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. 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
Page 1 and 2: E}4BRYOGENES IS OF EXPERIHENTALLY I
Page 3: MY PARENTS c.J.H. G. M.
Page 7 and 8: TABLE OF CONTENTS SECT ION PAGE 1 I
Page 9 and 10: },I.ATE R I ALS 3.1 THE CHICK EI4BR
Page 11 and 12: vt I I 6.3.2 Stage 10 Experímentel
Page 13 and 14: 9 APPENDTCES 378 APPENDIX A . 379 1
Page 15 and 16: 1.1 TERATOLOGY Birth defects have a
Page 17 and 18: 4 of the complexity of normal devel
Page 19 and 20: 6 flat, exposed plaque of neural ti
Page 21 and 22: 8 However, several early embryos in
Page 24 and 25: .1- 5
Page 26 and 27: l2 2.1 ErI0L0rL!! fïE qYs34t!1!!jr
Page 28 and 29: 4 which has rema¡ned fairly consta
Page 30 and 31: 6 and about 112 after two (Laure,nc
Page 32 and 33: l8 ln some very early human embryos
Page 35 and 36: 2l and kinky-ta¡l as disorders of
Page 37 and 38: 23 Vogel and McClenahan (1952) tnus
Page 39 and 40: I'IATE R IALS 25
Page 41 and 42: 27 lf necessary, eggs were stored a
Page 43: FÍg. 5. lncubator allowing precise
Page 46 and 47: 3l 4. r sELEcr I oN !F EGGq 4.1,1 .
Page 48 and 49: uler. Accurate Staging (Hamburger a
Page 50 and 51: .\lz '?. t
Page 52 and 53: Fis. I AsB, Windowìng followed by
Page 55 and 56:
Fis. 9 Aã8. t/indow?ng followed by
Page 57 and 58:
42 ( \--l /) ^ ) t0
Page 59 and 60:
44 serîal sectlonlng a camera luci
Page 61 and 62:
46 Thus the embryos selected for se
Page 63 and 64:
48 TABLE 3. srAGE t0 El,tBRyos (cno
Page 65 and 66:
50 TABLE 5 , Reg i ons srAGE 13:16'
Page 67 and 68:
52 4.8.4 HistologÌcal_De:plþtÍon
Page 69 and 70:
5 RESULTS OF TERAÎOLOGICAL PROCEDU
Page 71 and 72:
(c) deaths and defects in the survi
Page 73 and 74:
Fi g. Percentagcs of early deaths a
Page 75 and 76:
6o MoRTALtTY, AFTER ì,'tNpowtNq,AT
Page 77 and 78:
TABLE B.'DEVEIOPI{ENT'AFTER'WINDOIi
Page 79 and 80:
TABLE 9. ¡4ALFORMATIONS PRODUCED'B
Page 81 and 82:
N=231 ffil e ooyi ffi s oays [il t2
Page 83 and 84:
6B TABLEt loj lloRTALtTy AFTER VtBR
Page 85 and 86:
70 5,3.2 Pa-rôf i Idì ¿irid PIè
Page 87 and 88:
TABLE 13; MALFORI4ATIONS AFTER PLAS
Page 89 and 90:
41 13 0 23 No l^l i ndow Control s
Page 91 and 92:
Fíg. Percentages of early deaths a
Page 93 and 94:
78 ALBUMEN INTRODUCTION Analysís o
Page 95 and 96:
0 1 0 0 a o TABLE 17. I'lALF0RÌ1AT
Page 97 and 98:
N Eorly Deothi N=10ó N a ú, o d,
Page 99 and 100:
103 51 52 28 14 4 6 No W î ndow Co
Page 101 and 102:
Fi g. 14. Percentages of early deat
Page 103 and 104:
88 AtR qELL, REEXPANSI0N Analysls o
Page 105 and 106:
90 5.4 BACTERt4L CUTTURE 0F UlNpohr
Page 107 and 108:
92 TABLE 22. ¡¡UI'ISELTS OF PLATE
Page 109 and 110:
94 6.1 EMBRY0ûIIES I s 0L 0!!\- NE
Page 111 and 112:
Fis. I5 A¿8, Range of s izes (mm.)
Page 113 and 114:
Figs. 16 AaB.. Range of sizes (mm.
Page 115 and 116:
100 6.1.2 f4ortal í!y with Vsrying
Page 117 and 118:
421.. 26 25 I (3.85) 20 (76 .92) 1
Page 119 and 120:
00 N Eorly Deoths ,n -¡ F- ô- xu.
Page 121 and 122:
ì06 Degrees of Freedom 12 Chi Squa
Page 123 and 124:
hzE 53 42C z5 51(96.4)2\(96) 2(3.77
Page 125 and 126:
ffil Op"n Anterior Neuropore I I0 N
Page 127 and 128:
N=380 El Ovol Rhonnboid 'Sinus ì l
Page 129 and 130:
Øl Open Broin Defecls N=384 ffi F"
Page 131 and 132:
ligr. 21 - 30. Camera lucida drawin
Page 133 and 134:
Fi s. 22. lrregular open anteríor
Page 135 and 136:
lmm OPEN BRAIN DEFECT Á,NOPHTHALMI
Page 137 and 138:
lmm OVAL RHOMBOID S¡NUS 48 HOURS S
Page 139 and 140:
FiS. 26, Open neural folds extendin
Page 141 and 142:
t-l lmm IATER OPEN CORD DEFECT . RE
Page 143 and 144:
l2B t--l lmm EARLY OPEN CORD DEFECT
Page 145 and 146:
Fis. 30. Large caudal cyst Ìn an o
Page 147 and 148:
132 6.1 .4 DeveloÞñent of oÞen N
Page 149 and 150:
Fig. 31 . Percentêges of experinen
Page 151 and 152:
36 TABLE ?8. RH0I4B0lD stNUs ctôs
Page 153 and 154:
N=38o H ovol Rhomboid sinus l^ r38
Page 155 and 156:
F¡ S. 33. Percentages Õf open cor
Page 157 and 158:
142 6.1.5 Distríbution of 0pen Cor
Page 159 and 160:
Fi s. "l! Percentage distrìbution
Page 161 and 162:
lrregul ar (Z) somi te be low level
Page 163 and 164:
ì48 N=2ó4 ffi m Regulor Cord Defe
Page 165 and 166:
Fis. 36. Percentage distrîbution o
Page 167 and 168:
152 6.2 sPtIA! LFVELS 0F oPEN CORp
Page 169 and 170:
Fìgs. 37'38, Vertebral defecrs in
Page 171 and 172:
CERVICAT THORACIC LUMBAR FUSED CAUD
Page 173 and 174:
---l r r'I * 40
Page 175 and 176:
t59 a) spîna bif¡da occulta is se
Page 177 and 178:
:al . '**Ç- 4 ff, À(,) ftø 9-' o
Page 179 and 180:
162 lmnediately after closure, sepa
Page 181 and 182:
164 Hensenrs node and the primitive
Page 183 and 184:
-l :: l 48 I 49 i :. t: .lì 50 ì:
Page 185 and 186:
t67 somite area (through asymmetry
Page 187 and 188:
t69 ln both embryos the volume of n
Page 189 and 190:
171 The rhomblc roof became membran
Page 191 and 192:
173 At areas of myeloschisîs conta
Page 193 and 194:
175 neurêl tîssue wíth mesoderm
Page 195 and 196:
t77 e) encroachment of somitîc mes
Page 197 and 198:
ôt rf) , ,,. ì _ ^iiir¡:ù\. I r
Page 199 and 200:
l 55 56 57 58 59 ó0
Page 202 and 203:
Fiss. 65 Early myeloschisis in a St
Page 204 and 205:
fiSr. 71 - 76. Later myeloschisis i
Page 206 and 207:
Fiss. 77 -82. Early myelodysplesia
Page 208 and 209:
FÌgs. B3 - BB. Later myelodysplas
Page 210 and 211:
Figs, 89 - 91. Processíng artifact
Page 212 and 213:
ì86 6.3.10 SequenJ¡¿il ll lúSti
Page 214 and 215:
Fígs. 95 - 102, Sequentiêl drawin
Page 216 and 217:
l9o EVERsToN: srAGE rf (o r o+) o @
Page 218 and 219:
@ @@ "@ @ e q q a a 192 CoNTROL: ST
Page 220 and 221:
MYELOSCHTSTS: STAGE tZ (tZeïl OOG
Page 222 and 223:
coNTRO[: STAGE tó (ro c zz) . a@ Q
Page 224 and 225:
G (õ--==: \z-- HEMTMYELIA: STAGE t
Page 226 and 227:
Som i te Defects contact none sePa
Page 228 and 229:
Somite Som i te Contact Defects N'
Page 230 and 231:
Som i te Contact Som i te Defects c
Page 232 and 233:
none none N) o o\ B r8c 23 12 good
Page 234 and 235:
separat ion none contact/ none sepa
Page 236 and 237:
Regíon Embryo Stage Conditîon Neu
Page 238 and 239:
none none none none none none l'.J
Page 240 and 241:
18E 10 13- vëry poor closed 0 cove
Page 242 and 243:
separãtion none contact none conta
Page 244 and 245:
Regíon Embryo Stage Condition Neur
Page 246 and 247:
separation none sepâ ra t îon non
Page 248 and 249:
separâtion none sepa ra t ion sepa
Page 250 and 251:
Reglon Embryo Stagd cond¡t¡on Neu
Page 252 and 253:
none none none none N) NJ .{ A A A
Page 254 and 255:
contact/ none separatlon contact no
Page 256 and 257:
Region Embryo Stage Conditlon Neura
Page 258 and 259:
TABLE 38D. STAOE 17.20 CONTROL AND
Page 260 and 261:
Reglon Embryo Stage Condition Neura
Page 262 and 263:
428 72 20 closed covered none conta
Page 264 and 265:
239 neural tissue ât the later sta
Page 266 and 267:
241 The control group (Table 99 ) s
Page 268 and 269:
TABLE 39. APPEARANCE OF I,'HOLE EI4
Page 270 and 271:
TABLE 40. APPEARANCE OF }/HOLE EI"I
Page 272 and 273:
closed closed c I osed c I osed c I
Page 274 and 275:
249 6.6 pEVEL0PMENT 0F rH!_¡Ho|4gl
Page 276 and 277:
Emb ryo Stage Rhombîc Roóf Neural
Page 278 and 279:
CDE D'E DE CDE DE DE DE D BCD BC BC
Page 280 and 281:
TABLE q6,. STAGE 17-20 CoNTRoI AND
Page 282 and 283:
Figs- 10J - i11. Development of the
Page 284 and 285:
Fî s. tub. Thick rhombic roof in 5
Page 286 and 287:
Fig. 109 Hernbranous rhombic roof w
Page 288 and 289:
260 6.7 HrqroLoctcAL, cHANgË Assoc
Page 290 and 291:
TABLE 47. CONTROL EMBRYoS.HISTOLOGI
Page 292 and 293:
0 0 0 0 0 0 0 0 0 0 o 0 0 0 0 0 0 N
Page 294 and 295:
42c 21 zo B c D E 0000 00 00 00 1\.
Page 296 and 297:
l\t q\ 1oE 26 16 30E 59 16 3oE 77 1
Page 298 and 299:
0 0 0 0 0 0 0 0 0 0 0 0 N) .{ Embry
Page 300 and 301:
l\) \l N D E 00 00 0 0 13.79 0 100
Page 302 and 303:
N) \¡ \2E 72 20 E B c D' E 00 0 15
Page 304 and 305:
46. 15 76.92 0 20.51 14.83 2i,92 53
Page 306 and 307:
ECTODERM DISCONTINUITY IN EXPERIMEN
Page 308 and 309:
279 ECTODERM DISCONTINUITY IN EXPER
Page 310 and 311:
SOMITE SEPARATION IN EXPERIMENTAL E
Page 312 and 313:
283 NOTOCHORD SEPARATION IN EXPERIM
Page 314 and 315:
285 SOMITE DEFECTS IN EXPERIMENTAL
Page 316 and 317:
287 and distr¡bfrtíon in the cont
Page 318 and 319:
19.09 r8.95 r 1.84 14.82 15.20 0 17
Page 320 and 321:
0 15. 89 81 .08 100 100 ' 76.92 0 1
Page 322 and 323:
43. rB 100 100 0 12.53 74.07 r00 't
Page 324 and 325:
7.t6 16.03 100 100 160 7.90 18.22 r
Page 326 and 327:
Figs. 120'123. Percentage ie.ngths
Page 328 and 329:
299 O\TERLAP ZONE IN EXPERIMENTAL E
Page 330 and 331:
301 OVERLAP ZONE IN EXPERIMENTAL EM
Page 332 and 333:
303 ând every tenth sectlon was ne
Page 334 and 335:
30c 12 16 c 17707,61 2463.3\ 7.19 D
Page 336 and 337:
Ratio NT/N 4 .90 4.11 6.17 7.14 6.9
Page 338 and 339:
Rêtlo NT/N 4.go \.36 3 .44 3. 18 3
Page 340 and 341:
6.7t 6.lt 4.63 4.60 4.13 3.48 hzE 5
Page 342 and 343:
Fi9s, 124-127, Neurál tube-norocho
Page 344 and 345:
315 NEURAL TUBE -NOTOCHORD RATIOS I
Page 346 and 347:
317 NEURAL TUBE-NOTOCHORD RATIOS IN
Page 348 and 349:
319 TABLE 598 ' ANALYSIS OF VARIANC
Page 350 and 351:
321 TABLE 61., .NEURAL TISSUE/NOTOC
Page 352 and 353:
323 Apart f.rom the sîgnîficant d
Page 354 and 355:
t25 7. Dlscusst0N 1'/i th progressi
Page 356 and 357:
327 C¡osure of the anterior neurop
Page 358 and 359:
329 the mid-polnts of the defects w
Page 360 and 361:
331 'c¡osure of the neural folds w
Page 362 and 363:
333 . These findings s.ugges r that
Page 364 and 365:
335 Lendon (1968, 1975) and notos (
Page 366 and 367:
337 Ferm, 1965; l"larin-Padi r ¡a,
Page 368 and 369:
339 Kôplan (1965) and Kaplan and G
Page 370 and 371:
341 reach the floor-plate; asymmetr
Page 372 and 373:
343 took up the label, shovri.ng th
Page 374 and 375:
345 The defects in early human embr
Page 376 and 377:
347 after pred¡cted closure (Lemî
Page 378 and 379:
349 hours could provide evidence fo
Page 380 and 381:
351 t971), Gal lus and Xenopus (l'l
Page 382 and 383:
353 1962i 1965). Barth and Barth.(1
Page 384 and 385:
The result of any teratogen¡c insu
Page 386 and 387:
357 Uindowîlg at ear¡y stages of
Page 388 and 389:
SUHHARY AND CONCLUS I ONS
Page 390 and 391:
361 separatlon between the neural p
Page 392 and 393:
363 APPENDIX A Preparatlon of Early
Page 394 and 395:
365 APPENDIX B Stainl.ng of Carti l
Page 396 and 397:
367 10, BIBLIOGRAPHY, Alter, l.,l.
Page 398 and 399:
369 Braun, l'{. (1882). entwicklung
Page 400 and 401:
371 Crlley, B,B.(1969). Analysîs o
Page 402 and 403:
373 dqs anomal ies de lrorganizatio
Page 404 and 405:
375 Heath, H.D., Shear, H.H., lm€
Page 406 and 407:
377 Jel inek, R. (1960). Developmen
Page 408 and 409:
379 Laurence, K.14. (1964). Thé na
Page 410 and 411:
381 McKeown, T. and Record, R.G.(19
Page 412 and 413:
Penrose, L. S.(1957). Genetics of a
Page 414 and 415:
385 Russell, H.E. and A¡ tken' G.T
Page 416 and 417:
387 Spemann, H. (1938). Embryonic D
Page 418 and 419:
389 lJeed, ¡.H. (1937 -38'). M.nin
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