7 experîmentâl embryo,
42 hours afrer windowins (428 3) (H s E; x4o) :
FiS. 7.1 .
'
Synrmetrical neural canel with mï ios,es along ìu en.
SomÌtes dîspers!ng. Notochord vácuolated,
FiS. 72.
llide eversion of neural pìate material with some
mitótîc fïgures near exposed surface. l^lide separation
of notochord from neural pìaque. I'lo superficial
ecros I s
FiS. n ^ 7tt. Separation of neural plut" *"t.ri"l from tail-bud
material. Neural crest present. Notochord in close
contact wl th neural tissue. l"iitotic ì"ígures seen
along lumen of neural tube (derived from tail-bud
. material). Sonites show dîfferentiation.
Figs. 75 - 76. Neuraì tube composed of tail-bud mate¡-¡al, with mitatic
figures along the lumen. SomÌtes slightly reduced in
s ¡ze but well dîfferentÌated.

\l\)
ìr,jtr, r: : :;r:,irrliirqn
^ù): I

Fiss. 77 -82.
Early myelodysplesia in a St. 16 experinental ernbryo,
J0 hours after windovqing (¡OE 76) . lleural tissue
reduced irr rloìune and formíng a nårrow, open hemimyelia,
partly covered by ectoderÍ.. Neural tube
formed fronr taìl-bucl rnaterial in the caudal region.
A few scatterecl nitôt¡c f i5;ures. No neural crest.
Notochord v¡e I I -fcrnreci a¡rd in contact with neui.êl
tissue. Somites reduced ín volume and poorly
differentiated at level of the lesion (H s. E; x40).

:
F-
77

FÌgs. B3 - BB. Later myelodysplas Ìa in a St, lB experimental enbryo,
42 hours after window!ns (tlzË 50) . Notochord ìnelI -
formed and in coniact with neural.. tissue. l.lo neural
crest (H s E; .x40) :
,
Figs. 83 - 85. Progressíve reduction in neural vôlurne to a small,
flat plaque covered by ectoderm and so¡¡itic mesoderm.
Fig. 86,
0pen hemÌmyel ia. Somîtes reduced in volume and poorly
differentiated.
Figs. 87 - 88. Smal I neural tube formed by tail*buc{ material , and
covered by fused somites in the midline. Vessels
engorgàd with a probable local. hemorrhage.

''i
l.l

Figs, 89 - 91. Processíng artifacts in experimental ûnd control embryos
of different Stages (H a E; x40) :
FiS. 89. Separatìon of neu¡-al tissue from sornites at St. 10-
(oc 49) .
FiS, 90,
Separation of neurai tissue from pfotosomites and
notochord ar 5t. 1'l- (6E 28) .
Fig. 91 .
Reopening of neural tube with separation from
notochord and somites êr Sr. l3+ (1BE 25).
FiS. 92.
Separat¡on of area of myeìoschisis from protosomites
and norochord ar st. t4+ (lBE 54).
Fis. 93 - 94. Reopening of roof - platê ar st. 19 (4zc lt; hzl 31).

B9
90

ì86
6.3.10 SequenJ¡¿il ll lúSti¡it¡on6 óf Sèléctéd Eríbryos
To ¡l lustrate the events of normal and abnormal neural closure a group
of. sequent¡al. drawìngs of every tenth section are presented for eight
embryos. Serlal sections under a Leltz Dialux microscrope were projected
through a Sony DXC-1650 camera onto a Sony PYJ fO¡O micro-vìdeo monitor
and traced at an in¡tial magnifîcation of x88. Embryos selected for
illustration are shown ln Table J4 and Fiç.95to 102.
TABLE 34.
SEOUENTIAL ILLUSTRATI ONS
Embryo Stage Neural Ti ssue
18C 4 lt+ elevated (normal) posterior rhomboíd sinus
6f 34 1l+ everted (abnormal) posterior rhomboid sínus
18C 27 13+ normal neural closure
18E 36 14+ early myeloschlsis
\28 8 17 later myeloschlsîs
3OC 22 16 normal _neural closure
3OE 76 16 early hemimyel ia
428 50 18 larer hemlmyelia
At Stâge. 1l+ non-closure of the neural plate is first manifest as
eversion of the neural folds (68 34,Fîg.96 ) rather than elevation
(18C 4iFig.95 ) at the posterior rhomboîd sinus. Neither embryo shows
an overlap zone.
slightly later, a control embryo (18C 27,Fis,97 ) shows the neural
canaì traceable down to en open neural plate overlyîng one accessory
canal at the poster¡or rhomboid sinus. ln a comparable embryo with
early myeloschisis (l8e 36,Fig. 9B ) an open lrregular neural plate

187
overl îes three accessory canals at the rhomboid sinus. Establ ished myeloschlsls
(42E 8,f 19. 99) is assocîated with eversion of neural materîal
at the upper and lower ends of the leslon, separat¡on of neural sources
wîth progresslve reduction of neural plate materlal, and separation of
notochord from neural tÌssue at the cranial end of tbe leslon.
ln hemlmyel¡a (30E 76 ,428 50,Fl9s. lot s 102)the open defecrs extend
cranlally up from the ta¡l bud; there is no sign of an overlap zone
comparable to the control embryo (3OC ZZ,flg.100 ) showing accessory
canals and a fully closed neural plate. The cross-sectlonal area of
':
neural tîssue is reduced ln hemìmyel la and there is no separation
of neural tissue from notochord.

Fígs. 95 - 102, Sequentiêl drawings of every tenth seriâl sectÌon of a
group of control and experimental. embryos, to show the
development of. open cord defects. Drawings include
only neuraì tissue and notochord, witir brain region
ín first column, somite regíon of cord in seconcl
column, and caudal region of cord Ín thírd column:
Figs 95 - 96. Stage 11+ embryos (lBC 4; 6E 34) showing elevation
of neural folds in control embryo (arrow) and
eversion of neural folds in experímental embryo
(a r row) .

189
GoNTROL: STAGE ll+ (te c a)
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Fïgs. 97 " 99. Development of m)/eloschisis:
Fî S. 97.
Control embryo of 5t. 13+ (JrBC U) shor.,"ing an open
neural plate and one accessory canal at the rhomboid
sinus (arrow) but a closed neural tube above this
level .
FíS. 98,
Early myeloschisis in St. l4+ experimental embryo
(1BE 36) showing an open neural plate above the
rhomboid sínus (arrow) with several accessory canals.
FiS. 99.
Later myeloschisis in a St. 17 experimental embryo
(42E B) snowing ôn everted neural plaque separated
from notochord at the upper part of the defect
(arrow), separatîon of neural materials at the lower
. part of the defect (arron), and a normal neural tube
i.n the caudal region.

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193
MYELOSCHTSIS:STAGE ra.(re rea)
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Figs. 100 - 102, Development of myelodyspTasia:
Fig. 100.
Control embryo of St. 16 (30C ZZ), vtith a clcsed neural
tube and âccessory canals in the caudai region
(a r row) "
Fig. 101.
Early rnyelodysplasia in St. i6 experimental enib ryo
ßOf n.
Lower cord shows reduction in neural
volume, close contact with notochord, and hemimyel ia
in the caudal region (arrow).
Fig. 102.
Later myelodysplas ia in St. 1B experimenta.l embryo
(42E 50) w¡th reduction in neural volume, close
contact with notochord, and hehimyelia in the caudal
region (arrow). Taíl_bud has disappeared.

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6.4w
t99
The hlstologlcal study of neuruiation (Section 6.3.9) revealed
certaln dlfferences between experímentar and contror embryos. The changes
in the representatlve appearance of each region in every embryo are shown
ln Tables 35 -38,. The assessment of the separation of neural tìssue
from adjacent nobchord or somites was complicated by artifactual splittíng
in some embryos. However, when experîmental (g) and control (C) embryos
were compared in groups, some consîstent differences were evident.
Each regíon of each embryo is presented cnanío-caudal ly, so that
more than one description recorded for a region indicates a change in
the appearânce of that region from above downwards (Tables 35 _ 3g ).
Regions A,B,C,D and E of each embryo are described in terms of:
(a) cond¡tion of embryo after processing
(b) progress of neural closure
(c) number of accessory canals
(d) morphology of neural defects
(") cover of neural tissue by ectoderm
(f)
cover of neural tissue by mesenchyme (neurar crest or somitic mesoderm)
(s) contact.of notochord with neural tissue
(h) contact of somitic mesoderm with neural tissue
(l)
abnormal ities of somitîc nesoderm.

Som i te
Defects
contact none
sePa ra t ion none
separation none
contact none
contâct none
separat¡on none
contact none
contact none
contact none
contêct none
contact none
N'
o
Region Embryo Stage Condition Neural
Cl osure
Access.
Cana I s
Ectoderm Mesenchyme Notochord Somi te
Cover Cover Contêct Contact
6c 20 lo- good
c I osed/
closing
covered/
open
contêct
0C 49 t0- fai r
clòsed/
closing
covered/
oPen
contact
0C 52 10- good
c I osed/
cl os ing
covered/
open
con têc t
6c 21 10 good
c I osed/
closing
covered/
open
con têct
0C46 t0 fair
closed/
clos i ng
covered/
open
contact
6E 15 10' fair
cl osed/
closing
.0
covered/ none
open
contact
6E 8 t0 fair
c I osed/
clos ing
covered/ none
oPen
contact
6E 30 i0 faîr
c I osed/
closing
o
covered/ none
open
con têct
6E45 10 faìr
cl osed/
closîng
covered/ none
open
con tact
A
6E 18 t0+ poor
c I osed
covered none
contact
6E 41 10+ fair
c I osed
covered nìone
con tact

contact none
contact none
contact none
contact none
sepa ra t lon none
contact
contact
contact
con tac t
con tact
contact
none
none
none
none
none
none
NJ
o
6c 20 10- good
clos i ngl
i nverted
open
contact
B
0c 49 10- faîr
i nverted
0
open
none
con tact
B
0C 52 10- gocd
I nverted
0
open
none
contact
B
6c 21 10 sood
c I osed/
closing
0
cove redl
open
none
contact
0C46 10 fair
clos i ngl
i nverted
open
con tact
6E 15 to- faír
closing/
i nverted
open
contact
6E I l0 fair
closing/
i nverted
open
none
con tact
6e 3o to faîr
closîng/
i nverted
open none
contact
6E 45 to fair
c I osed/
closing
covered/ none
op9n
contact
6E 18 lo+ poor
c I osed/
clos ing
cove red,/ none
open
contact
6E 4t lo+ fair
closed/
closîng
covered,/ none
open
contact

Somite Som i te
Contact Defects
N'
o
N)
TABLE 35B.STAGE.IO CoNTRO.L AND EXPERIMENTAL EMBRYoS (GRoUP I)
Region Embryo Stage Condltlon Neural Access. Ectoderm Mesenchyme Notochord
Closure Canals Cover Cover Contact
c
6C 20 'l 0- good
i nverted
0 open
contact contact none
c
0c 49 10- fair
i nverted
0 open
contact sepa rat ion none
c
OC 52 10- good
inverted
0 open
none
contact contact none
c
6C 21 10 good
inverted
0 open
none
contact contact none
c
0c 46 10 faír
i nve rted
0 open
none
contact sepa ra t Îon none
6E 15 10- fair
inverted
,
0
open
none
contact separât¡on none
6E 8 10 falr
elevated
0
open
none
contact sepêrat¡on none
6E 30 lo fair
î nverted
0
open
none
con tact sepa rat ¡on none
6E 45 10 fai r
.closîng/
inverted
0
oPen
none
sepa ra t ion separation none
6E 18 10+ poor
inverted
0
oPen
none
contact contact none
6E 41 10+ poor
i nverted
0
open
none
contact contåct none
D
\
6C 20 10' good
i nverted
0
open
none
contact sepa ra t ion
D
0C 49 10- fair
i nver ted
0
open
none
contâct separatîon
D
OC 52 t0- sood
I nverted
open
none
eontact separat¡on

none
none
N)
o
6c 21
0c 46
10
10
good
fair
¡ nverted
i nverted
0 open
0 open
none
none
contåct contact
contact sepêratïon
D
6E 15 10- fal r
i nverted
0 open
none
cohtact
separatlon Rone
D
D
D
6E 8 to faîr
6E 30 10 fair
6E\5 to falr
e I eva ted
Înverted
closing/
inverted
0 open
0 open
0 open
none
none
none
contact
con tact
sePärat¡on none
separation none
separat¡on sepa rat lon none
D
6E 18 10+
POOr
í nverted
0 open
none
con tâct
D
6E 41 10+
poor
I nverted
0 open
nonè
contact
contact none
sepa ra t lon none

Som i te
Contact
Som i te
Defects
contact none
sepa ra t Ìon nonè
contâct none
separatlon none
sePa ra t Ion none
separat¡on none
separat ion nonè
separation none
separation none
sepêration none
separat¡on none
¡\J
o
Regîon Embryo Stage Conditîon
Neura I
C losure
Access.
Ca na I s
Ectoderm
Cover
Mesenchyme Notochord
Cover Con têct
E
6c 20 10- good
fl attened
0 open
E
0c 49 t0- fai r
el evated
0 open
E
OC 52 10- good
el evated
0 open
none
E
6C 21 10 good
e I eva ted
0 open
none
E
oc 46 10 faìr
f I attened
0 open
none
E
6E 15 10- fai r
f I attened.
0
open
E
6E I to fair
fl at tened
0
open
E
6E 30 t0 falr
flattened
0
oPen
none
E
6E \5 10 faír
everted
0
open
none
E
6E t8 10+ poor
el evated
0
open
none
E
6E 41 l0+ fai r
e l eva ted/
everted
,0
open
none

Som i te
Defects
contact none
contâct none
contact none
contâct none
contâct none
contact none
contact none
contact N)
o
AND EXPER I
Region Embryo Stage Conditlon
Neu ra I
C I osure
Acces s .
Cana I s
Ectoderm I'lesenchyme Notochord Somi te
Cover Cover Contact Con tact
lBc 4 t1+
gqod
cl osed
covered none
separation contact none
18C 23 12
good
c I osed
covered crest/
none
seÞâ ra t ¡on contact none
18c 7 1z+
good
c I osed
covered cres t/
none
sepa rat íon contact none
18C 22 12+
good
cl osed
covered crest/
none
separatíon contact none
A
6E t3 11- good
closed
0
covered none
separat ion
A
6E 28 11- poor
closed
0
covered none
separat ion
A
6E 31 11- fair
c I osed
0
covered none
sePa rat ¡on
A
6E 38 11- sood
c I osed
0
covered none
separat¡on
A
6E 44 11- good
closed
0
covered none
seParation
A
6E 2\ 11 sood
c I osed
0
covered none
sepa ra t ion
A
6E 34 l1+ good
c I osed
0
covered none
separation
18c 4 11+ good
c I osed
covered none
6on tact

none
none
N)
o
o\
B
r8c 23
12 good
c I osed
0
covered none
contêct contäct
B
18C 7
12+ good
c I osed
0
covered none
contact con têc t
B
18C 22
12+ g'ood
closed
0
covered none
contact contact
6E 13 lt- good
closed/
closing
covered
contact contact none
B
6E 28 11- poor
c I osed
0
covered none
separation separatíon none
B
6831 n- faîr
c I osed
0
covered none
contact separation none
B
6E 38 11- good
closed/
closing
0
covered none
contâct sepa ra t ion none
6E 44 11- good
c i osed/
closing
covered none
contact con tact none
6E 24 rl
good
closed
covered none
contact contact non"
6E 34 tl+
good
closed
covered none
contact contact none

contáct
contact
con tâc t
contêct
Som í te
Defects
sepa rat ion none N'
o
\
TABLE 368. STAGE 11-12 CONTROL AND EXPERIMENTAL EMBRYOS (
Regìon Embryo Stage
Condl tîon Neural Access.
Closure Cana I s
Ectodêrm Mesenchyme Notochord Som i te
Cover Cover Contact gon tact
c
18c 4 1t+
good
closed
0
covered none
contact
c
18C 23 12
good
c I osed
0
covered none
cùntact
18c 7 12+
good
c I osed
0
covered none
contact
c
18C 22 12+
good
c I osed
0
covered none
contact
/c
6E 13 rr-
6E 28 1.1-
good
poor
closlng
clos i ng
0 open
0 open
nonè
none
contact contact none
separation separation none
c
6E 31 11-
falr
closlng
0 open
contact separation none
c
61 38 1r-
good
clos í ng
0 open
contâct separatîon none
c
6E 46 11-
good
I nverted
0 open
contact seParation none
c
6E 24 11
good
clos I ng
0 open
contact separation none
c
6t 3\ il+
good
clos i ng
0 open
contêct separatïon none
t8c 4 t1+
goód
closlng/
lnverted
covered/
open
con tact
separatlon
18C 23 12
good
cl osed
cove red none
contact

separat ion none
contact/ none
sepa rêt.¡ on
none
none
NJ
o
oo
D
18C 7
12+ good
closed
covered none
con tact
D
t9c 22
12+ good
c I osed
covered none
contact
D
6E 13 11-
good
i nverted
0
oPen
none
separation separatÎon
D
6E 28 11-
poor
i nverted
0
open
none
separation s epa ra t ion
D
6E 31 lt-
faír
i nverted
0
oPen
none
contaçt sepa rât ion
D
6E 38 r1-
good
i nverted
el evated
0
open
none
con tact sepa rat ion
6E 4\ 11-
good
e ! eva ted
open
none
contact sepa rat ¡ on
6E 2\ 11
good
closing/'
eIevated.
open
none
contact sepê ra t ¡on
6E 3\ 1r+
good
e I eva ted
oPen
none
contact seParêt ion

Son i te
Con têct
sepa ra t ¡on
sePa ra t ion
separatíon
sepa ra t íon
Som i te
Defects
sePe rat Íon none
sepê rat ion none
sepa rat lon none
separatîon none
sepå rât îon none
sepâration none
separation none
l\)
\o
TABLE ?6C- STAGE 1I-12 CONTROL AND EXPERTMENTAL E¡4BRYOS (GROUP II)
Region Embryo Stage CondÎtion Neural Access. Ectoderm l'lesenchyme Notochord
Closure Canals Cover Cover Contact
l8c 4 11+
good
el evated
open none
18C 23 12
good
closed/
closing
covered/ none
open
18C 7 12+
good
c I osed/
closîng
covered/ none
open
18C 22 12+
good
closed/
closlng
covered/ none
open
E
6E 13 11- good
è I eva ted
0
oPen
E
6E 28 11- poor
e I eva ted
0
open
E
6E 31 il- fair
e I eva ted
0
open
E
6E 38 tt- sood
f I a t tened
0
open
E
6E 44 11- good
fl attened
0
open
E
6E 2\ 11 good
e I eva ted
0
oPen
none
E
6E 34 'l t + good
everted
0
open
none

Regíon Embryo Stage Conditîon Neural Access. Ectoderm Mesenchyme Notochord Somite
Closure Canal s Cover Cover Contact Contact
Som i te
.Def ect s
none
none
none
none
none
none
n orie
none
none
none
NJ
none ã
TABLE 37A. STAGE 13-16 CONTROL AND EXPERIMENTAL EI'IBRYOS (GROUP III)
A
A
A
A
A
18C 11
18C 10
18c r4
r8c 21
18C 27
30c 2
13-
13
13
l3+
13+
16
30c 3 16
30c 15 16
30c 12 16
good
good
good
good
good
poor
Poor
good
good
c I osed
c I osed
c I osed
cl osed
c I osed
cl osed
c I osed
c I osed
c I osed
0
0
0
0
0
0
covered
covered
covered
covered
covered
covered
covered
covered
cove red
crest
crest
crest
crest
crest
mesoderm/
crest
mesoderm
mesode rm
mesoderm,/
crest
sepa ra t i on con tact
sepa ra t ìon contact
sepa rat ion con tact
sêparat ìon con tac t
separat¡on con tact
separat¡on/ con tact
con tact
sepâ rat ion/ con tact
contact
sePêrat iony' con tact
contact
sepa rat ion/ con tâct
con tac t
30c 22 16
good
closed
covered
mesode rm/
crest
separat ion/ contact
contact
30c 25 16
900q
c I osed
covered
mesoderm/
crest
separation/ contact
contâct
18E 10 13- very poor closed
covered cres t
separat ion con tact

none
none
none
none
none
none
none
none
none
none
nonê
none
none
none
none
none
N)
A
r8E 6l
13
poor c losed
0
covered
cres t
sepa ra t ¡ Crn con tact
A
18E 25
13+
very poor c I osed
0
covered
crest
separat¡on contact
A
A
18E 13
r8E 28
14
14
Poor c I osed
verv Door ' .
c losed
0
0
covered
covered
crest
cres t
seParation con tact
sepa ra t ion contact
A
1BE 35
14
poor c I osed
0
covered
crest
sepa ra t ion contact
A
18E 47
14
góod c I osed
0
covered
crest
seParat ion con tact
A
18E 58
14
poor c I osed
0
covered
cres t
sePêratíon con tact
A
18E 36
14+
good' c I osed
0
covered
cres t
sePa ra t iÕn contact
A
18E 53 14+
poor c I osed
0
covered
crest
separatîon contact
r8E 54 14+
very Poor c I osed
0
covered
crest
separation. contact
A
tBE 59 14+
good cl osed
0
covered
crest
separation contact
A
18E 44 15-
good cl osed
0
covered
crest
sepa rat ion con tact
A
30Ê 4 15
good c I osed
0
covered
mesoderm/
crest
separat ion/ con tact
con tact
30E 25 15
fa î r cl osed
covered
mesoderm/
crest
separat Íon/ contêct
con tact
3oE 9 16
gool c I osed
covered
mesoderm
seParation/ con têct
con tac t
308 26 16
good c I osed
cove red
mesoderm
separatîon/ contêct
con tact
30E 35 16
faí r c I osed
covered
mesodeim
sepa ra t lon/ contact
contact

none
none
none
none
none
none
l'.J
l\)
3oE 56 16
good
c I osed
cove red
mesoderm
separation/ con tâct
contact
3or 59 16
good
c I osed
covered
mesoderm
separat¡on/ con tact
contact
30E 69
16
good
Cl osed
covered
mesoderm
separat lon/ contâct
rîon tact
3oE 76
16
good
closed
covered
mesoderm
separatìon/ contact
contact
3oE 52 16
good
c I osed
covered
mesoderm/
crest
sepâ ra t l.pn/ contact
contact
3ot 77 16
good
closed
covered
mesoderm/
crest
sepa rat ion/ con tact
contact

contact
con tac t
contact
con tact
contact
contact
contact
contact
con tact
contact
contact
Somi te
Defects
none
none
none
none
none
none
none
none
none
none
none
N)
TABLE 378. STAGE I3-16 coNTRoL AND EXPERIHENTAL'EMBRYOS (GROUP III)
Reg ì on Embryo Stage Cond I t lon Neural
C I osure
Access.
Cana I s
Ectoderm Mesenchyme Notochord Som I te
Cover Cover Contact Contact
B
r8c 11
13- good
c I osed
0
covered
none coñtact
B
r8c r0
13 good
closed
0
covered
none con tac t
B
r8c 14
13 good
closed
0
covered
none con tact
B
t8c 21
13+ good
c I osed
0
covered
none con tâct
B
18C 27
13+ good
closed
0
covered
none con tact
B
30c 2
16 poor
cl osed
0
covered
crest/ con tact
none
30c 3 16
POor
c I osed
covered
mesoderm/ con tact
crest
3oc 15 16
good
c I osed
covered
mesoderm/ ccn tac t
crest
30c 12 16
good
c I osed
0
covered
crest/ con tact
none
30c 22 16
good
c I osed
10
covered
crest/ contact
none
3oc 25 16
9o0d
closed
0
covered
crest/ contact
none

18E 10 13- vëry poor
closed 0
covered none
con tact
contact/ none
separatîon
B
18E 61 13 poor
closed 0
covered none
con tact
contact none
B
tBE 25 13+ very poor
closed 0
covered none
separat¡on/ separation none
con tac t
B
B
t8E 13 14 poor
t8E 28 14 very poor
closed 0
closed 0
covered none
covered none
contact
con tâct
contêct none
contact none
B
l8E 35 14 poor
closed 0
covered none
con tact
contact none
B
18E 47 14 good
closed 0
covered none
con tact
contêct none
B
B
18E 58 14 poor
18E 36 14+ good
closed 0
closed 0
covered none
covered none
contact
con tac t
contact none
contact none
B
18E 53
'I
4+ poor
closed 0
covered none
con tact
B
r8E 54
14+ u.ry poor
closed 0
covered none
contêct
B
18E 59
14+ good
closed 0
covered none
contact
B
188 44
15- good
closed 0
covered none
contact
B
30E 4
15 good
308 25 15 fai¡
closed O
closed/ 0/1
myeloschisis
J
covered crest/
none
covered/ crest/
open none
contact
con tact
contact none
contact none
contâct none
contâct none
contact none
contêct/ none
sepa rê t ¡on
30E 9 16 sood
closed 0
contact none
covered mesoderm/ contêct
crest N¡

Som i te
Con tact
Som i te
Defects
contact none
contâct none
contact none
contact none
contact none
separation none
con tact none
contact none
contact none
geparatlon none
contâct none
EXPERIMENTAL EMBRYOS
Region Embryo Stage Condltîon Neural
C losure
Access.
Cana I s
Ectoderm Mesenchyme Notochord
Cover Cover Con tact
¡^
l8C 11 13- sood
closed
0
covered none
contêct
c
18C 10 13
good
c I osed
0
covered none
contact
c
r8c 14 13
good
c I osed
0
covered none
con ta ct
c
18C 2t 13+
good
c I osed
0
covered none
contâct
c
18C 27 13+
good
c I osed
0
covered none
con tact
c
3oc 2 16
POOr
c losed
covered none
eon tact
c
3oc 3 16
poor
c I osed
0
covered none
contact
c
3oc 15 16
good
closed
0
covered none
contact
c
30c t2 16
good
cìosed
0
covered none
con tact
c
30c 22 16
good
c I osed
0
covered nonê
contact
c
3oc 25. 16
good
c I osed
0
covered none
con tact
c
18E 10
13- very Poor cl osed
0
covered none
separation separation
c
1BE 61
13 poor c I osed
0
covered none
contêct con tac t
c
188 25
13+ very poor
cl osed/
everted
0
covered/ none
open
separation separat ¡on 19
o\

separãtion none
contact none
contact none
separation none
sepa ra t ion none
contact none
contact none
separatlon none
contact none
contêct none
contact none
separati,on/ none
con ta ct
contact none
contact none
sepa ra t ion none
separêt ion/ none
con tsct
contact none
NJ
\
l8E 13 14 poor
c I osed/
everted
covered/ none
' open
con têct
c
18E 28 14 very poor
c I osed
0
covered none
con tact
c
18E 35 14 poor
c I osed
0
covered none
con ta ct
c
18E 47 14 sood
closed
0
covered none
contact
c
l8E 58 t4 poor
closed/
.myeloschlsls
1
covered/ none
oPen
con tê ct
c
tBE 36 14+ sood
c I osed
0
covered none
qon tact
c
18E 53 14+ poor
c l'osed
0
covered none
contact
c
18E 54 14+ very Poor
c I osed
0
covered none
contact
c
18E 59 14+
good
c I osed
0
covered none
contact
t
r8E 44 15-
good
cl osed
0
coveeed none
con tact
c
308 4 15
good
c I osed
0
covered none
contact
30E 25 15
fair
myeloschisis
2
open none
con tact
c
c
30E 9 16 sood
308 26 16 good
closed
c I osed
0
r0
covered none
covered none
contact
contact
c
3oE 35 16 fair
hemi mye I ia
0
covered none
contact
c
308 56 16 sood
myeloschisis/'
c I osed
2
open/ none
covered
con tact
308 59 16 sood
c I osed
covered none
con têct

sepa ra t lon none
contact/ cys ts
separatîon
separation none
contact none
NJ
@
c
3oE 69 16
good
myeloschîsîs
1
open none
contact
c
30E 76 16
good
hem i mye I ia
0
oPen none
con tact
30Ê 52 16
good
mye I os ch I s 1sl
c I osed
open/ none
covered
con tact
308 77 16
good
closed
covered none
con tact

Regíon Embryo Stage Condition Neural Access. Ectoderm Mesenchyme Notochord Somite
, Closufe Canals Cover. Cover Contact Contact
Soml te
Defec t s
separatlon none
sepa rat lon none
sepa rat ion none
separation none
sepâ ra t ion none
sepa râ t lon none
sepa rat ¡on none
separat ion none
separation none
separation none
I
\o
TABLE 37D. slAGE 13-16 GONTRoL AND EXPERIMENTAL EMBRYoS (GROUP III)
D
18C 11 13- good c'losed
0
covered none
contêct
separât¡on .none
D
18C 10
13
good
c I osed
0
covered none
contact
D
l8c r4
13
good
c I osed
0
covered none
contact
D
18C 21
13+
good
c I osed
1
covered none
con tact
D
18c 27
13+
good
c I osed
1
covered none
con tåct'
D
30c 2
16
poor
closed
2
covered none
contact
D
30c 3
16
poor
closed
3
covered none
contact
D
30c 15
t6
good
c ¡ osed
1
covered none
contact
D
D
30c 12
30c 22
16
16
good
good
c I osed
c I osed
2
'0
covered none
covered none
con tact
con tact
D
3oc 25
16
good
closed
3
covered none
contact
lBE 10 13- u..r
:oo. ":;::ij,
covered/
open
separêt¡on separat ion none
D
18E 61 13 poor myeloschisls
1
open
contact sepa ra t ¡on none
D
188 25
13+
very poor myeloschisis
3
open
sepa rat ion sepêration none
D
lBE 13
14
poor myeloschîsïs
\
open
contâct sepä râ t Îon none

N)
N)
o
r8E 28 14 very poor
18E 36 14+ good
r8E 35 18E 47 18E 58 14 r4 14 poor
good
poor
c I osed/
elevated
c I osed/
myeloschîsis
closed
myeloschîsîs
cìosed/
myeìoschisis
2
3
covered/ none
open
covered/ none
qpen
covered none
open nonè
covered/ none
open
separat ion separation none
contact separation none
contact sepa ra t ion none
contact
contact
separation ñone
separation none
1BE 53 14+ poor
c I osed/
myeloschisis
covered/ none
open
contact
sepa ra t ion none
t8E 54 l lt+ v¿¡t ooot
c I osed/
myeloschìsìs
covered/ none
open
sePa rê t ¡on
separatlon none
18E 59 14+ good
cl osed/
myeloschisis
covered/ none
open
con tact
cpntact none
18E 44 15-
good
c I osed
2
covered none
con têct
3oE \ 15
good
c I osed
2
covered none
contact
30Ê 25 1, faÎr
myeloschisis
open/ none
covered
contact
D
308 9 16
soo.d
c I osed
t
covered none
con tact
D
D
30E 26 16
30E 35 16
good
fair
closed
closed
2
0
covered none
covered mesoderm
contact
con tac t
D
3oE 56 16
good
c I osed
1
covered none
contact
contêct none
coñtact/ none
sepa ra t ion
contact none
sepa ra t îon none
separation none
contact cysts
contact none

separation none
sepâ ra t îon none
separat îon cys ts
separãtÎon none
separêtlon none
N¡
NJ
308 59 16
3oE 69 16
good
good
closed
myeloschisis
covered
open /
covered
contact
con tact
3oE 76 16
good
' hem î mye I îal
closed
open/
covered
contact
3oE 52 16
good
c I osed
0
covered
contact
3oz 77 16
good
c I osed
0
covered
contact

Som i te Som i te
Contact Defects
sepê ra t ion none
separatíon none
separation none
sepa rê t Ion none
sepa ra t ion none
separât ion none
sepêrat¡on none
sepa ra t ion none
separation none
sepa rat Íon none
separation none
N)
N)
TABLE 378. STAGE t3-16 coNTRoL AND EXPERII'ÍENTAL EMBRYoS (GRoUP III)
Regîon Embryo Stage Condí tion Neural Access.
Closure Canals
Ectoderm Hesenchyme Notochord
Cover Cover Con tâc t
18c 11 13- good
c I osed/
closing
covered/ none
open
18C 10 13 good
cùosed/
closing
covered/ none
open
18c 14 13 sood
c I osed/
closing
covered/ none
oPen
t8c 21 13+ good
c I osed/
closing
covered/ none
open
18c 27 t3+ good
c lbsed/
closing
covered/ none
open
E
30C 2 16 poor
c I osed
covered none
E
30C 3 16 poor
closed
4
covered none
E
30C 15 16 sood
closed
1
covered none
E
30C 12 16 good
c I osed
1
covered none
E
3OC 22 16 good
c I osed
ll
covered none
E
3OC 25 16 soo;
closed
3
covered noné

separâtion none
sepa ra t ion
separat ion none
sepa ra t lon nonE
seParatíon none
separation none
seParatlon none
sepê ra t ion nonè
seParation none
separat¡on none
separation none
sePa ra t lon none
sepa ra t ion none
seParat ion none
sepa rat ion none
sepa ra t íon none
sepa rat ion none
sepå ra t lon none
I\J
N
E
18E 10 13- very poor
everted
1
open none
E
18E 61 13 poor
myeloschisis
1
open none
E
188 25
13+
very poor
myeloschisis
0
open none
E
18E 13
r4
poor
myeloschisis
0
open none
E
18E 28
r4
very Poor
elevated
2
open none
E
18E 35
r4
poor
myeloschisis
2
open none
E
18E 47
14
good
e I eva ted
1
open none
E
t8E 58 t4 poor
everted
0
open none
E
18E 36 14+ sood
myeloschisis
2
open none
E
18E 53 14+ poor
myeloschisis
3
open none
E
18E 54 14+ very pôor
myeloschisis
0
open none
E
18E 59 14+ good
elevated
-0
open/ none
covered
E
18E 44 15- sood
closed
3
covered none
E
3oE 4
15 good
cl osed
2
covered none
E
30E 25
15 fair
c I osed
3
covered none
E
30E 9
16 9ooà
cl osed
2
covered none
E
3oE 26
16 good
closed
t
covered none
E
308 35
16 fal r
c I osed
covered none

sePârat Îon
sepa ra t ¡on
separatÎon
separation
sepa ra t ion
sepa rat ion
none
none
none
none
none
none
N'
N
.È-
E
3oE 56
16 good
closed
3
covered
none
E
30E 59
16 good
c I osed
2
covered
none
E
30E 69
16 good
closed
1
covered
none
E
3oE 76
16 good
c I osed
I
covered
none
E
3OE 52
16 good
c I osed
0
covered
none
E
30e 77
16 good
c I osed
0
covered
none

Reglon Embryo Stagd cond¡t¡on Neural Access. Ectoderm Mesenchyme Notochord Somite
Closure Canals Cover Cover Contact Contact
Somi te
Defects
none
none
none
none,
none
none
none
none
none
none
none
N'
¡\'
TABLE 38A. STAGE f7-20 CONTROL AND EXPERIMENTAL EHBRYOS (GROUP IV)
42C 4 18
POor
c i osed
covered
mesoderm
sePa ra t ion/ contact
con tact
\zc 7 18
poor
closed
covered
mesoderm
separat Ìon/ con tact
con tact
42c z 19
good
c I osed
covered
mesoderm
separ€¡t¡on/ contact
contact
42c 6 19
poor
c I osed
covered
mesoderm
separêtion/ contact
con tact
42c 11 t9
poor
c I osed
covered
mesoderm
separat¡on/ con tact
contact
42C 3
20
poor
c I osed
covered
mesoderm
separation/ contact
con tac t
\2c 8
42c 21
428 I
20
20
t7
good
poor
good
c I osed
c I osed
c I osed
0
0
covered
cove red
covered
mesoderm
mesode rm
mesoderm/
crest
sepa rat ion/ con tact
con tact
sepâ ra t îon/ contact
con tact
seParat ion/ contact
con tac t
4zE 10 17
good
c I osed
covered
mesoderm/
crest
separat ion/ con tac t
con tact
\28 j2 t7
POor
c I osed
covered
mesoderm/
crest
separat icn/ contact
con tact

none
none
none
none
none
none
none
none
none
none
none
N)
none ts
428 1 18
good
closed
covered
mesoderm separat ion/ con tact
. contact
428 21 18
good
c I osed
covered
mesoderm separation/ contêct
contâct
\28 34
18
good
c I osed
covered mesoderm
separat íon/ con tact
contact
\2E htt
18
good
c I osed
covered mesoderm
separat¡on/ contãct
contact
\zE 49 rB
good
c I osed
covered mesoderm
sepâ!"at¡on/ contact
contact
\zE 50
18
good
c I osed
covered mes0derm
sepârat Îon/ contact
contact
hzE 5\
18
good
c¡osêd
covered mesoderm
sepa rat ion/ contact
contact
\28 56
18
good
c I osed
covered mesoderm
seParat ion/ con tact
contact
42E 26 19
good
c ¡ osed
covered mesoderm
separatâon/ contact
contact
\2E 3i
19
ooo:
c I osed
covered mesoderm
separation/ con tact
con'tact
428 57
19
good
c I osed
covered mesoderm
separation/ con tact
contact
\28 65
19
good
closed
covered mesoderm
sepa ra t ion/ contåct
gontact

none
none
none
none
N)
NJ
.{
A
A
A
A
428 69 19 good
\2E 73 20 good
428 17 20 good
4zE 72 20 good
c I osed
closed
c I osed
c I osed
0
0
0
0
covered mesoderm
covered resoi.rm
covered mesoderm
covered mesoderm
separation/ con tact
con tac t
sepârat ¡on/ contact
contact
sePa ra t ion/ contact
con tact
seParat¡on/ con tact
contact

Region Embryo Stage Condltîon Neural Access. Ectoderm Mesenchyme Notochord Somíte
Closure Canal s Cover Cover Contact Contêct
Som i te
'Defects
none
none
none
none
none
none
none
cysts
N)
l'.¡
oo
TABLE 3BB. STAGE 17-20 CONTROL AND EXPERIMENTAL EI'IBRYOS (GROUP IV)
\zc 4 r8
poor
c I osed
covered
mesoderm/
crest
contact contact
42c 7 18
poor
c I osed
covered
mesoderm/
crest
contact contact
4zc z 19
good
cl osed
coVered
mesode rm/
crest
contact coR tact
4zc 6 19
poor
closed
cove red
mesoderm/
crest
contact con tact
42c 11 19
poor
c I osed
cove red
mesoderm/
cres t
contact con tact
42C 3
\zc I
20
20
poor
good
c I osed
c I osed
0
covered
covered
mesoderm/
crest
mesoderm
contact con tact
contact con tact
\zc 21
20
poor
c I osed
0
covered
mesoderm
contact/ contact
seParation
\28 8 17
soo9
closed/
myeloschisls
covered/
open.
mesoderm/
none
contact/ con tact
sepa rê t ion
42E to 17
good
myeloschisls
covered/
open
mesoderm/
none
contact/ con tact
sePêration
\28 i2 17
POOr
c I osed
cove red
mesoderm/
cres t
contact contâct

contact/ none
separatlon
contact none
contact none
contact none
contact none
contêct cysts
contact none
contact cys ts
contact none
contâct nonê
contact none
contâct none
contact cys ts
N'
N¡
\.o
42Ê I 18
good
closed/
m),e¡oschisis
covered/
open
mesode rml
none
contact/
separatÌon
\zE 21 18
4zE 34 r8
good
good
c I osed/
myeloschisis
closed
covered/
open
covered
mesoderm/
none
mesoderm/
cres t
contact/
separat¡on
con tac t
\28 U+
18
good
c I osed
covered
mesoderm/
crest
con tact
\zE 49
18
good
c I osed
covered
mesoderm/
cres t
ccn tact
42E 5a
18
good
c I osed/
hem î mye I ia
covered
mesoderm
con tact
LzE 54
18
good
c I osed
cove red
mesoderm/
crest
contact
\28 56 18
good
c I osed/
hem i myel 1a
covered
mesoderm/
crest
contact
:
428 26 19
good
c I osed
cove red
mesoderm/
cres t
contêct
4zE 31
19
poor
c I osed
covered
mesoderm/
crest
contact
428 57
19
no.:
c I osed
covered
mesoderm/
crest
contact
4zE 65
19
good
c I osed
covered
mesoderm/
crest
contact
42E 69 19
good
c I osed/
hem i mye I ia
covered
mesoderm/
crest
con tact

none
none
428 17 zo
c I osed
covered
mesoderm/
crest
contact con tact
\2E 72 20
c I osed
cove red
mesoderm/
crest
contact con tact
428 73 20
c I osed
cove red
mesoderm/
crest
contêct/ con tact
sepa ra t ion

Region Embryo Stage Conditlon Neural Access. Ectoderm Mesenchyme Notochord SomÎte
Closure Canals Cover Cover Contact Contact
Som i te
Defec t s
none
nOrre
none
none
none
none
none
none
¡\)
TABLE 38C. STAGÊ 17-20 CONTROL AND EXPERIMENTAL EMBRYOS (GROUP IV)
c
c
c
c
c
42c 4 18
\2c 7 18
I+zC 21 20 poor
poor
poor
\2C 2 19 good
t+zc 6 19 poor
hzc 11 19 poor
\zc 3 20 poqr
\zc I 20 good
c I osed
c I osed
closed
c I osed
c I osed
closed
c I osed
c I osed
0
0
0
0
0
0
0
0
covered none
covered cres¡-/
none
covered crest
covered cres t
covered crest
covered crest
covered crest
covered crest
con tact
contact
contact
con tact
con tact
contact
contact
con tact
contact
contact
con tact
contact
contêct
contact
contêct
contact
\28 8 17
good
myeloschisis
open/ none
covered
contact sepêrâtlon none
\28 10 17
good
c I osed
l0
covered crest
con tac t 6êparat¡on none
\zE 52 17
poor
hem i mye I ia
0
open/ none
covered
contact sepa rat í on cysts
\zE 1 18
good
myeloschisis
open/ none
cove red
separêt¡on/ separêtion none
contact
428 21 18
good
myeloschisis
open/ none
covered
separat¡on/ separatîon/ cysts
contact con tact

contact none
contact/ none
separat íon
contact none
contêct none
sepa ra t ion none
contact/ cys ts
sepa ra t lon
contact/ none
sepa ra t ion
contact none
contact/ none
sepa rat ion
contact/ none
sepa ra t ion
contact cysts
contact none
separatlon cys ts
contact/ none
sepa ra t lon
N)
N'
hzE 3\ 18
good
cl osed
0
covered
crest
contact
\zE 44 18
good
myeloschlsls
0
covered/
oPen
crest/
none
con tact
\zE 49 18
good
c I osed
covered
crest/
none
contact
42E 50 t8
good
hem í mye I la
0
covered
mesoderm
con tact
\zE 54 18
good
myeloschisis
0
covered/
open
none
ccn tact
\zE s6 18
good
hem I mye I la
cove red/
oPen
mesoderm/
none
con tact
428 26 19
good
c I osed
cove red
crest/
none
contact
c
4zE 31 t9
poor
c I osed
0
covered
crest
contact
c
hzl j7 t9
good
c I osed/
myeloschisls
0
cove red/
open
crest/
none
contact/
separat¡on
4zE 65 t9
good
c I osed/
myeloschlsls
covered/
open
crest/
none
contact
c
\zE 69 19
good
hem i mye I ia
0
covered
mesoderm
contact
c
,+28 17 20
good
c I osed
0
covered
crest
con têct
c
\zE 72 zo
good
myeloschisìs
0
open
none
sepa rê t ion
c
t+28 73 20
good
c I osed
0
covered
crest
contact

TABLE 38D. STAOE 17.20 CONTROL AND EXPERII,4ENTAL EMBRYOS (GROUP IV)
Region Embryo stage Condltion Neural Access. Ectoderm Mesenchyme Notochord Somite somite
Closure Canals Cover Cover Contact Contact Defects
contact/ none
separation
con tact/ none
separat¡on
contact/ none
separåtion
contêct/ none
sepa râ t ¡on
sepa ra t îon none
sepa ra t ion none
sepa rat ion none
sepa ra t î on cys ts
sepa ra t i on none
1..)
D
D
D
\2c 4
ízc 7
\zc z
18 poor
18 poor
19 good
c I osed
c I osed
c I osed
0
0
0
covered none
covered none
covered crest
con tac t
con tact
contact
sepa ra t ion none
sepa ra t ¡on none
contact/ none
separation
\zc 6 19
poor
c I osed
covered crest
con tact
\2C 11
19
poor
c I osed
covered crêst
contact
\zc 3
20
poor
closed
covered cres t
con tact
\2c I
20
good
c I osed
covered crest
contact
4zc 21
20
POOr
c I osed
covered crest
contact
D
\28 I 17
good
c I osed
0
covered none
con tact
D
42E 10 17
good
closed
0
cövered none
contact
D
D
\zE 52 17
428 118
poor
good
c I osed
c I osed
0
covered none
covered none
contact
contact

contact cys ts
separation none
sepa ra t îon none
contêct. none
contact cys ts
separation none
sepa ra t ion cys ts
sepa ra t lon none
separation none
separation none
separatîon none
separation none
sePâ ra t ion none
seêpa rat ion none
N)
D
42E 21 18
good
hemîmyel ïa
0
covered
mesoderm con têc t
D
hzl 3\ 18
good
closed
0
covered
crest con tact
D
428 44 18
good
myeloschlsls/
c I osed
0
open/
cove red
nóne/ contact
crest
D
428 49 18
good
c I osed
0
covered
crest con tac t
D
\28 50 18
good
hem ì mye I ia
0
covered
mesoderm con têc t
D
\28 5t+ t8
good
myeloschisis
0
open/
covered
none con têct
hzE 56 18
good
hem i mye I îa
open/
covered
none con tact
D
\28 26 19
good
c I osed
0
covered
none contact
D
\zE 31 19
poor
closed
0
covered
crest con tac t
D
\zE 57 19
good
myeloschîsis/
c I osed
0
open/
covered
none/ con tact
cres t
428 65 19
good
myeloschlsls/
c I osed
open/
cove red
none con têct
D
D
\zE 69 19
428 17 20
good
good
amyel îa
closed
'o
covered
crest contact
D
\zE 7z 20
good
c I osed
0
covered
none contact
D
\2E 73 20
good
c I osed
0
covered
crest contact

Reglon Embryo Stage Condition Neural Access. Ectoderm llesenchyme Notochor

contact cysts
sepãration none
separation none
sepa rat Ion none
contact cysts
sepa rat ion none
sepa ra t lon cysts
separat¡on none
separat¡on none
sepêratlon none
sePa ra t Íon none
sepê ra t ¡on none
N)
\, o\
42E 21 18
good
hem î mye I ía
covered
mesoderm contact/
\28 34 18
good
closed
covered
crest con tact/
428 \4 18
good
c I osed
covered
crest con tact/
428 49 18
good
c I osed
covered
crest con tact/
\zE 50 18
good
hem Ìmye I îa
covered
mesoderm con têct/
-.
\zE jU tB
good
cl osed
covered
nonè contact/
\28 s6 18
good
dlplomyel ia
4
covered
mesoderm
\2E 26 i9
good
closed
0
covered
none contact/
qzE
31 19
poor
c I osed
covered
crèst coñ tact/
\2E 57 19
good
c I osed
covered
none con têct/
4zE 6i 19
good
ciosed
covered
none contact/
E
\28 69 19
good
amyel ia
E
\zE 17 zo
good
c I osed
covered
none contâct/

428 72 20
closed
covered none
contact/ sepa ra t lon none
hzE 73 20
closed
covered none
contact/ sepê rât ion none

238
This revlew of the histologlcal appearance of every sectloned embryo,
after al lowing for some cracking durÌ.ng processing, reveals a series of related
events during neurulation. The flattened neural plate, protonotochord,
and fused somltic mesoderm lying adjacent to Hensenrs node show
progresslve changes as they are fol lowed cran.ially.
Formatlon of the notochord ís accompanied by development of unsegmented
mesoderm and elevation of the neural folds. Cranial to this,
development of protosomites is accompanied by ínversion of the neuraì
folds. As the somites develop, with încreasing differentiat¡on ¡nto
centrêl and perlpheral regions, they show progressive expansîon which
ls accompanied by further inversion and then closure cif the neural folds.
After fusion of the neural folds the closed tube is inîtlally in
contact with the overlyîng newly-fused ectoderm. At stage l2 neural crest
cells begin to infiltrate between neural tube and ectoderm, beginning with
the point of inltial closure at the hindbrarin. By Stage 1!, mesenchyme
cells from locally-dispersing somites then. migrate between neural tube
and ectoderm, also beginning at the hìndbrain. ln embryos with myelodysplasia
a local reduction in neural volume allows the adjacent somites
to fuse across the midl ine, dorsal to neural tissue.
ln experimental and control embryos until Stage l! the notochord îs
separated from developing brain but lies in close contact *¡tn tÁ" developing
cord, except for the upper part of establ ished myeloschisis tesions
in most cases of myeloschisis.
}Jhlle fused and unsegmented somít¡c mesoderm are separated from neural
tlssue durlng early neurulation, the protosom¡tes are in contact wlth

239
neural tissue ât the later stages of closure ¡n most control and normal
experlmental embryos. Hany embryos wÌth myeloschisis, however, show
some loss of con.tact between neural tissue and protosomìtes (stages 13-16)
or somìtes (Stages 17-20'). Embryos with myelodysplasia general ly retain
contact between somite mesoderm and neural t¡ssue, but the mesoderm often
shows cystic changes and reduced volume.
Even though the distrlbution of Stages is not perfectly mêtched,
there ls no major difference ín the number of accessory canals between
experimental and control groups, though embryos wîth myeloschisis and
myelodysplasla show some delay in the disappearance of accessory canals
at Stages I 3- 16.
When the development of neural defects is followed, myeloschisis
appeårs ât an eêrlier Stage ênd at a slightly higher level than the myelodysplaslas
(see Tables 37 è 3B). Thus myeloschisis is first detectable as
a wide eversìon of the neural folds, in smooth cont¡nu¡ty wíth ectoderm at
Stage 10 (6E 45), leading to non-closure and separation of the two sources
of neural material by Stage 13 (18E 61). ln Stage 1/-20 embryos the lesíons
lie in regions B, C and D, gíving way to a normal cord developed from
tail-bud material in region E.
tlyelodysplasía fîrst appears âs a narrow eversion of the neural folds
at Sta.ge 16 (3OE 35, 30E 76) wittr no separatîon into rwo sorr".s of neural
materlal and partial ectoderm cover. At Stages 17-20 the lesions occupy
regions B, C, D and E, gîving way to a rather small cord or to diplomyel ia
or amyel ia in region E.
0n the basls of these findings several aspects of neurulatîon were
analysed quantltatlvely to determine the significance of differences
between experimentat and control embryos (see Sections 6.7, 6.8 and 6.9).

240
The histological features selected for analysis were:
(a) pr.ogress of normal neuraI closure
(b) development of myeloschisis
(c) development of mye I odysp I as ia
(d) length of the overlap zone (rather than merely the number of
accessory canals)
(e) cover of neural tíssue by ectoderm (though not by mesenchyme)
(f)
contact of neural tissue with notochord
(g) contact of neural tissue with somites
(h) cys.tlc changes and reduced volume of somites.
6.5 coMpêRlsoN 0J !l!r!!qcrEÂL FrNprNGs r^,rrH AppEARAtlqE_eL r,/!e!Elf4!R\roå
The histologîcal review of normal neural closure in controì embryos
(Sectîon 6.4)may be compared with the appearances of the same whole
embryos recorded by camera lucida drawings before seríal sectioning
(Sectlon 6.t.¡),
ln Tables 39-42, sectioned embryos of Gròups llland
lV are divíded into four categories, based on neural defects:
Stage 13-20 control emb ryos
Stage 1l-20 experimental embryos w¡thout neural defects
Stage 13-20 experimental embryos with Íryelosch¡sís
Stage 13-20 experimental embryos w¡th myelodysplasia.
The morphology of each whole embryo is compared with the histological
appearance of:
(a) the neural tube at somite and post-somite levels
(b) the rhomboid s i nus.

241
The control group (Table 99 ) show no neural defects; an oval
rhomboid sinus corresponds to ínciplent histologìcal closure. ln
exper¡mental embryos without neural defects (Table 40 ) there are again
no cord defects, but in two cases (l8E 44, 30E 4) a closed rhomboid sinus
ln serial sectlons was recorded as open in the whole embryos. Embryo
l8E 10 should probably be regarded as an example of early myeloschisis,
0f the embryos wÌth histologîcal myeloschisis (Table At
) two were
recorded with irregular, rather than regular, defects în the whole embryos
(30E 56,42E 8). At the rhomboid sinus one embryo (lBE 59) exhibited an
oval rhomboid sinus and elevated neural folds caudal to the lesion, and
another (3OE 25) showed a triangular rhomboid s¡nus and closed neural folds.
Apart from these four exceptions there is close âgreement between the
histological findings and the camera lucida drawings, with a trîangular
rhomboid sinus corresponding to early myeloschisis at Stages lJ-14, and a
regular cord defect correspondíng to establ ished myeloschisis at Stages
16-20.
l'lyelodysplasîa (taUle 4Z ) is characterized by an irregular cord
defect wìth only one exception (\ZE 69). At the rhomboid sinus, apparent
closure in the whole embryos corresponds to a closed neura¡ tube or to
ectoderm covering the defects in the serial sectîons.
This comparîson of the appearances of the neural tube and ifromUoia
slnus before and after seriaì sectioning reveals that:
(") an oval rhomboid sinus is followed by a normal neural closure
(b) a triangular rhombold sinus precedes an open neural defect
(myeloschisis)
(c) a regular neural defect corresponds to hístological myeloschisis

242
(d) an lrregular neural defect represents histologìcal myelodysplasia.
As no embryos earlìer than Stage 16 show nyelodysplasìa no comment
can be made on the appearance of the rhombold sînus. Thts is consistent,
however, wlth the suggestÌon that myelodysplasîa does not ar¡se by abnormal
closure of the neural folds, but Lnvolves an absence of neurai plate
material and development of the dysplastic cord from tê¡ l-bud material
alone after Stage 15.

TABLE 39. APPEARANCE OF I,'HOLE EI4BRYOS COI4PARED TO HISTOLOGY OF RHOMBOID SINUS AND OPEN CORD DEFECTS,AT
STAGES l3-20 (CoNTRoLS)
closed/closing
closed/closing
closed/closlng
closed/closing
c I osed/cl os ing
c I osed
c I osed
cl osed
c I osed
. c I osed
cl osed
cl osed
cl osed
c I osed
closed N)
Embryo
S têge
lJhol e Emb ryo
Les i on
H i stol ogy
Les i on
l/hoI e Emb ryo
Rhomboid Sinus
Histology
Rhomboîd S ìnus
18C I r
13-
none
none
ova I
18C 10
13
none
none
ova I
r8c 14
13
none
none
ova I
t8c 21
l3+
none
none
ova I
18c 27
13+
none
none
ova I
30c 2
16
none
none
c I osed
30c 3
16
none
none
c I osed
3OC 15
16
none
none
cl osed
3OC 12
16
none
none
c I osed
3OC 22
16
none
none
c I osed
3oc 25
16
none
none
cl osed
42c 4
\2c 7
\zc 2
\zc 6
18
18,
19
19
none
none
none
none
none
none
none
none
cl osed
c I osed
closed
closed

c I osed
closed
closed
c I osed
NJ
42c 11
\zc 3
19
20
42C 8
20
l42c 21
20
none
none
none
none
none
none
none
none
c I osed
c I osed
c I osed
c¡osed

TABLE 40. APPEARANCE OF }/HOLE EI"IBRYOS COI-IPARED TO HISTOLOGY OF RHOIIBOID SINUS AND OPEN CORD DEFECTS,AT
STAGES 13-20 (EXPERIMENTALS WITHOUT DEFECTS)
everted
el evated
e I eva ted
c I osed
c I osed
c I osed
cl osed
c I osed
c I osed
c I osed
c I osed
c I osed
c I osed
c I osed
c I osed
N)
Lh
Emb ryo
S tage
VJhole Emb ryo
Les ion
H ìstol ogy
Les i on
lJhole Embryo
Rhombold Sinus
H i stol ogy
Rhombo id S inus
t8E 10
13
none
trianjulêr
f8E 28
14
none
none
ova I
lBE 47
t4
none
none
OVä I
18E 44
15
none
none
ova_l
'30E 4
15
none
none
ova I
30E 9
16
none
none
c I osed
308 26
16
none
none
cl osed
308 59
16
none
none
c I osed
3aE 77
16
none
none
closed
\28 34
18
none
none
c I osed
42Ê. \9
r8
none
none
c I osed
428 26
19
none
none
c I osed
4zE 3t
19 '
none
none
cl osed
428 i7
20
none
none
c I osed
428 73
20
none
none
c I osed

TABLE 41. APPEARANCE OF ì^IHOLE EMBRYOS COI',IPARED To HIsToLoGY OF RHoMBOID sINUS AND oPEN CORD DEFECTSI*AT
STAGES 13-20 (EXPERI},lTNTALS WITH NYELOSCHISIS)
mye I osch i s is
myeloschisis
myeloschlsis
myeloschîsîs
everted
myeloschisìs
mye I osch ls is
myeloschlsis
el evated
cl osed
c I osed
c I osed
. c I osed
closed
Emb ryo
Stêge
l^/hole Embryo
Les i on
H I stology
Les ion
l{hol e Embryo
Rhomboid S ìnus
Histology
Rhomboid S inus
18E _6r
13
none
myeloschísis
triangular
188 25
13+
regu lar
myeloschisis
tr i angu I ar
188 13
14
none
myeloschisís
triangular
188 35
14
nohê
myeloschisis
triangular
18E 58
14
none
myeloschisis
triangular
18E 36
14+
none
myeloschisis
trlangular
18E 53
14'
none
myel osch ls I s
trlangular
18E 54
t4+
none
rnyeloschisls
tr¡angular
18E 59
14+
none
myeloschisîs
ova I
30E 25
15
regular
myeloschisls
trîãngulêr
30E 56
16
i rregu I a r
myeloschisls
closed
30Ê 69
16
regular ì
myel osch i s îs
'closed
308 52
16
regular
myeloschisls
c¡osed
428 8
17
î rregular
myeloschisls
c I osed

closed
closed
c I osed
c I osed
c I osed
c I osed
c I osed
covered hem I mye I ia
N
.{
42E 1o
17
regul ar
myeloschlsis
cl osed
4zE 1
r8
regul ar
myeloschisis
c I osed
4zE 4\
r8
regular
myeloschisis
-closed
\zE 54
r8
regular
myelosch¡sÎs
c I osed
42E 57
19
regular
myelosch¡sis
c I osed
hzl 6,
19
regular
myeloschlsîs
closed
hzl 72
20
regu la r
myeloschls¡s
c I osed
42E 21
18
irregular myeloschlsls/
closed
hem i mye I ia

TABLE 42. APPEARANCE OF I,'HOLE EMBRYOS COMPARED TO HISTOLOGY OF RHOI'IBOID SINUS AND OPEN CORD DEFECTS,AT
srAGEs r3-20 (rxpenHrnrnls t,/trH i.,tyELoDyspLAstA)
covered amye I ia
covered hem i myel la
N)
F
Emb ryo
Stêge
30E 35
16
30E 76
16
428 Sz
17
428 50
18
\zE 56 18
Whole Embryo H í stology
Les ion Les ion
i rregul ar
î rregul ar
I rregular
I rregular
i rregu I ar
hemlmyella
hemimyella
hem imye I la
hemi mye I la
hem imye I ial
d iplomyel la
t{ho I e Emb ryo
Rhomboîd S Ìnus
a¡osed
cl osed
c I osed
c I osed
c I osed
Histology
Rhomboid Slnus
closed
c i osed
c I osed
covered hem i mye I ia
covered dlp!omyel ia
I42E 69
19
none
hem i mye I îa/
amyel la
c I osed
\zE 21
18
I rregular. myeloschlsis/
hem imye I ia
c I osed

249
6.6 pEVEL0PMENT 0F rH!_¡Ho|4glc R00F
Changes in the structure of the rhornbic roof were also evaluated for
Groups I - lV. They are presented, together w¡th the norphology and levels
of neurâl defects (from Section 6.2), in Tables 43 - 46. and Flgs. 103- I11.
Tables 43 - 46 show that the rhor¡rbic roof undergoes progressive
th i nn îng after neural closure:
Stages l0 - 11+
Stages 12 - 15
Stâges 16 - 17
Stages 18 - 20
thick
thin
very th¡n
membranous
The choroid plexus of the fourth ventricle is not present before
Stage 18 (f¡S. lO9 ) considerably later than the first appeêrance of
myeloschîsis and myelodysplasia. ìlîthin each g¡-oup there is no difference
in rhombic development between experimental and control embryos. ln this
ser¡es of chick embryos, the formatíon of open neural defects cannot be
secondary to excessive pressure within the cerebro-spinal fluid system.

RHOMB IC ROOF DEl/ELOPI,IENT
N'
o
Emb ryo
Stage
Rhombic Roof
Neural Defects
Regions of Defects
6c 20
t0-
èlosíng
none
oc 49
t0-
clos i ng
none
0c 52
10-
closing
none
6c 21
t0
thick
none
0c 46
10
closlng
none
6E 15
6E8
t0-
t0
closing
thick
none
none
6E 30
6E \s
t0
10
th ick
thick
none
early myeloschisis
E
6E 18
10+
thick
none
6E 4r
10+
thlck
early myeloschlsís
E

Emb ryo
Stage
Rhombîc Roóf
Neural Defects
Regions of Defects
18C \
11+
thick
none
18C 23
12
thin
none
lBc 7
12+
thîn
none
18C 22
12+
th in
nonê
6E 13
il-
thlck
none
6E 28
t1-
th ick
none
6t 3t
t1-
thick
none
6E.38
11-
thick
none
6E 44
I t-
th i ck'
none
6E 2\
11
thîck
none
6e 3\
11+
thick
early myeloschisls

Emb ryo
Stage
Rhombic Roof
Neural Defects
Regions of Defects
18C 11
13-
thin
none
18C 10
13
th in
none
r81 14
13
th in
none
t8c 21
13+
thin
none
18C t7
13+
thin
none
30c 2
16
vèry thÎn
none
30c 3
16
very th ln
none
30c 15
16
very th¡n
none
30c 12
16
very th¡n
none
30c 22
16
very th¡n
none
30c 25
16
very thin
none
t8E l0
13-
)
thin
myeloschIsîs
DE
18E 6t
13
thin
myel osch i s is
DE
18E 25
13+
thîn
myeloschisis
DE

CDE
D'E
DE
CDE
DE
DE
DE
D
BCD
BC
BC
BCD
N)
188 13
r4
thin
myeloschísls
18E 28
14'
thin
none
r8E 35
14
thÎn
myeloschisls
188 47
14
thin
myeloschlsis
18E 58
14
th in
myeloschisis
18E 36
14+
thin
myeloschisis
188 53
t4+
thln
myeloschisis
18E 54
l4+
thin
myeloschisis
l8E 59
l4+
thin
myeloschisis
18E 4.lr
15-
th in
none
30E 4
15
thin
none
3oE 25
15
thin
myeloschisis
30E 9
16
very thin
none
3OE 26
16
very thin
none
308 35
16
very thin
hem i myel ia
3oE 56
16
very thin
myeloschisis
30E 59
16
very thin
none
30E 69
16
very th ln
myeloschlsls

B C DE
BC
1..)
30E 76
16
very thin
hemi myel i a
30Ê 52
16
very th¡n
myeloschîsis
308 77
16
very thin
none

TABLE q6,. STAGE 17-20 CoNTRoI AND EXPERI¡IENTAL EMBRYoS (GROUP lV)-çpnMRrî RooF DEVEL0PT'îENr
Embryo Stage Rhombic Roof Neural Defects Regîons of Defects
BC
B
c
BC
BCDE
N)
42C 4
42C 7
18
membranous
t8
memb ranous
42c 2
19
membranous
none
42C 6
19
membranous
none
\zc 11
19
memb ranous
none
\zc 3
20
membranous
none
hzc 8
20
memb ranous
none
42C 21
20
memb ranous
none
428 I
17
very th¡n
myeloschisis
42E to
17
very thin
myeioschisîs
\zE Sz
17
very th in
hem i mye I i a
hzE l
18
memb ranous
myeloschisis
LzE 21
r8
membranous
myelosch I s i slhemlmyel la
\28 34
18
membranous
none

CD
BCD.E
CD
BCDE
CD
CD
BCDE
c
NJ
\¡r
o\
428 \\
r8
membrênous
myeloschisis
42E \9
r8
membranous
none
hzE 50
18
membranous
hemimyel ia
4zE Sh
18
memb ranous
myeloschisis
\2E 56
18
membrânous
hem i mye I i a/d i pl omye I îa
\zE 26
19
membranous
none
\zE 3t
19
membranous
none
\28 57
19
membranous
myeloschisls
4zE 65
19
membranous
myeloschlsis
hzE 69
r9
memb ranous
hemimyel lalamyel ia
428 17
20
membranous
none
\28 72
20
memb rênous
myeloschisls
42E 73
20
memb ranous
none

Figs- 10J - i11. Development of the rhombic ioof În control and
experínental emb ryos (H ¿ f; xt6) :
Fig. 103. Thick rhombic roof in St, 11+ control ernf ryo (tBC 4).
Fís. 104. 'fhîn rhombÍc roof in St. l3+ control emOryo (1BC Z7) .
Fis. 105.
Very thin rhonrbic roof in St. 16 con¡rol embryo
ßoc zz)'.

7-
4.
103
r05

Fî s.
tub.
Thick rhombic roof in 5t.
11+ experìmentai ernb ryo
wi th neural folds everted
at the rhombo rd srnus
(6E 34)"
Fis.
147.
Th in rhcrnb ic roof i n St, 14+
experimental emb ryo
with earì;, mye losch is i s (tBE
3ó )"
Fig.
loB.
Very thin rhombic roof in St. 16 experimental
embryo with early nryelodysplasia (3or 76).

.t
l
l]]
80t
¿ot
rfÞ'.
!-d...---
901

Fig. 109
Hernbranous rhombic roof with choroid plexus in St, lB
cónrrot embryo (4ZC 7),
Fig. ll0.
Membranous rhombic roof wÍth the first sign of
development of a choroíd plexus in St. 1B experimcntal
embryo with myelodysplasia (Aze Sg),
Fis. 111.
|4embranous rhombic roof with the fírst sign of
development of a choroid plexus în St. 17 experimental
embryo with myeloschisis (428 B).

'Ì
1ú
111

260
6.7 HrqroLoctcAL, cHANgË AssoctArEp tltrH NEURAT p;FEcrs_
To assess the h¡stological differences between normal embryos
and those with neural defects, the numbers of f0 mlcron sections
showlng a part¡cular feature in each reglon were counted, and expressed
as percentage lengths of each reglon and of the entire embryo.
. Counts were confined to Groups lll and lV (Stages 13-20), excluding
the embryos with amyet ia and myeloschísis/myelodysplasia
(\2Ê69'\2E21)andthoseínverypoorcondítionafterprocessing
(l8E 10, 188 28, 18E 25, t8E 54). Cnly regions B,c,Dand E (coverÌns
the spinai cord) were counted, though their boundaries differ în Group lll
and Group lV.
For iomparison of the lengths of neural

261
ln these tables the contrors and the experimentar embryos w¡thout
neural dèfects show extensive separation between somites and neural
material; there Issome separation between notochord and neural material
by Stêge 20 in two embryos ('ZC Zl , 428 73). The embryos wìth neural
defects also show extensive separat¡on from somites, with separation
from notochord (especially ln myelosct isis after Stage ,t7) ¡and somite
defects (especial ly ín myelodysptasia after Stage l6). There is no
close association between the rength of ectoderm discontinuíty from
neural tissue and the type of neural tesion
.
Figsi. 112-119 compare the percentage lengths of neural defects in
regions B,c, D and E of each âffected experimental embryo with the correspondîng
percentage I engths of:
(a) ectoderm d iscont inu ity
(b) somite separation from neural tîssue
(c) notochord separation from neural tissue
(d) local somi te defects.
These figures demonstrate the percentage distribution in the four
regions of embryonic cord of myeloschisis irom Stage lJ and myelodysplasia
from Stage 16. Horvever as region B is much larger than all the other
regîons the size of each region in relation to a whole embryo is disproportionate.
The assocîation of myeloschisis with notochord sãparation
after Stage 1/,and of myelodysplasia with somite defects after Stage l6
are clearly i I lustrated.
.An analysis of variance could not be performed with these figures
as so many of the histologîcar features showed zero varues in both
exper imenta I and control embryos.

TABLE 47. CONTROL EMBRYoS.HISTOLOGICAL ANALYSIS
Embryo Stêge Regions of Regions of *"r."
Lesions Measurements Lesi'on
- Díscontinu¡ty Separatlon s"p"i"iiån ó;f¿;r.
ZZZZZZZZZ.Á
region embryo règion embryo regíon embryo region embryo ."gion urbryo
o 9.80
0
o 15.08
0
0
0
0
0
0
0
0-
00
0
0
0
0
0
0
0
N)
o\
N)
t8c rl 13
B
0
0
0
00
9.63 0
c
0
0
0
0
D
0
0
0
6 .12
E
0
0
0
t00
18C 10 13
B
0
0
0
c
0
0
0
D
0
0
0
36.36
E
0
0
0
.t 00
r8c 14 13
B
0
0
0
c
c
0
0
D
0
0
0
76.92
E
0
0
0
100
lgc 21 13+
B
0
0
0
o 12.56
c
0
0
0
0
D
0
0
.0
28,57
E
0
100

0
0
0
.0
0
0
0
0
0
0
0
a
0
0
0
0
0
0
0
6.76
0
0
0
100
12.28
100
100
100
0
88.89
B5
'0
0
55 .88
28.57
100
2.22
3.5
19.15
22.38
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
.0
12.32
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
7 .8r
0
0
0
0
0
0
0
0
0
0
0.
0
0
0
0
0
0
0
0
N)
o\
5.87
B
c
D
E'
B
c
D
E
B
c
D
E
B
c
D
E
B
c
D
18C 27 13+
30c 2 16
30c 3 16
30c 15 16
30c 12 16

0
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
NJ
o\
E
0
0
42C 7 r8
B
0 )0
0
B
c
ó
E
B
c
D
E
\2C 4 18
B
c
D
E
0
0
0
0
0
0
0
0
0
o
0
0
0
0
0
0
0
0
0
0
0
0
0
0
30c 22 16
30c 25 16
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
3.17 10.56
10
77.7e
100
4.18 11.67
32
59.\6
100
o '21 .61
82.8r
100
100
0 20.25
c
0
0
0
82.76
D
0
0
0
100
E
0
0
0
100
0 5.48
42C 2 19
B
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
35.29
100
0
0
17.39
100
0
0
6o
0
.0
0
100
100
0
0
16.67
100
4. ol
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
,0
2.34
0
0
0
0
0
0
.0
0
0
0
0
0
0
0
0
0
0
0
0
7. 88
0
0
0
0
0
0
0
0
0
0
0.
0
0
0
0
0
0
0
0
N)
o\
4.59
c
D
E
R
c
D
E
B
c
D
E
B
c
D
E
B
c
D
E
42C 6 19
\zc 11 19
4zc j zo
20
4zc I

42c 21 zo B
c
D
E
0000
00
00
00
1\.62 12,23 o 6.70 o o
0 6.25 0
o 100 o
0 100 0

00
0
0
0
0
0
0
0
0
0
0
0
0
0
N)
o\
\j
TAB
S,HISÎOLOGICAL ANALYS I S
Embryo Stege Region of Region of Neural Ectoderm
Les ion Measurements Lesion Discontinuity
ZZ7.'Á
, regîon embryo region embryo
Notochord Soml te Soml te
Separatïon Separat ion Defects
zzzzzz
region embryo region embryo regíon embryo
18E 47 ttt
80000
c00
000
ouo
12.50
18E 44 15
30E 4 15
30E 9 16
DO
EO
BO
c0
DO
EO
BO
c0
DO
EO
BO
c0
DO
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
100
100
0
,0
36.36
100
0 1.48
0
14.29
33.33
0 4.33
0
20
4.2\

l\t
q\
1oE 26 16
30E 59 16
3oE 77 16
428 34 18
h2E \9 t8
EO
BO
c0
DO
EO
BO
c -0
DO
EO
BO
c0
DO
EO
BO
c0
DO
EO
BO
c0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
r00
o 6.15
0
4o
100
0 7.74
33.33
28.57
t00
| .87 6.77
0
53.s5
100
o r1.96
12,77
100
100
o \.22
0
0
00
0
0
0
00
0
0
0
75
73.08
26.32
0 5.95
00
0
0
0
00
oco

N
o\
\o
DO
0
0
65.38
0
EO
0
0
100
0
4zE 26 t9
BO
c'0
DO
0
0
0
0
0
0
.,0
61 .32
80
17.1\
0
0
0
EO
0
0
100
0
4zE 31 19
BO
c0
DO
EO
0
0
0
0
0
0
0
0
0 20.09
73.55
100
100
0
0
0
0
428 17 20
\28 73 zo
BO
c0
DO
EO
BO
c0
DO
EO
0
0
0
g
0
0
0
0
0
0
0
0
5.64
0
0
0
3 .86
0 3.61
.0
21 .05
r00
0 19.39
44.58
100
100
0
0
0
0
0
0
0
0

0
0
0
0
0
0
0
0
0
0
0
0
N)
.{
Embryo Stage Regions of Reglons of
Les íons Measurements
HISTOLOGICAL ANALYSIS
Neural Ectoderm Notochord
Les ion Di scont inu i ty Separation
%zzzzz
reglon embryo regíon embryo region embryo
Somí te Som ì te
Sepa ra t ion Defects
zzzz
regÍon embryo reg ion embryo
18E 61 13 DE
B
c
0
0
10.97 0 0
0
00
0
0 7.04 .o o
00
D
100
0
0
,5.82
0
E
190
0
0
r00
r8E 13 14
CDE
B
0
8.55
0
00
27.61 B.\6
c
22.72
0
0
25
D
100
0
0
100
E
100
0
0
100
t8E. 35 l4
B
0 2.35
0
0
20,24 ß,65
c
o
0
0
0
D
0
0
0
21 .t+3
18E 58 14
CDE
E
B
8o
o 9.29
0
0
0
0
100
16.67 22.\4
c
27.59
0
0
100
D
100
0
0
r00

0
2.67
0.
N'
!
18E 36 14+
DE
22.22
0 5.32
100
,0 r .82
c
D
0
69.7o
'0
12.12
18E 53 14+
DE
60
0 7.35
loo
0 8.90
0
0
63.83
80 .85
E
100
100
18E 59 14+
DE
B
0 2,85
c
'0
308 25 15
BCD
64.71 o
4s.45 o
5.52 12.75 0
23.53
r00
3.07 4.ll
100
,
0
33.33
79.07 0
00
0
100
3oE 56 - 16
BC
1\.4\ 12185 o
600
1 0 .90 14 .92
7\.29

l\)
\l N
D
E
00
00
0
0
13.79 0
100 0
30E 69 16
30Ê 52 16
BCD
B
c
D
E
B
c
D
E
10.48 ß.93 o o
190 0
5\.17 0
00
14.7t 12.25 14.71 12.25
60.87 60.87
oo
oo
0
0
0
0
0
0
0
0
4.s6 lo;07 o o
100 0
35.\2 o
100 0
12.75 18.55 2.70 2.81
100 0
r0o 28.57
100 0
t+28 I 17
42E ro 17
.B
B
c
D
E
c
D
E
29.81 23.01 0 0
31 .87 o
00
gro
13,\4 9.24 o o
00
00
00
\3.\3
0
0
0
35.40
0
0
0
29.27
2\.33
0 15.63 0 0.88
65.93 o
100 22.22
100 0
0 20,96 0 0.92
77.46 o
100 13.64
100 0

N)
.{
\28 1 t8
42Ê 44 18
\zE 54 t8
BC
B
c
D
E,
B
c
D
E
B
4.8 13.85 1.77 1.zo 18.40 19.43 3.10 26.06 o
75 o \2.92 1oo o
o o o 1oo o
0 0 o loo o
o 8.73 o 0.63 o ,.s\ o 13.97 o
39.80 4.8g 16.33 43.88 o
55.17oo1ooo
0 0 0 100 o
0 14.55 0 0.61 o o 18.45 18î45 o
\28 s7 19
428 65 t9
c
D
E
B
c
D
E
B
c
D
69 J1 3.96 o 61 ,3g o
58.14 o o roo o
o o o ìoo o
o 17.02 o o.3o o 8.66 o 16.26 o
78.57 1.79 50.89 5\.\6 o
100, 001000
0 0 0 loo o
0 8.06 0 1.10 O O O 16.65 0
22.99 6.90 o 49.40 o
82.76odtooo

N)
\¡
\2E 72 20
E
B
c
D'
E
00
0 15.81 0 0
80..21 o
00
00
0 100 o
0 12.73 0 2\.2\ O 0
64.58 77.08 o
0 100 o
0 loo
0

16.76 13.52
0
29.57
100
0 14.04
49.02
89 .66
33.33
4¡.gA 38.31
87.95
44.77
35 .71
35.97 36.71
61.5\ \ \JI
100
TABLE 50. EXPERII,TENTAL EI"IBRYOS I^'ITH I4YELODYSPLASIA. HISTOLOGICAL ANALYSIS
Embryo Stage Regions of Regions of Neural Ectoderm Notochord Somìte Somlte
Lesions I'leêsurements Lesion Dlscontinuity Separation Separation Defects
zzzzzzzT"zz
region embryò region embryo reglon embryo regÍon embryo regîon embryo
308 35 16
308 76
16
BCB
c
D
E
BCDE B
c
D
E
10.99 13.9
100
0
3.05 16.64
100
100
26.67
3.30 8. 13 0
10.81 0
00
00
0 r.21 0
12.74 o
00
00
7.14 11.19
86.49
0
50
0 4.68
11,76
20.69
!00
\2E rz 17
B
o.09 13.68
0 6.52
0
c
D
r00
0'
49.40
0
0
0
o 7.80
3.61
8\.21
AzE jo
18
E
B
0
r0.46 4.68
0
o 4.34
0
lì
100
15.05 16.09
c
100
36.92
0
1 8.46
D
100
0
0

46. 15 76.92
0 20.51 14.83 2i,92
53.33 65.33
o 48.28
100 5\.05
NJ
\¡ o\
E
\zE s6 B
't00
11.92
32.51
0
0
0
2.O2 8,72 7 .93
c
f00
16.00
14.67
D
100
0
0
E
64. 86
0
0

Figs. 112-119. Percentage lengths of histological changes associated
with myeloschisis and myelorlysplasia in experimental
embryos of St. 13 to St. 20. Each dou!:le bar
rePresents one emb ryo I
Fig. 112.
Ectoderm díscontinuity wíth myeloschisis,
FiS. 113.
Ectoderm discontinuity with myelodysplasia.
FiS. 114.
Somite separation wi th myeloschisis,
FiS. 115.
Somite separatÌon with myelodysplasía.
FiS. 116.
Notochord separation with myeloschisis.
Fig. 117.
Notochord separêtion rvith myelodysplasia,
Fis. 118.
Simite defects with myeloschisis.
FiS. 119.
Som¡te defects wïth myelodysplasia.

ECTODERM DISCONTINUITY IN EXPERIMENTAL EMBRYOS WITH MYELOSCHISIS
N=l9
E NEURAT LESTON N ECTODERM DTSCONT|NUITY
l8
19 20
REGION B
IrNl En
Ë
T
N
t-
Ë¿E
REGION C
o
zul
*Ë
-t
\o o\
lIIE
EE
REGION D
ã'¡
E
REGION E
3
5
tó
STAGES
17

ECTODERM DISCONTINUITY IN EXPERIMENTAL
EMBRYOS WITH MYELODYSPLASIA
N_Ã I NEURAL LESION
N ECTODERM DISCONTINUITY
t IT
S
I
tó 17 l8
STAGES
REGION B
REGION C
REGION D
REGION E
279 ::
J-
o
zt¿¡
ñ

279
ECTODERM DISCONTINUITY IN EXPERIMENTAL
EMBRYOS WITH MYELODYSPLASIA
N_Ã
I NEURAL LESTON
N ECTODERM DISCONTINUITY
REGION B
I
s
IT
N
J-
l-
o
zt¡¡
ñ
REGION C
REGION D
I
REGION E
tó
17 l8
STAGES

SOMITE SEPARATION IN EXPERIMENTAL EMBRYOS WITH MYELOSCHISIS
E NEURAL LESION NI SOMITE SEPARATION
16 17,
STAGES
EGION C
:tr
t-
o
zu¡
J
\o
o\

SOMITE SEPARATION IN EXPERIMENTAL EMBRYOS
WITH MYETODYSPLASIA
N=5
E NEURAL LEsloN
N SOMITE SEPARATION
REGION B
R\-
Nhr
-
o
ztl¡
ñ
NN
N
REGION C
REGION D
N
REGION E
17 l8
STAGES
\

NgTOCHORD SEPARATTON rN EXPERTMENTAI- EMBRYOS- W|TH MYETOSCHTSTS
N=I9 E NEURAL LESION øNOTOCHORD SEPARATION
!¡
13 14 l5 ló 17 r8 19 20
STAGES
I
REGION B
REc,oN D
ã'H E REGION E
Erl
W.
I v6%fu*"'*'
-¡-
o
z¡l¡
,-t
às
EE
¡t¡¡Ë

283
NOTOCHORD SEPARATION IN EXPERIMENTAL
EMBRYOS WITH MYELODYSPLASIA
¡ NEURAL LESTON
YZ NOT OCHORD SEPARATION
REGION B
I
()
ztt¡
REGION C
àe
REGION D
REGION E
STAGES
17 l8

REGION E
N=|9
SOMITE DEFECTS IN EXPERIMENTAL EMBRYOS WITH MYETOSCHISIS
E NEURAL LESIoN NSOMITE DEFECTS
:tr
t-
o
zt¡¡
-t
ã¡E* Et
HH
\o o\
REG¡ON B
tu
Eå
NN
t¡
!tE
! REG,.N D
13 14ì l5 16
. STAGES
17 8r t9, 20

285
SOMITE DEFECTS IN EXPERIMENTAL EMBRYOS
WITH MYELODYSPLASIA
N=5 f NEURAL LEsloN
NI SOMITE DEFECTS
N S^
REGION B
.L
l-
o
zt¿r
N
i\
N
N REGT.N c
àq
N
REGION D
N
NN
NN
$
REc,oN E
16 17 18
STAGES

286
6.8. EXTENT OF THE OVERLAP ZONE
The overlap zone is characterized by multiple accessory.canals
wlthln the tail-bud nater¡al, lying deep to a closing or closed tube
derlved fron neural plate material. Even without accessory canals ìts
presence is revealed by asymmetry of the definitive neural tube derived
from both sources (Figs. 51 - 60).
Uslng this asymmetry to lndicate the extent of the overlap zone, the
numbers of sectlons containing neurêl plate material and taíl-bud materiaì
were counted în each region of Group lll embryos. Values were expressed ås
percentages of each region and of each whole embryo .at Stages 13-,l6,
coverlng the developmental period in which the overlap zone ís most prominent.
The results could not be expressed ¡n terms of somite levels as the
definitive number of èomîtes has not differentiated at Stages lJ-16.
Group lV embryos could not be included because the overlap zone ís
obscured by complete fusion of the two sources of neural material after
Stage 16 in the controls and în experimental embryos without neural defects.
Tables !l - 54 show the percentage lengths of neural plate material,
tai l-bud material and the overlap zone ln Group lll
embryos, rearranged
into four catçgories:
Stage 13- 16 control embryos
Stage 13-16 experlmental embryos wîthout neural defects
Stage Il-16 experimental embryos with myeloschisîs
Stage 1l-16 experimental embryos wíth myelodysplasia ,
Figs. 120 - 123 compêre the lengths of overlap zone with the lengths of
neural defects in the four categories. They show that the length and distrlbut¡on
of the overlêp zone in myeloschísls closely resembles lts length

287
and distr¡bfrtíon in the controls and ín experimental embryos wlthout
neural les ions.
The two embryos wlth myelodyplasia show a very d¡fferent pattern.
The upper boundary of tail-bud materiar lies at a dimilar revel to that
seen in the controls at Stage ,16. The lower boundary of neural plate
materlal, however, lies at.almost the sême level, due to the absence of
neural plate material ln Regions C, D and E.
statist¡cal analysîs of, the resurts was not performed because of the
smal I number of embryos with myeiodysplasia.

100
o 20.79
47.50
100
,N'
oo
100 æ
ZONE IN ST
Emb ryo Stage Type of Regions of Regions of
Les ion Les ion Measurements
E.
93.33
r 8c '¡4 13
Neural Plate I'laterial
66
reg ion emb ryo
18C 11 t3'
100 62.08
c
100
D
100
r8c 10 13
E
95.8¡
B
100 69.95
c
100
D
100
Þ
c
D
100
100
'!00
59.54
Ta îl -Bud Material Overlap Zone
zzzz
regîon embryo regíon emb ryo
0 22.6'
100
100
100
0 18.62
27.03
100
100
0 '19.09
29.\1
r00
0 22.27
100 .
100
95.83
0 18.23
27.03
100
100
0 19.09
29.41
r00
E
)
100
100
18C 21 13+
B
100
70.62
o 20.79
c
100
47.50
D
t00
100
E
100
100

19.09
r8.95
r 1.84
14.82
15.20
0
17.76
11.65
14.17
14.20
t\,
@
\o
18C 27 13+
B
100
69,26
0
19 .09
c
100
7\.50
7\.50
D
100
100
loo
E
100
100
100
30c 2 16
B
100
73.43
6'52
6.s2
c
100
100
100
D
100
100
100
E
78.95
.r 00
78.95
30c 3 16
B
100
81.57
0
0
c
100
55.560
55.560
D,
100
100
r00
E
94.74
100
94.7\
3oc 15 16
B
r00
81.76
0.72
0.72
c
D,
100
100
100
t00
100
100
E
78.95
100
78,95
30c 12 16
B
100
' V5.t46
5.32
5.32
c
100
100
100
D
100
100
100
E
53.33
100
53.33

t4.09
13.94
0 1).59
100
100
88. 89
o 1 3.07
88
r00
76.19
N)
\o
3OC 22
16 none
B
100
77.18
0'
c
100
100
D
100
r00
E
88.89
100
30c 25
16 none
B
r00
BoJz
0
c
f00
88
D
f00
t00
E
76.19
ioo

0 15. 89
81 .08
100
100
' 76.92
0 14.38
100
70.59
0 10.04
15.79
100
f00
o 9'66
35.09
1oo
È
88.24
Embryo Stage Type of
Les ion
IMENTAL EMB
Reglons of Reglons of
Les lon l',leasurements
Neural Plâte MaterÍal
.t ø'
4tô
reglon emb ryo
Ta I I -Bud Haterlal Overlap Zone
zzzz
region embryo reglon emb ryo
18E r0 13-
B
r00
65.65
0 15.89
c
100
81 .08 .
D
1oo
100
E
100
100
lBE 28 r4
Þ
100
64.04
o 15'24
c
100
76.92
100
100
E
70.59
100
r8E 47 14
B
100
76.33
0 10 .04
c
100
15.79
D
E'
loo
100
r00
100
18E 44 15-
B
'100 :
68.8r
0 10.00
c
100
35.09
D
100
100
E
88.24
100

13.85
10. 84
14'5 t
15.97
11.06
1.53
100
100
91 .67
0.67
100
100
100
3. 86
100
100
91 .67
2'9t
100
100
87.50
1 .40
100
100
30E. 4 15
30E 9 16
308 26 16
308 59
308 77
16
16
B 100
c 100
D 100
E 9r.67
B 100
c 100
D 100
E 100
B 100
c 100
D 100
E 91 .67
B 100
c 100
D 100
E 87.50
B 100
c 100
D 100
77.27
8\.67
90.61
80. oo
78.55
1.53
i00
100
100
0.67
100
100.
100
3 .86
100
100
100
2'9t
100
100
100
r .40
r00
100
13.68
10.84
14. r6
1j.49
10.23
N)
to
NJ
E 73'68
100.
73.68

43. rB
100
100
0 12.53
74.07
r00
't00
l\)
\o
0s l1| ITH t'lYEL0ScH r s I
Embryo Stage Type of Regîons of Regions of Neural Plate Mater i a I
Les i on Les i on l'leas urements zz
reg ion ernb ryo
l8E 6l ß nyeloschisis DE
B
c
D
E
100 't00
100
r00
66.87
188 25 13+ myeloschlsis CDE
B
100 69.52
c
D
E
100
100
50
B
100
68.59
Tai l-Bud Material Overlap Zone
zz%z
region embryo reg ión embryo
o 13.87
\5.16
100
100
0 1 3.90
J6.67
t00
100
0 10.34
0 18.87
\5.t6
100
100
0 12.78
16.67
r00
100
o 10.34
c
100
4:.tB
D
gl
100
100
100
100
18E 35 14 myeloschls,ls E
B
100
57.97
0 12.53
c
t00
7\.07
D
100
100
E
100
100

0 12.18
62.07
100
77.78
o t3.72
78.38
100
100
0 i1.4r
25
100
100 .
0 11.02
66.57
ï00
75
0 8.00
36.5\
NJ
loo B
81 .82
18E 58 14 myelosch¡s¡s CDE
B
100
60.73 0 12.57
c
100
62.07
D
t00
100
E
77.78
. 100
18E 36 1¡+ myeloschlsls
DE
B
100
69.87
0 13.72
|.
100
78. 38
D
100
100
E
100
100
18E 53 l4+ nyeloschîsls DE
B
100
69.79
0 11.41
c
100
25
,D
t00
100
E
100
100
18E 54 t4+ myetoschisis
B
r0o
65.67
o 11.65
c
100
,t6.67
D
I
100
100
f8E 59 14+ myeloschl'sls DE
E
B
75
100
7?.06
100
o 8.36
c
100
36.5\
D
'100
100
E
8r .82
100

7.t6 16.03
100
100
160
7.90 18.22
r00
1oo
75
r 1 .85 20 :30
100
100
83.33
16.9t 21 .02
100
100
87 'so
t\,
\o
308 25 15 myeloschisis BCD
B
100
71 .0\ 7.36 17.30
c
100
100
D
100
100
E
6o
' 100
308 56 l6 nryeloschlsls BC
B
100
78.\7
7.90 18.92
c
100
100
D
100
100
E
75
.l00
308 69 16 myeloschísis BCD
B
100
85.24
11.85 20.63
c
100
100
D
100
100
e
83.33
100
30E 52 16
myeloschlsis BC
B
t00
77.15
16.9t 21 .36
c
100
100
D,
100
100
E
87.50
100

Embryo Stage Type of Regîons of Regions of Neurêl P¡ate Haterial Tai l-Bud Material Overlap Zone
Lesion Lesion l'leasurements Z Z Z Z .Z Z
region embryo region embryo region embryo
0
o
0
2.54 1.73
0
0
0
N¡
lo
c
I4-ale ¡q,ovERLAp zoNE tN srAGE t3-16 ExpERtMENTAL Er,tBRyos t^ltrH HyELoDyspLAStA
30E 35 16 hemlnryel la BC
B
9\.51 62.09 12.36 20.08 6.87 4.5r
c
0
100
D
0
100
E
0
100
308 76 16 heml mye I ia BCDE
B
95.42
64.99
7.12 21 .32
c
0
100
D
0
100
E
0
r00

Figs. 120'123. Percentage ie.ngths of the overlap zone in control
and experìmental embryos of St. l3 to st, 16. Each
double bar represents one embryol
FiS. 120.
Overlap zone in control embryos.
FiS. 121 .
FiS. 122,
0verlap zone in experimêntal embryos wîthout neural
. defects.
Overìap zone in experimental embryos with
r'ryeloschisìs.
FiS, 123.
Overlap zone in experimental embryos with
myelodysplas ia"

298
OVERLAP ZONE IN
CONTROL EMBRYOS
N=11
EÐ OVERLAP ZONE
REGION B
:tr
Þ-
o
zllJ
REGION C
\oo\
REGION D
REGION E

299
O\TERLAP ZONE IN EXPERIMENTAL EMBRYOS
WITHOUT NEURAL DEFECTS
N=9 Hl ovrnrAP zoNE
REGION C
T
l-
o
zu.t
-t
\o o\
REGION D
REGION E
15 ló
STAGES

OVERLAP ZONE IN EXPERIMENTAL EMBRYOS
WITH MYELOSCHISIS
N=13
tr NFURAL LESION
@ OVERLAP ZONE
o
zllJ
\o o\,
14
STAGES

301
OVERLAP ZONE IN EXPERIMENTAL EMBRYOS
WITH MYELODYSPLASIA
N=2 f NeuR,qL LEsloN
ffi oveRrap zoNE
REGION B
.L
o
zt¿¡
REGION C
àe
STAGES

302
6.9 ANALYSIS OF NEURAT VOTUHES
Examination of the serial sections showed that the cross-sectional
area of neural tissue was greatly reduced at the slte of all myelodysplasla
lesions. ln rnyeloschisis leslons the sectional area of neural tissue was
nelther much reduced (as ln myelodysplasia) nor much lncreased (as would
be expected in neural ttovergrowthtr). The sectional area of notochord în
all experlmental and control e'mbryos, however, appeared to be fa¡rly
un lform.
For direct comparlson of indivldual embryos, the rêtio of rn""n n"rr"l
tlssue to mean notochord was thus calculated for Regions C and D. Region B
was not lncluded because of the considerable length of normal spinal cord in
the upper somite areas of abnormal embryos.
Region E could not be lncluded because neural tlssue ând notochord
were not fully dífferentíated. The embryos with amyel ¡a (42E 6!) and myeloschísis,/myelodysplasia
(\28 21) were excluded
calculation of the ratios of neural t¡ssue to notochord allowed pool in9
of embryos in Groups lll and lv (stages l3-20) desÈite differences in their
regional boundaríes. This províded comparison between:
(a) embryos. of different sizes
(b) reglons of different sizes
(c) sect¡ons cut in different planes
(d) well- or poorly - processed material.
A Leltz - ASI'I lmage Analyser was used to measure the cross-sectional
area ( ,z ) of neural tube, neural canal (when present), and notochord ¡n
every tenth sect¡on. The mean areas of notochord and neural tíssue (neural
tube mlnus neural canal) were obtalned by dlviding the sum of'area measurements
by the number of sections measured. As a!l sectíons were cut at lo microns

303
ând every tenth sectlon was neasured, these mean areas refrect the vorumes
of notochord and neural tissue in each region.
The mean areas of notochord and neural tissue, ù¿ith the¡r respect¡ve
ratlos for Regions C and D of embryos with and w¡thout neural levions,
are gfven in Tables 55-5g and Figs. 124 _ 127.

Rat io
NT/N
6.53
6.zB
6.78
5.02
8.26
6.92
6.96
7 .07
8.00
5. 40
6.6\
7.60
9.04
8.59
6,zo
4. 89
o
.F.
Embryo Stâge Regions of Regions of
Les ion Meas u remen ts
Hean
Neural Tissue ( p2)
l'lean
Notochord ( u2)
18c 11 13
c
17695.11
2709.94
D
1\935.79
2376.88
r8c 10 13
c
15611.88
2301 .21
D
13027 .13
2593.52
18c 14 13
c
12045.92
1\57 .91
D
11209.23
1619.58
t8c 21 13+
c
14257 .44
2A\7.\0
D
15221 ,70
2151 .7\
tïc 27 13+
c
r9424.14
2\27.12
D
16616.94
3078. 31
30c 2
16
c
18191.20
2740.36
D
17t472.7\
2299.40
30c 3
16
c
21\70.54
2375.\6
D
186il.46
2167.35
30c 15
16
c
13680.02
2207.07
D
12667.02
2592.33

30c 12 16
c
17707,61
2463.3\
7.19
D
15\21 .59
2384.81
6.47
30c 22 16
c
r 8261 .88
2049.27
8'9 t
D
17035.18
2396.82
7.11
30c 25 16
c
22011 .75
1985.70
1r.09
D
21911.\z
1849.85
1r.86
4zc 4 tB
c
23075.86
283\.57
8. 14
D
15344.00
2134.61
7.19
42c 7. t8
c
2\007.6\
2938.7\
8.47
D
1q1 39 .01
2113.3\
6.69
\2C 2 19
c
D
23270.21
r 8448. r I
5410.r4
3zb8. r o
4. 3o
5.61
4zc 6 19
c
31037.73
4085.82
7.ê0
D
22594.20
3574.01
6.32
42C 11 19
c
30579.56
34\9.9\
8 .86
D
21679.28
3308.64
6.55
4zc 3 zo
c
36390.6\
4664. 48
9.93
D
31835.22
4\36.03
\2c I 20
c
3j809,99
57t+6.95
7.17
(,
o
6.23 \¡
D
27096.33
5297.28
5.12

7.30
6,5\
o
C'\
\2c 21 29763.8\
25897,29
4079 .80
3960.70

Ratio
NT/N
4 .90
4.11
6.17
7.14
6.93
7.75
6.48
4.85
8.27
8.99
6.62
5.71
6.08
5.85
8. 3l
8.06
\¡
Emb ryo
Stage Reg lons of Reglons of
Les ion l4eas u remen ts
Hean
Neural Tí ssue (u2)
l'1ea n
Notochord (u')
18E 10 13-
c
r 0104 .53
2062.53
188 28 rq
D
c
13431 .45
14\1\.26
3271.63
2336.59
D
16120.72
2257.72
18E 47 14
c
12063.37
1740.59
D
1 3504 .85
1 838. 54
18E 44 15-
|^
1820\.95
2808.93
D
20011.18
\122.8\
30Ê 4 15
c
21176,52
2561 .63
D
20323.61
2260.43
30E 9
16
c
15361 .89
2320.80
D
1B8o.S3
25?0.40
308 26
r6
c
15355.11
2526.26
D
1 3308.84
2276.53
308 59
t6
c
20740.19
2495.89
D
17038.79
2119.57

3.97
4. B0
8. 3g
5.81
6.37
5.53
8.88
6.8r
7.99
6.27
7.63
4.96
3.95
3.52
o
@
308 77
r6
c
8480.76
2134.62
D
6682.87
1392.67
428 34
18
c
28229.64
3365.52
D
16386.13
2822.43
428 \g
18
c
3389\.29
5318.37
D
2?835.72
\126.zs
4zE 26
r9
c
24723.1\
2785.70
D
16361 .79
2\02,93
\zE 31
t9
c
35685.93
\462.92
D
25229.\6
4ozz.19
428 1l
20
c
29134.62
3816.02
D
19326.99
3895.g2
42E 73
20
c
19937.55
50ll3 .48
D
r 4190 . 97
\oz7.S3
l

Rêtlo
NT/N
4.go
\.36
3 .44
3. 18
3.76
4. 18
4 .09
4. 66
4.58
5.38
5. oo
5.\9
7.2\
8. 99
5.71
6.59
5.90
Embryo Stage Regions of Regions of
Les ion Meas uremen ts
l.lea n
Neural Tlssue ( u2)
Mean
Notochord ( u2)
t8E 61 13
DE
c
11106.91
220\.99
D
I 1 306.04
259a.85
rgE 25 13+
CDE
c
10409.27
3024.80
D
951\.25
2987.77
r8E 13 14
CDE
c
10699.99
2842.79
D
15929,87
3809.03
r8E 35 14
D
c
1105r.83
2702.77'
D
10319.69
2212.\6
18E 58 t4
CDE
c
11542.98
2517.75
D
16496.84
3068. 54
18E 36 14+
DE
c
12232.23
244i.\1
D
14082.28
2545.31
i8¡ l¡ 14+
DE
c
16313.7t+
2254.53
D
18221 .77
2025,31
18E 54 14+
DE
c
10902 . 68
1909 . 05
D
11705.42
1776.51
18E 59 t4+
DE
c
1\129.61
2394.39

5.33
q.40
3.14
3.88
4.50
4.75
\.65
3.22
3.10
5 .84
7.29
3.79
5 .04
8.62
7.02
8.36
7.10
6.69
7.57
o
qzl 54 CD
c
308 25 15
BCD
D
c
D
r6170.00
13531 .40
9795.97
3oE 56 30E 69 308 52 4zÊ 8 42E ro LzE l \zÊ \4 16
t6
16
17
17
t8
r8
BC
BCD
BC
BC
B
BC
CD
c
D
c
D
c
D
c
D
c
D
c
D
c
D
10979.95
10675.14
14075. 30
13692,85
7\30.63
8059.26
18472.31
10836. 53
10123.53
868S.1¡
21369.09
9664.56
32564.19
2r685.00
r8
20747.83
13576.92
3035.20
3073.92
3118,57
¿8zB.7t
2371.50
2965.91
29\4.03
2304.\9
2599.22
3162.93
1\87.37
2671 .83
1626.2\
2480. I 4
1376.51
3903.74
3c54.28
3103.44
1792.39

6.7t
6.lt
4.63
4.60
4.13
3.48
hzE 57
428 65
CD c
D
c
D
c
D
4oqo9. r 3
2\661 .93
18039.2\
13392.88
17414.\2
12851 .82
6020.97
3908.29
3897.63
?914.10
4216.91
3694.39

(u2 )
Ratio
NT/N
1.72
2,33
2.19
2.79
1.99
3. t9
2.22
1 .92
r .47
2.6\
Embryo
S tage
Reg ions of Regions of
Les icin l'leasu remen ts
Mean
Neural Tissue (u2 )
Hean
Notochord
30Ê. 35
308 76 16
16
hzl 52 17
4zE So 18
\zE 56
l8
BC
BCDE
BC
BC DE
BC DE
c
D
c
D
c
D
c
D
c
3815.57
52\3.32
5674.14
6299.43
7921 .52
7090.52
9066.77
181\.45
7177.\7
2219.02
2252.72
2595.16
2261 .37
4077.35
222\.38
4084 .47
4060.84
4890. 1 I
D
10131 .55
4r 46.40

Fi9s, 124-127, Neurál tube-norochord ratios in control and
experimental embrycs of St. 13 to St, 20. Each bar
rep res en ts one emb.ryo i
Fis. 124. Ratios in control embryos.
Fís. 125.
Ratios in experimentar embryos with no neurar deflects.
Fig. 126.
Ratios in experlmental embryos w¡th myeloschis¡s.
Fig. 127.
Ratios in experimental embryos w¡th myeìodysplasia.

314
NEURAL. TUBE -NOTOCHORD
RATIOS IN CONTROL EMBRYOS
N=I9
¡ NEURAL LESION
E NO LESION
5
tn
o
tr0
É.
REGION D
ló l8 19
STAGES

315
NEURAL TUBE -NOTOCHORD
RATIOS IN EXPERIMENTAL EMBRYOS
WITHOUT NEURAL DEFECTS
tr Nrun,ql LEstoN
E No LEsIoN
REGION C
REGION D

316
NEURAL TUBE_NOTOCHORD
RAÏIOS IN EXPERIMENTAL EMBRYOS
WITI.I MYELOSCHISIS
N=21
I NEURAL LESION
trI NO LESION
ttt
I
e,
REGION D
15 ló t7
STAGES

317
NEURAL TUBE-NOTOCHORD
RATIOS IN EXPERIMENTAL EMBRYOS
WITH MYELODYSPLASIA
N=5
| NEURAL LEsloN
E No LEsroN
an
o
tr
ü,
REGION D
ló 17 18
STAGES

3t8
Figures fl:4-127 ' demons t ra te the ratios. in the four categorîes of
enbryos, distinguishi.ng between regions with and w¡thout a neural lesion
in affected embryos. The ratlos are highest ìn the control embryos and
sltghtly lower in the experimental embryos with no defects. l,,tyeloschisis
is associated w¡th a definite reductîon ln rptios,with no suggestion of
neural lrovergrowtht' before or after the stages of normar neural crosure.
All embryos wíth nryelodysplasia show a marked reduction of ratlos. ïhere
is no obvious difference ín ratio between an affected and an adjacent
unaffected region in either myeloschlsís or myelodysplasia, implying
that the neuraì tube adjacent to a leslon shows a similar reduction in size.^
Statistical analysis of the data in Tables 59-66 was restricred to
embryos of Stages 16-20, to provide a comparable distribution of Stages
within the four categoríes. The vatues for the mean notochord area were
f¡rst exam¡ned to test the assumption of uniform notochord s¡ze. An
analysls of varíance showed no signifícant differences in mean notochord
area between the four categoríes in both Regions C and D (Tables .59 ê 60).
TABLE 59A.MEAN NOTOCHORD AREA IN REGION D
Ca tegory Number l''lean S.D. s.D.H.
Cont ro I s
Norma I Exptls.
l4yeloschisís
llyelodysplas la
Totâ I
14
10
1t
5
4o
2985.9
2960.6
2524.\
2989,1
2853.1
1027.3 274.5
980.7 310.t
875.1 263.8
t017,9 455.2
958.4 15t..5

319
TABLE 598 ' ANALYSIS OF VARIANCE (NOTOCHORD) REGION D
Between
}llth¡n
Tota I
D. F. s.s. 14.s.
3 164377
54792
36 3\17713
94%6
39 3582090
t:
P.
0,577
NS
TABLE 6On, ileRì{ NOTOCHOnn Rn¡R.lH.rEctotii C.
. .. .... .. .,
Category Numbe¡ llean .S.0. S.D.l,.l.
Controls
Ilorma I Expt I s.
Mye I osch ls i s
Itlyelodysplas ia
14
3350.1
10
3\27.o
It
3412.2
5
3573.2
Toral 4o 341\.9
TABLE 6od" AüALysts oF vARtANcE (NorocHoRD)
1250.5
334.2
1173 .9 371.2
1061 .7
320.1
1122.5
502 .0
1123.5
177.6
REGION C
D. F. s.s. t'f. s . F.
Between
tlithin
3
36
1855 t
4904098
6184
136225
0. 045 N.S.
Totâ I
39
\9226t+9
As the neural t i s s ue/notochord ratios thus reflect the relative
volume of neural tissue in each regîon, the ratios were then.suU;ected
to analys¡s. An omnibus (Anova) analysis of variance showed significant
dlfferences (p . O.g) between the four categor¡es (Tables 62 and 65).
Fínally, multiple T - tests of all possíble pairs were performed
(Tables 63 arld 66).
The Bonferroni procedure to partition alpha was
used, to maintaln the error rate near the level employed in the Omnibus
test (qq
^â o.ot ).
llelcþrs procedure was appl led as a conservarîve

adjustment for nominal alpha level ln the presence of heterogeneity of
varlance (as revealed by Bartlettrs test) and unequal numbers în the
four câtegor ies.

321
TABLE 61., .NEURAL TISSUE/NOTOCHORD RATIOS IN REGION,C
Category Nurnbe¡. ... Mean . .... ..s. D, s. D.t'|.
Controts t4 7.g't\29 1,7345g0
0.4635861
Normal Exptls. t0 6.gtgooo 1.767074 0.5587979
lilyetoschisls 11 5.510909 f:g66g60 0.5628795
l.lyelodysplasia 5 1,907999 O,31g1{;;z o.144176
Totat 40 6.206750 z.hgoos3 0.3937120
TABLE 62. ANALYSIs oF VARIANCE (RATIos) REGIoN c
D. F. s.s. 1.1 . s - F.
Between
lr¡ rh in
3
36
1 39.340
102.474
\6.\\7
2.8\7
16.317 < 0.05
Tota I
39
241.814
BARTLETTIS TEST FOR REGION C
Chi qq.
D. F.
P
TABLE 6
Categor i es
= 9.25\
=3
< 0.10
I'{ULTIPLE T- TS RAT I
t
G ION C
D; F.
Controls/Normal Expt¡ s. r.419
Contro I s/l'lye losch i s i s 3,209
Con t rol s/Mye I odyspl as i a 12.279
Normal Exptls./t'tyeloschísis 1,649
Normal Exp s. /Myelodysplas ia 8.502
Ìlyeloschlsís/l.tyelodysplas i a 6.1g1
19
21
15
r9
t0
1t
N.S.
P < 0.0,|
P < 0.01
N.S.
P < 0.01
P < 0.01

322
TABLE .64. ¡leunn|ilssue/norocro*o *ot'or''* REGtoN 0..
Category Number Hean . S. D. s . D. i,f.
Cont rol s
Normal Expt I s.
Hye I os ch 1s i s
l4yelodysplas îa
Tota ¡
TABLE 65, ANALYSIS
t4
t0
t1
5
4o
OF VARIANCE
6.979286 1.704087
5,732000 1.2r5180
5.51\545 1.599928
2.593999 0. 478989
5.713999 1.959256
(RATros) REGtoN D
0,4554363
0.3842739
0.4823966
0.2142107
0.3097856
D. F. s.s. r't. s . F.
P.
Between
3
72.152
2\.051
1r.164 < 0.05
l'/i th i n
36
77.556
2.154
Tota I
39
149.709
BARTLETTIS TEST
chí ôq.
D. F.
P
FOR REGION D
= 6.3\8
-)
< 0.10
Categor ies
Control s/Norma I Exp s.
2.o97
22
- N.S.
Control s/Myel osch i s i s
2.218
22
N.S.
Cont ro I s,/Mye I odysp ì as i a
8.779
t7
p < 0.01
Normal Exptl s. /Myeloschisis
o.356
l8
N.S.
Norma l Expt I s. /l'lye lodysp I as i a
7.161
13
P < 0.01
Ìlye losch i s i s,/l'lye I odysp I as i a
5.570
13
P < 0,01

323
Apart f.rom the sîgnîficant difference between embryos with
myeloschisìs and the controls in Reglon C, there is little distinction
between the control embryos and the exper¡mental embryos with myeloschlsls
or wîth no defects. This suggests that neural rrovergrowthrt is not
an essential component of myeloschlsis between Stages l6 and 20.
There are however slgniflcant differences in neural t¡ssue/notochord
ratlos between embryos wìth myelodysplasia and each of the other
three categories in both Regions C and D. Myelodysplasia ls therefore
characterized by reduction ln the volume of neural tlssue.

Dlscusst0N
324

t25
7. Dlscusst0N
1'/i th progressive control of infectious diseasesi congenitar defects
have become an inportant cause of ¡nfant mortal ity and morbidity. Open
defects of the central nervous system form a signifrcant proportion of
the major malformations. Anencephaly ls uniformly fatal, but the effect
of spina bifîda varles ¡¡îth the exrent and level of the cord lesion (Barson,
1970).
ln an êttempt to lnvestlgate the embryogenesis of anencephaly and
splna bifida, ên exper¡mental method has been developed for produclng
open defects of the brain and splnal cord in the chlck embryo, by a simple
physlcal procedure.
The dlscussion ls llmlted to cons¡deration of the malformations
obtained by thls technlc - open neural lesions, skeletal defects of the
vertebra¡ column, and a range of associated non-neural malformations.
Anterîor spina bifida and neuro-enteric connectlons, hydrocephalus and
the Arnotd-chlari nalformation, syringomyelia and myelocystocele were not
diagnosed in the experimental enbryos, and so êre not consîdered in this
d i scuss íon.
A wlde renge of neural malformatíons has been produced in domestic
and faboratgly animals by a plethora of agents - vitamin and míneral
deflciences, stêrvat¡on, hypervítaminosis A, ionizing radîations, infections,
hypoxla, hypothermia, hypertherrnía, and many drugs, dyes, hormones and
chemîcal nateriêls (Kalter, l!68; Shepard, 1976; persaud, 1977).
0pen neural defects have been produced in the chick embryo by x-rays,
ultraviolet light, ultrasound, víruses, hypoxla, hypercarbía, and a variety
of drugs, hornones and chemicals (see Section 2.3.2 for references).

326
Spontaneous neural defects have been reported in mice, rats, gulnea
plgs, rabbits, cats, dogs, pigs, cows, horses, sheepr. goats and non-human
prlnates, with occasional reports in other animals (Kalter, t96g). By far
the most extensive investigations have beèn performed ln mice, where armost
a hundred genes can be impr icated in neurar marformation syndromes (sidman
et al ., 1965) .
ln the present study statistical anarysis was performed for the overa
malformatlon and mortår ity resurts after windowinq at 14,26, and lg hours,
and fol lowlng remova I of the lntroduced alr space at varrous lntervals after
wlndowlng at 26 hours. Anaìysrs showed that windowíng is highry teratogen¡c,
w¡th ¡ts maxlmun effect at the earl rest stages. tn embryos windowed at 14,
26 and 38 hours,the 14 hour group showed a high early mortal ity, while the
26 hour group showed a high lncldence of neural mêlformatlons.
After wlndowing at 26 hours, the mortar ity increased steadi ry when
embryos were recovered at progesslvely longer perlods of incubation.
0bl iteration of the rntroduced aîr space, however, substantia y reduced
the teratogenic effect of windowing when performed immediately.
lndívidual malformations observed at 3, 5, and 12 days were not
analysed statlst¡cally, because the hí9h rncidence of earty and rater
deaths reduces the value of any such analysis.
Despite the high mortal ity of windowing in.the first lg hoúrs, it
is clear that with prolonged culture of the survivîng embryos the range
of malformations increases. Rump and rimb defects were not apparent
at three daysrand ectopia víscerum wâs not seen ât five days.
ln the major experiment to investigate the development of open
hêurll dèfêets after wlndowing at 26 - 30 hours, 4t! embryos were used, of
whlch 90 were selected for serl.al sectionlng.

327
C¡osure of the anterior neuropore was completed by Stâge lJ in the
control emhryos, apart fiom one Stage l/ embryo with an open anterior
neuropore (regarded as an open braln defect by thls Stage). Several
experimental embryos after Stage 12, however, showed open anter¡or neuropores
(regarded as an open brain defects)at the Stages lmmediately following
Stage 12, provlding evidence of non-closure rather than closure and
reopenlng of the braln. These defects were not seen ln large enough numbers
to allow detai led hîstologlcal study. The appearance of open braÌn defects
was very similar at 3 days and at 12 days, and closely resembles the welli
preserved.human exencephal lc embryo l l lusrrated by Hunter (,|934-35).
Closure of the rhombold sinus occurred at Stage 15-f6 in both experlmental
and control groups. A trlangular rhombold slnus, however, was
seen only in experlnental embryos of Stages 11-16. Open cord defects
first appeared at Stage 13, and were seen at all Stages after this, agaîn
suggesting that they arose by non-cloSure rather.than by reopening of the
closed neural tube.
0n comparlng the drawings of whole embryos with their subsequent
histologlcal appearânce, It became apparent that a tr¡angular rhomboid
slnus is the precursor of myeloschisis. The fact that the development of
rryeloschlsis can be predícted from the shape of the rhomboid sînus before
the perlod of normal closure is strong evidence that myeloschîsís arîses
by non-cl osure.
Skeletal stalning of l1-12 day embryos revealed an increasing
sever¡ty of axial defects from cervlcal to caudal level's. Spina bifîda
occulta was seen mainly ln the cervical r.egion. Spina blflda manifesta
occurred (wlth open cord defects) from the lower thoracic to the upper

328
caudal regions. rrregurar or dereted vertebrae were a¡most all rocated
in the caudal region (rumplessness).
Spontaneous rump defects were seen in ll of the 62 control embryos,
and were much the cornmones t spontaneous defects observed ln these exper_
lmen ts .
Rumplessness nây occur ln fowls as a dominant, recessive, or sporadic
character (Landauer and Dunn, '|925; Dunn and Landauer, ,|934; Landauer, 1945);
the enbryogenesis of each type is different (Zwilllng, 1942i 19\Ð.
Experirnental ly, rumpressness has been produced by injection of insul-in
ín ot¡o (Landaue r and Bllss, '|946), and by vlbration of unopened eggs
(Landauer and Baumann, l!41). ln both cases the lncidence of rump defects
varled with the genetíc background of the frock and wrth the t¡me of year.
ln the present exper¡ments rvíbratlon of unopened eggs was not found to be
signlficantly teratogeníc (for the smal number of eggs used) when compared
to windowing. Although seasonal variatíon was not specif ica.l ly tested,
again no significant trend courd be detected when compa red to windowing.
Because of continued embryonic Arowth, open cord defects were found
at both somite and post-somite levels in Stage l3_16 chick embryos, but
only at somite. levels by Stages l/-20.
The posterior neuropore closes êt the 20-21 somite stage,at a level
that later lies opposite son't tes 27/29 after addition of a further 6 somites
(Hami I ton, 1952). As the first four permanent som¡tes contrlbute to the
formation of the occípitar regíon, the poster¡or neuropore thus coincides
with a future spinal levet of vertebrae 23/24 (in the lumbar reglon). Uhen
the distr¡bution of open neural defects ln lZ hour embryos was plotted,

329
the mid-polnts of the defects were found to lie essentially between
somltes 21 and.31, correspondi.ng to future vertebral levels of T.4 to S.2.
ln the 12 day expêrimental embryos rÌrost lesions of spina biflda manifesta
were lndeed centered at the lumbar r.egion. Those lylng at more caudal levels
may have been nye I odysp I as ias , rather than myeloschisls, though the two
defects were dlfficul t to distinguish.
The less serious defects of spina bifida occulta in 12 day embryos,
were mainly locâted in the cervical and upper thoracic regions, and showed
very llttle overlap w¡th sp¡na blflda manifesta.
A slmllar d¡stríbutlon of lesions emerges from the study of human
dysraphism. 0f 601 dysraphic infants admitted to hospital and examined
by radlology and necropsy, skeletal defects lay mainly în the lumbar and
sacral reglons. The low incidence of cranial and uppercervical defects
was probably due to abortîons and stiltbirths caused by associated anencephaly.
Skeletal defects at the cervico-thorac¡c and lumbo-sacral areas
were quite local ized, but bony lesions ¡n the thoraco-lumbar regîon and
those involving anencephaìy were more extensive. This suggest that there
are two types of dysraphic lesions in man - major defects (anencephaly and
thoraco-lumbar. spína bifida) and more minor defects in other regions (Barson,
1970') .
- Rump defeòts observed in the chick embryos may be compared to sacral
agenesîs in man, which varies in severity from loss of coccygeal sêgments
to partíal reductîon of the.sacrum or even absence of all sacral and lumbar
vertebrae. Extensîve sacral agenesis may be accompanied by neurological
involvement and anal or genito-urlnary defects (Blumel et al., '|959; Russel I
and Altken, 1963). ÌJhereês rumplessness is one of the commones t spontaneous

330
defects seen ln fowls, human sacral agenesis is rare. This may be
because phylogènetlc reductlon of caudal segments, already evident in
chlckens, has been carried further in the hur¡an sprne (Hughes and Freeman,
19741 .
A revlew of the histological differences between experimentat and
control embryos was complîcated by shrlnkage of ,or" embryos during pro_
cesslng, producing sp!ittlng of the neural tube roof ln older embryos,
and wide separation of notochord, somites and neurar trssue rn earry embryos.
These art,¡ facts could probably be avoided by using dloxane for processing._
Examination of the control emb ryos by serîal sectlons revealed a co_
ordinated sequence of changes in chorda-mesoderm and neural tissue during
¡eufolation, though the description is stat¡c rather than dynamic.
Duríng Stages l0-12 at the posterior rhombold sinus, neural plate
dlfferentiated ín the region of Hensenrs node and the neural folds were
flattened or elevated, while the chorda-mesoderm was fused l¡to an undiffer_
entlated cell nass. At the anterior rhomboíd sfnus the neurar fords showed
further elevatíon, and accessory canars were present ín the tat-bud materiar;
The notochord was estabr ished and somitic mesoderm became separated, though
not segmented. lmmediately above the rhombold sinus the mesoderm showed
separation intå club-shaped protosom¡tes, while the neural plate was in_
vertèdr. closing, or crosed. cranially the brain was crosing or irosed.
During stages r3-20,Hensenrs node {ån¿ r"t.. the primitive streak)
gave bray to a tail-bud by Stage 16, from which the caudal regíon developed,
The rhomboíd sinus was crosed by stages l!-r6, but neurar materiar from the
ta¡ l-bud contributed ro the sp¡nal cord of the tail until Stages 1!_20.
l'llth the onset of neurulatlon, thickening, elevation, folding, and

331
'c¡osure of the neural folds were closely integrated with formation of the
notochordr and the developnent of nature somites from undifferentlated
mater¡al of the streak and node, and later the tail-bud.
ln the experlmental embryos the development of lnyeloschîsis was
preceded by everslon of the neural folds at the rhomboid sinus in
serial sections, producî.ng a trlangular shape on examining the whole embryo.
ln the'earl iest myeloschisis leslons the rhomboid sinus was s.tlll open.
Exanlnation of the serlal sect¡ons showed that myeloschlsis consisted of
an open defect of the lower part of the neural plate material, extending
caudally to involve the upper part of the ta¡l bud materlal. ln older
embryos an appêrently normal neural tube formed at a more caudal level.
Establ ished myeloschisis lesions on hlstology showed a flat surface
plaque (continuous with neural plate), overlyíng neural materîal containing
accessory canals (derived from the têíl-bud). The two sources of neural
material were clearly separated,.wíth dîfferent orientation of their constituent
cells. The majority of mitotîc figures were seen at the luminal
surface of the closed neural tube, and along the dorsal surface of the
exposed plaque. The everted neural plate showed smooth continulty
wi th adjacent .ectoderm.
Neural crest cells were seen at the margin of most myeloschîsis lesions,
and adjacent structures were wel I deveìoped. The notochord was uniformly
normal ln appeârênce, but wldely separated from neural tlssue at the
cranlal end of most myeloschisis lesions after Stage ,l6. The somîtes
appeared normal , and the impressíon of separation of somites from affected
areas of neural tube wâs not confirmed quantítatively.
I'leasurements of neural tlssue/notochord ratios provided no evidence
of local overgrowthrr of neural tlssue, which could therefore not be

332
lrnplicated in the pathogenesls of nryeloschlsis. lt has to be concluded
that myeloschisis în the present series of chick embryos treated by
wlndowi.ng .arises by simple non-closure of the neural folds, representing
a fai I ure of neurulation.
The development of rrúélódvsolasia was not preceded by any characterístic
shape of the rhor¡bo ¡ d sînus, and did not occui befor. Stage 16. The neural
canal could not be traced ilrectly înto the leslon, and there was no
separat¡on ¡nto neural plate and tai l-bud mater¡als. l',lyelodysplasia dld
not coexist wlth an open rhombold sinus, and formed an lrregular open
defect in whole embryos at /2 hours.
ln serlal sectionsof myelodysplasia the neural tube at the upper
end of the lesion was triangular (due to reduced neural plate material),
giving way.to a narrowly-everted or f lat plague(derived from tail-bud
material, and partly covered by ectoderm). Caudally, there wês an
apparently normal neural tube (derived from tail-bud materlal), or a
disrupted region forming diplomyel ia or amyel ia. The myelodysplasia lesions
were partly covered by ectoderm, and nowhere so smoothly contínuous with
ectoderm as the myeloschisis lesions. l'lîtoses were not restricted to
the surface of the plague.
The notdchord was uniformly ¡n contact with neural tissue, except
in one embryo that showed a combinatíon of myeloschisis and myelodysplasia.
Somites în the area of myelodysplasia were reduced in volume, and often
reduced in densityrdue to a loose arrangement of cells suggesting edema.
ln some areas blood vessels were dflated., with hemorrhages into the local
mesoderm. l'lhere neural tissue was very reduced, the somites fused dorsal ly
lnto a slngle mldline mass. All myelodysplasia lesions showed reduction of
neural volume, both on lmpressidn and by measurement.

333
. These findings s.ugges r that myelodysplasia does not ar¡se by simple
non-closure of the neural folds, but develops from the tail-bud materiar
after St¿ge 15, in the absence of neural plate materlal.
Histologically, deveropment of the rhombic roof showed no difference
ln enbryos wi th and wÌthout neurar defects. The choroid prexuses did not
âppear unt¡l Stage rB in either control or experîmental embryo, after the
establ lshment of myeloschisis and nryeIodyspIasIa. rn these wlndowed chick
embryos, open neural defects cannot be attrlbuted to delayed passage of
cerebro-splnal fluld across the rhombic roof as suggested by Gardner (r!6r,
1964, 1972).
The role played by the ¡14çip¡¡L in neurulation ls stl I I not clear,
desplte many investigat¡ons. Elongation of the notochord appears to be
an essential componen t of neural plate formation (Holtfreter,
1955). Jacobson and Gordon (1976) snowe¿ by cell counts in Tri turus
that the extending notochord does not creave through the neurar prate
cells, but dísplaces them anterîorly to contrlbute to the future brain.
ln several mutant mice such as Danforthrs short taí1, brachyury,
anury and trgncête, open and closed neural defects occur sporadical ly,
but are probab.l y secondary to abnormarities of the notochord or pr¡m¡tive
streak (Grüneberg, rg63). These mutants show extensive vertebrar defects
of the splne and tail, as well as some vîsceral defects, "rro"i"t"d
*¡th
the notochordal malformatîons. Thei r neurar defects may represent myerodysplas
ia rather than nryeloschisis.
The slze of the notochord is reduced in amphibia by treatment wrth
l¡thlum chlorlde (Lehmann, 1937), and enlarged by treêtmeñt wÍth sodium
thlocyanate (Ranzri and Tanlnl, 1939). These changes can be explained by
act¡on of the postulated mesodeimal¡z¡ng factor (Tolvonen, l96l; Tolvonen

334
et al. 1961). tríth the single exception of a severely affected embryo
wlth myelodysplasia (showi.ng loss of all structures due to cystic
changes ln the caudal region), no notochordal abnormal i ties were seen in
the present series of chick embryos.
Howeve r the embryos with establ ished myeloschlsis showed separation
of the notochord from neural tissue at the cránial end of the leslon. ln
the looptal I mutant mouse open neural defects are a predomlnant expresslon
of the gene, and appear to arise by non-closure representlng a myeloschlsis.
Embryos lllustrated by Stein and Rudin (t953) sho" separat¡on of notochord
from the open neural defect at 10 days. Dav¡s (1942, 1944) by ,ultraviolet
lrradlation of Stage 7-9 chick embryos produced nryeloschlsls, similar to
the defects in the present embryos, also associated wìth notochordal
separation. A similar finding was reported by Ancel (1946-\7,1956), who
suggested that the separat¡on arose by incomplete separation of mesodern
lnto somltes at the end of gastrulation. .ln the.present embryos, however,
the gap was filled by a loose mesenchyme after the establ îshment of myeloschisis,
rather than by fused somitlc mesoderm before the formation of
the neural' defect.
Notochordal separation from the neural tube occurs as a normal
developmental process upon somíte díspersal and migration of sclerotome
cells. Even ôt Stage 10 in the present enbryos ¡t was seen at tbe
cephal ic end of the notochord, and by Stage 20 had extended into the
sornite region. Separatíon,rjid not occur in the early stages of myeloschisls,
and so appears to follow rather than cêuse non-closure. This
suggests a reduced adhesion between notochord and neural plate, but
it mlght reflect the fallure of some essent¡al inductlve process êt
an earl lei srase r¡jllán (1968).

335
Lendon (1968, 1975) and notos (1976) both described fiuid
accumulatlon deep to the neu¡:al plaque, which they regarded as a sequel of
separat¡on, leadîng to the later elevation of the plaque and stretchlng
of the spinal nerves.
Some indlcatlon of the lnfluence of the notochord on early neurogenesi's
ls provided by studîes of the rrovêrgrowthlr phenomenon. Bergquist
(1959) and rãlán (1965) found .that remova I of the fourth neuromere of the
chlck braln produced marked overgrowth of local braln tissue only when
the underlylng notochord was removed or damaged by the operatlon. They
suggested that an ¡ntact notochord may exert some controll îng influence
over the ôdjacent neural tube. Refinement of the technic' to allow
separatlon or remova I of the tip of the notochord and replacement of the
overlying rhombencephalon at stðge 11-12 (Burda, 1968), also produced
local overgrowth of brain tissue. Thís was accompanied by increased cell
dlvision and the distribution of mitotic figures throughout_ the braln
wall. Autoradlography showed that both experimental embryos with overgrovrth
and normal controls lost the abilîty to lncorporate H3 - thymldîne
by the fourth day'mafking the onset of differentiation (Bsrda-l,lilson, 19/1)'
Furthermore the ânterior notochord'efter experimental . s.eparatîon from
the rhombencephalon showed earlier vacuolation, nuclear pycnosls , and
accumulatÍon of P.A.S. - posl tive material than the notochord oí control
emb ryos .
Examlnatlon of somitic mesoderm in control embryos of the present
serles showed close contact of -g.j-!gg with the neural tube, whereas
the unsegmented and fused mesoderm of the rhomboid slnus was general ly
separated from the neural plate by a smal I. gap. Somites had formed down
to the t¡p of the tail by Stage 20.

336
The inpresslon of somlte separât¡on in embryos wlth myeloschisis
was not confirned by further analysis, as the lengths of somite separatîon
dld not correspond to the revers of the defects, and extensîve somite
separation occurred in control embryos.
ln , aaurans (with a bilaminar neural plate), analysis of the mech_
anlsm of neurulatlon revears that as well as'lntrinsic forces within the
neural plate, folding involves elevation of the differentlatlng somites,
ln the presence oi tight adhesion between neural plate and notochord
(Schroeder, 1!/0). Somite elevation does not appear to be împortant ln
the chick embryo, as disruptron of neuroepither iar mîcrotubures (by corchrcine)
and mlcrofllaments (by cytochalasin B) înhiblts or even reverses neurulation
(Karfunkel , 1972).
ln mammals, open neural defects have been produced by maternal
treatment wlth varlous agents, încludíng trypan blue ln rats (Gillman et
al., 1948; l,/arkany et ai., l95B; Lendon, t96gt 1915; Rokos et al., t97O;
1976| or mice (tJaddington ênd Carrer, 1953; Hamburgh, 1954): and dímethyl
sulfoxlde or high doses of vrtamin A in hamsters (Marin-padi a and Ferm,
1965; Marin-Padilla, r966; 1966; Ferm t966). rn each cêse rhe development
of the neural defects was crosery associated with edema, cyst formâtion,
and hemorrhages in local mesoderm. Rokos et al. (,|970) also descríbed
extensive cell death ín mesoderm, heart, gut and neural plate.
ln embryos recoùered within 48 hours of maternal injection with
vitamin A or dimethyl sulfoxide, accumulation of f .luld and dilation of
local slnusoids was observed in unsegmented nesoderm at g-10 hours, followed
by necrosis and col lapse of somítes after 24 hours (l4arin-padil la and

337
Ferm, 1965; l"larin-Padi r ¡a, r966; 1966). As these mesoctermal changes preceded
the development of open neuiar defects, they were regarded as the cause
of neural dys raph.i sn.
The relevance of these observat¡ons to the production of neurar
defects by maternal lnjection of trypan blue is complîcated by agent and
species dlfferences. Trypan.blue êppears to ""t
ln rats and rnlce by
Interfering with fetal n.utrition at the yolk sac (Beck et al., ,l967;
l{llllams et al., 1976), and signlflcant levels of the dye have not been
detected within the embryo (Waddlngton and Carrer, f953; tji lson et al.,
1963't. Vitamin A, however, appears to cross the placentê when given in
hlgh doses (cîroud and I'lartinet. , 1957), and the low molecular weight
of dimethyl sutfoxide suggests that placental transfer mí9ht occur (Ferm,
1966). A more direct action by trypan brue wîthout prâcentar intervention,
however, has also produced fluid accumulation, hematomas, and tlssue
damage in amphîbia (Waddíngton and perry, 1956) and chick embryos subjected
to ¡ntravascular injection at J days (Kaplan and Johnson, i970) , or
explantation on the f¡rst day (üurherkar, 1960) . rn each câse the brunr
of the damage was borne by mesodermal tissues, wíth less severe involvement
of neural tissue. The interpretation of trypan brue actívrty is further
conpllcated by the presence of various impurities în different commercial
samples, and the existence of three fractìons withd,n the dye (BecÉ and Lroyd,
r963) .
ln the present experíments' sectíoned embryos with myeroschîsis showed
no abnormal ities of notochord or somites, apart from the notàchordal
separation already discussed. l,lyeloschlsis could not.be regarded as
secondary to somî te defects.

338
Embryos wîth myelodysplasia however, showed changes crosely resembríng
thos e produced by maternal hypervitaminosîs A or trypan blue ln rats - fluid
accumulation, vascular dllatlon, henatomas, and somlte reductions. As a
large number of embryos showi.ng cystic changes were not serected for
serial sectionlng, the histologlca¡ descriptions covered embryos showing
the smallest nesodermal invorvement. Thrs suggests that mye¡odysprasia,
accompanled by extensive trunk and rump defects, resulted from extenslve
tlssue damage after wîndowing, for rowed by a variabre degree of embryonic
rggulation. Myeloschisîs, by contrast, resulted from a much more selective
actlon, causlng non-closure of the neural plate but often no other defects.
separation of notochord from establ ished myeloschrsls reslons lndicates
loss of adheslon between neurar plate and notochord, but rs not sufficient
evidence to postulate fâiture of neurulation due to a loss of inductive
interactlon.
Formatlon of the avian tail from the ta¡l bud was clearly demo_
strated by Zwill¡n9 (r942) who excised the taH-bud åt Stage r2-16 and
obtalned embryos wlth no tail, which ended àbruptly just posterlor to
the hlnd l[mbs when the entire tall-bud was removed. Zwilling (1942;
1945) also followed the embryogenesis of dominant and recessive rumplessness.
He found that the dominant gene produced extensive celr death
withln the taÍl-bud and tail anlage by the Jrd day, whereas recesgíve
rumplesSness arose by masslve necrosls within a formed tall on days
4-5. lnvestigation of rnsur in-induced rumplessness showed a varrabre
development, wlthout massrve ce death but w¡th frequent inversron of
the tail lnto the hindgut (ourentery) or defects of varlous caudal elemenrs
(l4oseley, 1947).

339
Kôplan (1965) and Kaplan and Grabowski (1967,t , afrer treatment of
48 hour chiòk embryos wlth trypan blue, observed extensive blisters and
hematomas of the trunk and rump, associated with vascurar dîration and
rupture. These changes were fol lowed by rumplessness in older embryos,
and dlrect observation through a glass coverslip over the shell window
revealed that enbryos that d¡d not form hematomas did not later shour
rump defects.
The present series of wîndowed embryos recovered at 12 days were
not subdivlded on the basÌs of myeroschrsls or myerodysprasra resrons.
It seems reasonable to suppose that embryos with an irregurar neurar
defect and extenslve vertebral .rrregurarlty and deletions showed myerodysplasla,
associated with cysts, hemorrhages, and somlte reduct¡on at
72 hours. However, embryos with regular defects at 12 days also showed
rump defects, în splte of the absence of assoclated mesodermal defects
ât 72 hours. The embryogenesls of rump defects after wrndowíng crearry
requi res further lnvestigation.
ln many stud¡es of neural dysraphism, continuity of the open neural
t¡ssue with adjacent ectoderm has been taken as evídence of non-closure
(Glroud and I'lart¡net, 1957; Dékaban, 1g6r. ln an investlgatîon of the
regulatlve abi'l lty of ectoderm in the early chíck embryo,. Rokos and
Knowles (1976) split open the roof of the neuraì tube dur¡ng the thírd
day of lncubatÍon. This produced an open defect of the neurar tube, w¡th
close apposîtlon of the. cut edges of ectoderm and neural tissue w¡thin
two hours. Thls demonstrates that neura r -ectode rma I continuity ls not
clear proof of non-closure. ln the present windowed embryos, however,
there was a perceptlble difference between smooth contrnurty of ectoderm
wlth neural tissue in myeroschisís, and ress rntimate contrgurty between

340
ectoderm and neuraì tlssue ln myelodysplasia.
The s.uggéstion that caudal levels of the neural tube, formed after
closure of the posterlor neuropore, are derived from the tail-bud was
flrst made by Biaun (1882), who described a caudal mass of cells undergolng
cavltation in avian enhryos. Development of the terminal part of
the hur¡an spinal cord from the tail-buci was described by Keíbei and Elze
(1908). Schumacher (1927) showed that multiple cavìtation of caudal neural
tlssue ls a normal process in the chick embryo.
ln birds, Hensents node increases ln size and lncorporates the
remalnder of the primlt¡ve streak, to form thê ta¡ l-bud at the 18-22
somlte stage ($eevers, 1!J2). Wetzel (1929) regarded the actív¡ty of the
tall-bud ês the same as that of the streak and node of earller stages, but
Hunt (193.l) and Seevers (1932) showed that it has ìost the capacity for
primary induction, and ¡s restr¡cted to the formatlon of posteríor trunk
structures.
Criley (1969) demonstrated an overlap zone în the caudal region
of the chlck neural tube, between material derived from the neural plate
(lying dorsalty) and material derîved from the tall-bud (lying ventrally).
This overlap zone (of 192-280 microns) was detectable between Stages I1
and 18, with fìsion occurring maximal ly at Stages 13-15. The taîl-bud
materlal extended up to at least somite 35, and the neural plate material
down to at least somíte 33. Removal of the most caudal section of the
neural plate showed that an appârently normal segment of spÍnal cord can
develop from the tail-bud ín complete isolatlon from the neural plate.
ln the present embryos the extent of the overlap was revealed by
qulte subtle changes in outl ine of neural tlssue at. caudal levels. ldentifylng
features from cranial to caudal were: fallure of the neural canal to

341
reach the floor-plate; asymmetry of the neural canal; a pear-shaped external
contoür of the neural tube; an hour-glass external shape of the neural
tube; accessory canals în the tail-bud nraterial; and finally a solid tailbud,
deep to a foldi.ng neural plate. Using these features, rather than
sirnply the presence of accessory canals, the overlap zone was detectable
ln control embryos of Stage tl-16. After ttr¡s, complete fusion obscured
the extent of overlap in normal enbryos, but the zone was still recognisable
in myeloschisis lesions because of the contínued separation of the
tv'ro sources of neural materlal
The overlap zone could only be analysed quantltatively between
Stages lJ and 16, when it was promînent ín control embryos. There was
little dîfference in the extent of overlap between embryos wlth myeloschisis
and the experímental and control embryos wi th no neural defects,
showlng that myeloschisis does not arlse through changes ln the extent
of the zone.
The two embryos with myelodysplasía at Stage 16, however, showed
a very short overlap zone due to a greâtly reduced contribut¡on by the
neural plate. The lower boundary of the zone (marking the lowest extent
of the neural plate material) lay at almost the same level as the upper
boundary (markíng the h¡ghest extent of the taìl-bud material).
'
For the analysis of volúmetric changes associated with open neural
defects, measurement of the ratìo of neural tube to notochordal areas
allowed comparison of: embryos of different sizes; reglons of different
lengths; sections cut in different planes; poor I y-p rocessed and wellprocessed
material ¡ and groups of embryos with different reglonal
boundarles. Statistical analysis revealed greât constancy in notochordal
areas, wlth no evîdence of excesslve ¡eural tîssue in myeloschisls, but

3\2
marked reduction in myerodysprasia. possible sources of error m¡9ht
be attrlbuted toi inaccurate tracing of thu tirrr. outl ines, measurements
of only every tenth sectlon, and the extens¡ôn.of some lesions
into Regions B and E (which were not incruded). Future volumetric
analyses should: measure every section; include all four reglons (to
abolish the effect of different regìonal boun¿aries); and include
older embryos (to detect any overgrowth at later Stages).
lnterklnetic migration of nuclel in neuroe p ithel ial cells after
neural closure v/as suggested by F.C. Sauer (1935), and conflrmed by
llarrerson et al. (1956), H.Ë. Sauer and Walker (1959), and Fuj îra (,|960).
Hamburger (1948), by measuring mitotic densíty ln the neural tube of 2{ -
8* day chick embryos, found that the mitot¡c êct¡v¡ty in alar and basar
plates was quite d¡fferent. Ì,ritotíc density in the basal plates reached a
peak at 2å - 3 days and then fe stead y, whereas mítoses in the alar
plates rosê steadi¡y to a max¡mum at 6 days and then fell sharply. After
8* days, dlfferentiatron follows this prori.feratîve phase, and the enormous
lncrease in the size. of the cord is due to growth, rather than division,
of individual nerve cells. Corllss and Robertson (1959 1963) measured
mitot¡c dens¡ty in the chick neural tube before, duríng, and after neural
closure (at Stages 9, 11-15, and 19-26). They found that whíle the neuraì
plate was wide open there was no difference în mrtosis between arár and
basal plates; in regions of active fording, mitotic density wês tr{ice as
high in the basal plates, while after closure the ratio reversed, to be_
come twlce as high in the alar plates. Early neurogenesis is thus accompanled
by differential mltosls.
Autoradiography of l-2 day chick embryos gîven two doses of H3 -
thymldine, revealed that all cells of the recently-closed neural tube

343
took up the label, shovri.ng thêt differentiation had not yet occurred
(l'lart I n and Langman, 1965).
ln splotch and looptê¡ I mice the development of open neural defects
(which are a major expressîon of the mutant genes) is accompanied by a
prolonged cell cycle in the neuroe p ithelium, and an accumulatlon of
mitotic figures. This retardation îs followeó by a pericd of accelerated
cel I divislon, producing oúergrowth of the open neural tissue (Hsu and
Van Dyke, 1948; ì,/¡lson, t974; 1974). ln trypan blue - induced dysraphlsm
of rat enbryos, autoradiography at l0| days provided no evidence of lncreased
mltosls ln the neural tube (Lendon, l!/2).
Reductlon of mltosis, however, may not be essential to the development
of neural dysraphlsm. Davis (t942, 1944) found that in neural
dysraphîsm of chick embryos produced by ultraviolet irradiation, the
wavelengths that were most effective in reducing mitoses in the neural
plate were least effectíve in inhibitíng neurulation.
ln the present chick embryos m¡totic figures appeared to be restricted
to the exposed plaq,ue surface and the luminal surface of closed areas in
myeloschîsis; figures were more scattered through the ta¡ l-bud material in
nryelodysplasia. 14Ítotic dens¡ties in these lesions were not estimated
because the séparation of neural tissue into its two sources of origln
made ít impossible to count figures in relation to a constant sur.face area.
lilany of the changes associated wîth the development of open neural
defects in windowed chick embryos are relevant to the pathogenesis of
dysraphism in man. ln the human developmental horizons formulated by
Streeter (1942), the anter¡or neuropore closes during horlzon Xl (,l3-20
somites) and posterior neuropore during horlzon Xll (2.|-29 somites).
ln the chlck embryo the anterior neuropore closes at Stage ll (,|3 somites)

344
and .the rhombo¡d s f nus at the, 21.-22 $oarl1e..þer tod . {St¿ges 13"j14¡ ,
(Haril l ton,. 1952) ,
Dekaban (1963) and Dekaban and Barrelmez (1964) descrÌbed a t4 somite
(Horlzon xl) human embryo wlth complete neural dysraphism, a normal notochord,
and slight reduction in somlte density ât the perlod when the anterior
neuropore should be closi.ng. An older human embryo (of l3 mm) ,..¡! th exencephaly,
consistlng of everted cerebral hemispheres and exposed thalami
and choroid plexus (Hunte r lgli-sil, demonstrates that an open human braîn
can continue to deve I op.
Experîrnental exencephaly produced in rats by maternal hypervîtaminosis
A and followed rn a closely-spaced series of embryos, lr lustrates a very
simllar evolution of the brain defect. Exencephaly wâs not analysed
hlstotogically ln the present experiments (because of the smal'l number
of embryos), but the existence of open anterior neuropores in wlndowed
embryos at each Stage after Stage l2 Ís strong evidence of ?gn_closure,
Early human embryos with open neural defects of the lower cord
have been described at 8 mm (patten, 195r,7 mm (lngalls, 1932), and
5.5 mm (Lernire et al ., 1965). The smallest of these specimens, with a
regular 1 mm open cord defect opposite somite 25, wäs at Horizon XlV.
It showed a wiuely everted neurar defect (with ross of regurar or¡entation
of cells), normal notochord, and abnormal somites.
lrlarkany et al. (1958) in trypan blue - induced neural defects of rats,
found that open cord defects (rnyeloschisis) could be detected before the
expected closure of the neural plate. Simîlarly, in the present chlck
embryos, the development of myeloschisis - courd be predicted from the
shape of the open rhomboid sinus. rrlye r odysp r as i a however did not rnvolve
abnormallty of the rhomboid sinus, and deveroped from tafl-bud materrar in
the absence of the neurâl plate.

345
The defects in early human embryos, together with the f indi.ngs in
experimental dysraþhism, suppoFt,the assertiori that excencephaly, craniorachlschisIs,
and nyeloschisis arise by neural non-closure. Secondary
neural overgrowth and later degenerative châ.nges then produce the character¡stic
lesions of anencephaly and mye I omen i ngoce I e . Format¡on of the
caudal spinal cord fron thc ta¡l-bud wouid urpl"in sparing of the sacral
reglon, though complicated by relative shortening of the cord in the fetal
per iod.
Several forms of myelodysplasia can be explained as reductions în
the vo I ume
of neural plate material, tail-bud materîêl, or both.
Dipl'onryelía at the caudar rever. may arise by persistent cavîtation of taí r.-
bud material. This cannot apply to dlplomyel ia at hlgher levels, which
was not encountered in the windowed chíck embryos and so is not discussed
further.
Local overgrowth of neural tissue was suggested by patten (1952,
1953) as a posslble cause of non-crosure, because'¡t wês seen in embryos
w¡thout visíble externar defects, as well as in dysraþhism. Neural overgrowth
can fol low experimental incision of the roof of the closed avian
neural tube, at either the cord level (Fowler, 19531 or in the brain
(.lel ínek, 1960).
Harked folding of the ruminar surface of the chick neurar tr¡be has
been reported, w¡th and wíthout dysraphísm, after exposure to lead
chlorlde (Catlzone and Gray, l94l), tentanus tox¡n (Corl íss et al., 1966)
and several viruses (Hamburger and Haber, 19\7r Heath et ar"' r.956; rJir riamson
et al., 1956). However analysis of closed brain overgrowth produced by
influenza A vîrus revealed that the apparent folding and thíckening of

346
the braln wall were.due to marked reduction of ventricular volume(Robertson et
al .', 1967 ); the volume of brain tissue and the mitotic densíty were
actual ly reduced.. Neural overgrowth therefore cannot be the cause of
vIral-induced dysraphism. Similarly Bergguist (1960) found that over-
. growth of the chîck brain, produced by injury of the notochord wíth
removal of the fourth neurornere at Stages I1"I4, was assoc¡ated w¡th
lncreased mÌtosis but not increased neural volume.
Desplte Pattents description of overgrowth at early stêges, there
is no evidence that non-closure (myeloschîsis) is caused by íncreased
neural volume. ln the present series of chîck embryos,neural volume _dÌd
not lncrease durlng or after the establ ishment of myeloschlsis. lt
would be interesting to measure neural volumes in older embryos with
rryeloschîsis.
T¡s hypothesis that dysraphísm arises by ruÞture of the closed neural
tube due to hydromyelia (Gardner 1961 , 196\, 1972), was ba_sed on studies
by tJeed (1917¡ lgZZ; 1937-38) of the dynamics of cerebro-spinal fluid.
These experiments, however, relied on perfusion of the venticular system,
with consequent changes în hydrostatic pressure. As further evídence of
rupture of the rhombic roof due to hydromyelia, Padget (1968, l97o) quotes
studies by Bonnevie (1934) on the mouse mutênt later called myelencephalic
blebs.. Bonnevie belíeved that exencephaly and multiple congenîtál defects
assocîated with subepìdermal fluid blebs, were secondary to brain rupture
caused by hydronryelia. Reexamination of these mice by carter (1956, 1959) has
shown that exencephaly precedes bleb-formation, ând that th'e blebs (which
are responsible for some defects) are derived fron mesenchym¡l tissue fluíd.
Hydromyelia and rupture of the neural tube after closure does not
explaln the ex¡stence of dysraphism in human embryos of horizons lmmediately

347
after pred¡cted closure (Lemîre et al., 1965; Dekaban, 1963i Dekaban and
Bartelmez, 1964), and before the appearênce of the choroid plexuses.
lloreover, studies of the ernbryology of the human rhombic roof reveal
an actîve developmental process, rather than passive rupture of the
roof and dissection of the subarachnold space (Brocklehurst, '|969).
Experlnental exencephaly in rats appears at stages immediately
after normal brain closure (ciroud and l,lartinet, 1957; Langman and
lJelch, 1!66). Sinilarly in the present chick embryos, dysraphism is
present lmmediately after normal neural closure, and before the rhombic
roof shows a membranous structure or formation of a choroid plexus.
The other hypotheses of human neural dysraphism mentioned in the
lntroduction can be excluded as causes of open neural defects in chïck
embryos. Prlmary vascular defects (Vogel and ttcClenahan, l!!2)
cannot be impl icated in the chick embryos, whose neural tube is not
vascularized until 72-84 hours (Feeney ênd Vatterson, 1946). Abnormal
flexion of the brain (teuedeff, 188l; Frazer, 1921) or cord (Browne, 'l934)
cannot be responsible, as no flexures had appeared by the period when
dysraphîc lesions were al ready establ ished.
Birth trauma (Pol îtzer, 1954) is riot appl icable to a non-mammalian
model. nmniotîc adhesions can be dismissed, as open neural defects were
present before the formation of the amníon. lnfection was excluded as a
cause by performing bacterial cultures. Finally, abnormal fusion of the
tv,ro sources of neural material in the overlap zone cannot be excluded as
a factor in human cord defects (Lemlre, .1969), but wês not the underlying
cause of myeloschisis and myelodysplasia in the windowed chick embryos.
ln this díscussion of dysraphism, spîna bifida occulta has not been
nent¡oned. Splna bifida occulta may be subdivided lnto two types (James

348
and Lassman, 1972). ln the sirnple form there is a local ized absence of
spinous processes for a few ségments, usually without neurological ínvolvenìent.
The spina blfida occuìta syndrome consists of neural arch defects
overlylng an abnormal (dysplastic) cord or nerve roots.
The relåtion of occult to cystic forms of spÌna bifida is not clear.
Lorber anc Levtck (r967) found that parents oi chirdren with myelomeningocele
showed an lncreased frequency of spina bifida occulta, suggest¡ng
some etlologlcal connection. several mutant mice wîth open neural defects
may also show spina bifida occulta (Gruneberg, I963).
ln the present chick embryos mesodermal defects, which m¡ght later
result ln spina bifida occul ta,. were seen in experimental embryos with
rryeloschlsis or closed cords. Many 12 day chick embryos exhibited spina
blfida occulta but the pôthogenesis of the lesion was not investigated.
Trypan blue-induced dysraphism of rats can result in exencephaly,
spina bifida manifesta and spina bifida occulta in older embryos. The
early development of the defects is associated with extensive blebformatlon
and hematomas which later disappear, so that small open defects
mîght possibly close ât .later stêges. (Rokos et al., .|970; 1975; Lendon,
1968; 1976).
Rokos ani Knowles (1976) demonstrated a high regulative abílîty in
chlck embryos after opening the roof plate of the neural tube on the third
day. Smal I incísions showed rapid closure and reconstitution. Larger
înclslons produced an everted neural lesîon, at which the cut edges of neural
tube and ectoderm often fused together in 2-4 hours. Thus spìna bífida
occulta could originate e¡ ther as a primary mesodermal defect, or by closure
of a smâl I neural defect. lnvestigation of windowed chick embryos after 72

349
hours could provide evidence for one or the other mechanîsrn.
Use of a non¡.placental embryo for the experìmental nodel avoids
problems associated with maternal health and diet during gestatìon,
the sltes of lmplantation, and functlon of the placenta. ln the chÌck
enbryo, the close series of morphological stages al lows comparîsons of
lndlvidual embryos. Regia,ral subdi.¡lsion facil itates analysis of a
Particular developmental process ln embryos of different Stages oÉ through
different regions of the same embryo.
ln the case of early neurogenesis, the chíck is an excel lent experi;
mental model for human malformations. Hughes and Freeman (1974) compared
the development of the caudal region of the spinal cord in rat, mouse,
oppossum, plg, chick, and human embryos. 0f these embryos only the chick
and man show development of the caudal region of the neural tube by cavi t-
at¡on of tai l-bud material, after closure of the neural plâte.
ln amphibia, repti les, bìrds, and mammals the neural plate forms as
an ectodermal thickening overlyíng the cho rda -mesode rm, "nO
fold, to form
a tube. At caudal levels, however, development of the cord ís not so uníform.
l'lost work on neurulation has used amphibia, though studies on anurans
(with a bilaminar neural plate) are not str¡ctly comparable to other groups
(schroeder, t 97o) .
With the advent of electron microscopy, investigations of lgglglg1lq
,
have revealed. the existence of intracellulêr structures involved in neural
closure. Previous work attempted to explain neurulatlon în terms of extr¡ns¡c
forces, or by changes secondary to cell division and migrat.ion.
Hls (187q) suggested that the change ln shape of neuroeplthel ial cells,
from cuboidal to columnar, might be due to cell compression after a period

350
of rapid ectodermal mitosis. However Gillette (1944) found a reduction
in the sl2e of. neural plate cêlls in Amblystoma during folding. Glaser ('l9.l4¡
19l6) sugsested that neuroepithel iar ceüs change from cuboidar to corumnar
and then to pyramidal shapes, due tc differential uptake of water at the
basal parts of the ce s. This was shown to be unrikely by the detect¡on
of only negliglble changes in the denslty of ,,uur"t plate cells during folding
ln Rana and Amblystoma (Brown et al., l94l), and by Gillettés finding of a
reduction ln cerr size. Derrick (1937) reported a hígher mitotic index in
neurectoderm than ín adjacent ectoderm of chick embryos during neururation,
but Bragg (1938) courd not detect a simirar differentiar mitosís in Bufo.
clllette (1944) and Holtfreter (1943) postulated a contractile surface
coat at the free ends of neuroepither iar ceus, responsibre for changes rn
cel I adhesion and shape.
ln a detai red analysis of neururation ín the bilaminar neurar prate of
xenopus, Schroeder (1970) described myotome erevation and epidermar expansíon,
as well as changes of shape in both layers of neuroepithel ial cells. Similar
changes have not been detected in the uniraminar neurâr prêtes of other
vertebrêtes, whích continue to show fording and crosure when cur tured rn ¡solatlon
(Roux, 1885; Boerema, 1g2Ð. The assertion by C. O. Jacobson (1962)
that neural elevation and folding in the Axolotl is produced
by forces generated in the underrying chorda-mesoderm has now been refuted by
Karfunkel and Burnslde independently (Karfunkel , 197Ð. Thus intracellular
mechanisms must be capable of generatlng the forces needed for neurar closure.
I'licrotubules running rn the long axis of neuroepither iar celrs have
been reported in embryos of Triturus (Waddington and perry, 1966¡ Burnside,

351
t971), Gal lus and Xenopus (l'lessier, 1969; Kêrfunkel , 1971). These nicrotubules
are dlsrupted by vînblastine and colchicine (Karfunkel, 1971; 1972) ,
w¡th arrest of neurulation and flattening of neuroepÌthel ial cells. Burnside
(197t) foun¿ that the m¡crotubules were distributed obl iquely, suggesting
that they might produce cell elongatÎon by displacing cytoplasm towards
the expanding cell bases, rêther than by'direét elongation.
A system of contractíle microfilâments has also been described in
the apical cytoplasm of neuroepithel ial cells in Hyla and Xenopus (Baker
and Schroeder, 1967) and in Rana, Ambìystoma, and Gallus (Schroeder, 1969)-'
Neurulatîon is associated with a short períod of contrêction in these microfl
laments. Disruptìon of the microfilaments in neuroepitheliaì¡ cells of
the chick embryo, (by vinblastine or cytochâlasin B), inhibits neurulation,
v,rt th loss of apical wedging but no reduction in columnar heíght of the
neural plate cells (Karfunkel, 1971 ; 1972)'
Ambellan (1955; l95B; 1962) showed that treatment of frog neurulae
with A.T.P., A.D.P., and A.l'1.P. - 3 caused accelerated n"ut.ul"tion, in direct
proportion to the number of phosphate groups in the nucleotides' Microfi
lament contraction may be ca I c i um-dependen t (Gingell , 1970, ì,tessels 1971).
It would be of great înterest to examine the uìtrastructural changes in
the neural tis'sue of chick embryos after windowing.
ln wîndowed embryos with hemimyel ia, the neural plate was absent
caudal ly. As only five embryos were examined histologically ît was not
clear whether this couìd be attributed to a faîlure of neural induction at
the caudal level, or to necrosîs of presumptive neurectodermal cells'
ln amphibîan embryos, differentiation of neural epithel ium resul ts
from the actlon of t¡ssues ín the archenteric roof on overlyÎng ectoderm
(Spemann, 1938). Experîmentally, however, neural ijìduction can be produced
by: larval or adult neural tissue; certain other tissues such as kidney,

352
liver, and muscle (but not gut or skin); and even by chemical agents.
ln ãrnnlotes, the archenteron is either absent or much reduced; ln
birds neurål ìnduction appears to be related to the activity of Hensenrs
node and the primitîve streak. Neural plate first forms in the chick
embryo in the future braln region duri.ng. rnid-gastrulat¡on, and different¡ation
continues as the.node moves posteriorly with streak regression
(Hamilton, 1952). After formation of the neural tube, the anterior
region expands to form the braîn, and the poster¡or part lengthens to
form the cord. Hunt (.|931) and t/addington (1932) demonsrrôted the
ablllty of Hensenrs node to induce axlal formation at the def¡nítive
streak stage, but restrictíon of this abil ity at Iater stages, VJaddîngton
(1fi2) showed that a transplanted node can st¡lI produce axial
lnductlon, while Shoger (1960) found that disaggregated. node tissue retains
Its lnductíve abllity until the early somite stage. Crabowski (1957)
suggested that the node acts as a regîonal organizer during streak
regress lon.
Tissue induction can be achieved across a millipore fílter with
pores down to 0.8 microns (Grobs¡ein, 1!!J), suggesting the êction of some
diffusible material. Niu and Twírty (1953) found that inductor tissues
in uitro released materials into the culture medíum, capable of promoting
differentiation of neurones and melanophores in smal I fragment. áf u*-
planted ectoderm.
lnvestlgations of the nature of the prímary organizer have proved
inconclusive. ln some experlments R.N.A.rs from different sources have
produced specìfîc promotion of notochord, neural tlssue, kidney, and
heart (Hillman and Niu, .l963; Sanyal and Niu, 1966). Other workers report
neural 'tnCuctlon wlth D.N.A. plus A.T.P., but not wîth R.N.A. (Butros,

353
1962i 1965). Barth and Barth.(1974) susgest that inducing ðgents may
act by aliering the propertles of cell membranes in the reactî.ng tìssue,
to promote red¡str¡bution of intracellular ions.
Fai lure of înductiori might, occur byl restriction of contêct
between the two interacting t¡ssues; a defect in the înductor; incompetence
of the reacting tissue; or imperfect tíming of the contact
between the two t¡ssue components (Saxá, 1975).
Cell death occurs as a normal phenomenon in many embryoníc processes'
part¡cuìarly those involvîng morphogenesis or regression (Glucksmann, 1951) '
Regressive phases ln embryogenesis are programmed to occur in a specifíc
sequence that suggests genetic control. Mutant genes cên enhance or
reduce the normal patterns of celì death (Saunders, 1966) ' Experimental
treatments such as x-rays' drugs, viruses, hormones, vitamins, and hypoxla
can promote cell death (l4enkes et al. 1970); Janus green can Prevent the
normal cell death in the interdÎgital clefts of the chíck foot (Saunders,
r966).
Necrobiosis in the neural tube shows peaks preceding neural groove
formatíon, foldîn9, fusion, and separatíon îrom surface ectoderm
(Glucksmann, l95l). Käl lén (1955) des.cribed another peak of cell deaths
ln the rabbí t brain, associated with a period of ce'l I differentîatíon at
14 days, Rokos et ê1. (1976) described cell deaths in mesoderm,'heart,
gut, and neural plate tissues of rat embryos after maternal injection of
trypan blue; they suggested that th¡s was an exaggerated form of the nor-
¡na I cell death seen in early neurogenesis.
Cell death is accompanled at early embryonic stêges by a ttj!h-regu-.
.làtli¿è àbititv, which is responsîble for rapid lncorporation of grafts

354
(Rose,nqu i s t,l !66) , and rest¡tution of excísed areas (Criley, '|969). The
dramatlc regulat¡on shown by.the nervous system of amphibian embryos
(Harrlson, 1947) ìs not so pronounced in the chìck. However, încísÌons
ln the roof plate of the chick neural tube close spontaneously when
local lzed, and fuse to.ep¡dermis in 2-4 hours when more extenslve (Rokos
and Knowles, l!/6). Lendon (t975) and Rokos ér al. (1976) found that rhe
extensive blebs produced ln early rêt embryos by naternal trypan
blue lnjectlon later resolved. ln the present windowed chick embryos,
superflcîal cells of the open neural plate ln early myeloschisis showed
necrosls, whlch was not present at later ståges. Embryos with myelodysplasia
showed extensive degenerative changes ín mesoderm, but ít is
not clear whether these accompanied or caused the neural defects.
Stockardrs prlnciples of teratogenesis proposed that: malformations
ln different ,o""ffilar
agentsi a given defect
in one specíes may result from a wide range of treatments; the in¡tial
act¡on of a teratogenîc agent is to retard the rate of development; and
the type of defect is determined by the developmental stage ât wh¡ch
the embryo was treated (Stockard, 1!21). ore recent work has shown that
these prlncíples do not make sufficíent allowance for: species differences ;
agent specifièity¡ dosage of the agent; the metabolic pathways of the agent;
and the nêture of the embryological process involved.
lllth more detai led knowledge of embryological mechanisms it is no¡¿
clear that the importance of developmental êrrest was overemphasized by
Stockard. lndeed there are some malformation that arise by excessive
growth or excessive resorption (Patten, 1957). The principle that remêîns
most valid ls the împortance of tlming (Hughes, 1976).

The result of any teratogen¡c insult depends on the site of action
by the agent, and the developmenta¡ stage of the embryo. Thís appl les
to both the expression of genes, and the action of environmentâl agents
(saxá, t976).
ln general, the first period of embryonic development (up to the
formation of germ layers) shows little tendenöy to malfcrmations, with
embryonic death at high dose levels. lJîth the onset of morphogenesîs,
the embryo become¡ very suscept¡ble to teratogenlc influences, resultíng
ln major malformatlons. 0n reaching the fetal stêge, only structures
still undergoing differentlatlon (such as the braln, palate, and major
vessels) are stlll susceptlble. to abnormal development (falter, l968).
ln mammals, there ls the additional problem of whether an êgent
acts directly on the embryo or indírectty through the placenta, as
exempl ifíed by the activity of trypan blue. For experimenta¡ teratology,
the use of physical agents provîdes accurate control of timing and dosage,
though some effects may be secondary to tissue damage or alteration of
fiorphogenet i c processes.
X-ray treatment has revealed the sequence of crÌtical periods ín
development of the rat(.:lob,et €jL, 19352 Kàven, l!J8; Hicks,.t954), the mouse
(Russel l, 1950; 1956; r,rilson, .|954; Hícks, 1954), and the chick (Reyss-Brion,
1956; Hadj I tsky,l!62;¡1¡ rrmann and t/olff,1964). Hicks (1954, 1954) found
that irradíation of pregnant rats: at 9 days produced an open brain; at
l0 days produced forebrain, hindbraín, cord, and facial defects; at ll
days produced hydrocephalus, with braínstem and cord dèfects; êt l2 days
caused reductÌons of the brain and eyes; and after thls reduced the size
of fiber tracts. Hicks found that embryos showed a high regulative ability
before the onset of d i ffe ren t i a t i on, so that extensive t¡ssuè damage might
355

356
resu¡t ¡n only minor defects a few days later. He concluded that the
nature of. the defect was related to the time of tréatment, wh¡le the extent
of the defect was rerated to the dose. After irradlation of chíck embryos
Hadjiîsky UgAù: at O hours produced brain and eye defects; êt 22 hcurs
produced nicrocephaly, mîcrophthalml", op"n cord defects; "nd
at 4g-96 hours
obtaîned limb defects; and at r68 hours found'rocar ized dîgltar cefects.
Kirrmann and t/olff(1964) after local îzed irradiation of chick embryos concluded
that: undîfferentiated cells are the rnos t sens¡tve to a teratogen¡c
ðgent; t¡ssue damage during morphogenesis does not suppress dîfferentiatign,
whlch goes on to produce an abnormar organ; and the parts of the earry embryo
have cons î derab le autonomy.
The importance of timîng and dosage has been confirmed by experiments
wlth other physícal âgents, such as: hypoxia in mice (Murkami and Kameyáma,
'|963) and in chícks (Gar rera, r95r); urtrasound in avian embryos (Lutz et ar.,
1955; Lutz and Lutz - Osterag, 1957); ultraviolet lîght in the chick embryo
(Hinrlchs, 1j2J; Ðavis, 1942; 19\4); and hyperthermîa in the chick (Deuchar,
1952) and rhe hamster (Kilham and Fërm, l9i6).
Despite the uncertainty of maternal metabolism and placental trênsport
in mammals, the action of chemical agen ts also depends on the timing of maternal
treatment.' ln rats, treatment on days 7-10 of gestâtion has produced
exencephaly, spina bîfida, hydrocephalus and other defects: with trypan blue
(i,/arkany et al., .l958i Lendon, 1968i 197Ð: wirh sal ícylates (Warkany and
Takacs, 1959): and with hypervitaminosis A (Giroud and l4artinet, 1957;
Langman and t{elch, l!66). ln hamsters hypervitaminosis A, dimethyl sulfoxide,
and sodium arsenate have all produced exencephaly after maternal
înjectlon on the 8th day of gestation (Marin-padi I la and Ferm, l!6!; Ferm,
1!66; Harln-Padilla, 1966: Ferm and Carpenter, l968).

357
Uindowîlg at ear¡y stages of avian development can be regarded
as another physícal teratogenÌc procedure. The standard windowíng technic
at 26-30 hours produced a high incldence of mortal ity and marformatíons.
This effect was almost abol ished by obliteration of the introduced air
space, if performed i mmed îa te I y.
The teratogenlc actlon of windowing at êarly stages of development
has been reported by Ancel (19\6-47 t '1956),Hinsch and Hami lton (1956),
l4cCalllon ånd Clarke (1gSÐ, ,"nn et ê1. (i973). ln many publ îcations
"nO
no attempt to obl iterate the a¡r space is reported, so thât any nalformations
recorded mlght be caused by windowing as well as by the agents
emp I oyed .
ln the presen, "rp.rlr"ntr,
embryos at the t¡me of treêtment ranged
from Stage 5 to Stage 10. The effect of wîndowing is not confined to
a short period like other physical agents, though formation of the amnîon
by Stage 1B protects the embryo after about 60 hours. Some defects (such
as neural dysraphism, eye defects, and trunk cysts) can be attrÍbuted to
an early effect; others (such as rumplessness, ectop¡a viscerum, and limb
defects) arise later. A¡though windowing exposes the dorsal surface of
the chick embryo, and neural defects originate dorsally, other defects
(such as ecto¡iía viscerum) involve ventral structures.
ln the neural tube, four degrees of involvement resulted from
windowing. l4any embryos developed quite normally. Embryos with early
myeloschisis showed necrosis ín the superflcial cells of the neural plate,
which later resolved. Hyelodysplasia, due to absence of neural plate
materîal,was comblned with mesodermal cysts and hemorrhages. The most
severely affected embryos (not examined histological ly),showed early death
associated wlth open neural defects, severe trunk dlstorsion, reduced

358
braln and cord volume, and often extensive cysts.
Wlndowlng at 26^30 hours, like other teratogenic lnsults, thus
appears to have eîther a moderate or a severe effect on the chick embryo.
This gradation of response enables myeloschisis and myerodysprasia to be
distlnguished as dlstinct pêthological processes, each of which requires
further I nvest igat lon.

SUHHARY AND CONCLUS I ONS

360
I sutlt'lARY ANp coNcLUsto]s
1. The slmple pþysical procedure of wîndowlng.e.ggs at early stages of
lncubatlon (with removal of 2 ml . of albumen) proved to be highly teratogen
I c.
2. Wlndowing at 14 hours caused a very high earìy mortal ¡ty, w¡th severe
malformations ln the survlvlng embryos. fr."ar"na at 26 hours produced a
lower nprtal lty, wlth a hÍgh incidence of defects (predominantly involving
the nervous system). Exposure at 38 hours was much less teratogen¡c.
3. Remova I of the lntroduced alr space, by reexpansion of the aír-cell
or by the.addltlon of albumen or F 12 medlum, almost abol ished the teratogenlc
effect of windowing, îf pêrformed immediately.
4. The lncidence of malformations produced by windowing at 26-30 hours
increased with extended periods of incubation. Open brain and cord defects,
nlcrocephaly, eye defects and trunk and rump cysts were present by 3 days.
Facial defects and rumplessness appeared by ! days, but ectopia viscerum
and limb defects were not promingnt unt¡l after 5 days.
5. Examination of skeletal defects at 12 dêys showed that vertebral
lesions varîed ín severity according to the region. Spina bifida occulta
occurred largely in the cervical and upper thoracic regions. Spina bifida
manifesta was seen between the lower thorac¡c and sacral regions. Vertebral
irregularities and deletíons were almost confined to the caudal region.
6. open braln defects occurred at every Stage after the expected closure
of the anterior neuropore, suggesting that they arose by non-closure.
7. open cord defects were of two distinct types.
8. Hyeloschlsis was preceded by a characteristic triå.ngular shape of
the rhombold sînus. Serial sectlons revealed regular open defects, wlth

361
separatlon between the neural plate and taîl-bud sources of neural
t¡ssue, but coirtinuity of the neural plate ¡nto the caudal region.
These findings lndicate that myeloschisis arises by non-closure of the
neural folds. îhe establ ishment of myeloschisis was fol lowed by local
separatlon of the notochord from the open area of neural tube, but not
by overgrowth of neural t¡ssue.
9. lilye I odysp I as I a appeared at about the time of expected closure of
the rhomboÌd sinus. Serial sections revealed irregular open defects,
wîth complete absence of neural plate material and formation of the
cord tlssue from tal l-bud materlal alone. The lesions were accompanied
by extensive cyst¡c and hemorrhagic changes in local mesoderm, with
reduction ln somite volume. The¡:e was no assoc.¡êtecL notochordal separêtíon,
but the volume of neural tls.sue was greatly reduced.
10. lllndowing can be compared to other physical terêtogenic agents,
whose effects depend on timing and dosage. The hígh incide-nce of neural
defects was the result of treatment at the. perîod of axis formation and
neurulatíon. Depending on the degree of embryonîc involvement,wíndowing
produced two different types of open cord defects - myeloschisis and
mye I odys p I as ì a.
11. Because of the símílar development of neural tube f rom neural plate
and tall-bud materials, with an overlap zone showîng cavitation and fusion,
the chlck embryo provídes a good model for experimental investigatlon of
neural dysraphîsm ln man.

APPEND I CES
362

363
APPENDIX A
Preparatlon of Early Chlck Embryos for Serial Sectîoning.
1. Tlp contents of egg lnto a dlsh of warm Howardrs chick saline.
2, Cut vitelline r¡ernbrane around the equator of the yolk and peel
membrane and blastoderm off the yolk.
3. Transfer to another dlsh of Howardts sal ìne, remove vitell íne
membrane wlth flne forceps, and pipette off most of the saline to flatten
the embryo ln a thln fllm of saline (wîth ventral surface facing upwards),
4. Add fresh Howardrs sallne dropwlse to ventral surface of the embryo'
to wâsh off the yolk,
5. Remove salîne and add Boúints fíxative dropwise to wash off remainlng
yolk partlcles and fix the embryo. Hold the dish at an angle to prevent
the embryo floating lnto a deep pool of fixatíve and then curl ing at
the edges.
6. Remove yolk - laden fixative and add just enough fixative to cover
the embryo but prevent curling (for 15 mins.).
7. l{ith a section-ll fter transfer to a fresh dish of Bouin's fluid,
cover with a disc of filter paper to prevent curlîng, and leave for
several hou rs..
8. Decolorize with several changes of 70? alcohol containing 2?
amrhon¡a (for several hours each).
9. Leave ln 702 alcohol overnight, and examine for vísîble defects.
Draw embryo with camera lucida.
lO. Sta¡n with saturated eosln-bluish in 702 alcohol (to improve
vlslbll Ìty after embeddi.ng),for several hours.
1t. llash briefly with 709 alcohol and separate the embryo from area
vasculosa wlth a cork - borer.

364
1i. Dehydrate with changes of 802, 90? and 95? alcohols for t0 nins.
each.
13. Take embryo and a smal I pencil-wrìtten label through amyl acetate
for 10 mlns.
14. Take embryo and label through three changes of hot wax, for: 10 mins.
each.
15. Embed enbryo in fresh wax in a plastîc capsule, with ventral surface
facing upwards, trunk parallel to long axis of capsule, and label at taíl
end of emb ryo
16. Cool capsule rapîdly and leave overnight ín fridge.
17. Remove plastîc capsu'le and trim wax around embryo with a razor blâde
until a very smal I segment containing the embryo is left with a buttress of
wax beh î nd it.
18. Cut in as long a ribbon as possible, trimming the buttress of wêx
behind the embryo several times during the cutting process.

365
APPENDIX B
Stainl.ng of Carti laginous Skeleton ât 11-12 Days.
l. Flx for 48 hours in a mixture of:
608 absol ute ethyl alcohol
303 ch I oroform
101 glaclal acetlc acid.
2. Overstaln wlth 0.052 alcían blue ín a solutîon of /02 alcohol ,
containing 5? acetíc acld, for 12 hours.
3. Destain wi th 702 alcohol , containing 5% acetic acid, for 48 hours
(using several changes of solution).
4. Dehydrate in 90? and 100? alcohol for 12 hours.
5. Pass. through xylol and clear fully in benzyl benzoate.
NB, Embryos are still hard enough at the end of th¡s þrocess to take
back into absolute alcohol, and section with a hand-held razor blade.

366
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