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Clin Genet 2005: 67: 143–153 Copyright # Blackwell Munksgaard 2005
Printed in Singapore. All rights reserved
CLINICAL GENETICS
doi: 10.1111/j.1399-0004.2005.00404.x
Developmental Biology
Section Editor:
Jacques Michaud, e-mail: jacques.michaud@recherche-ste-justine.qc.ca
The developing limb and the control of the
number of digits
Talamillo A, Bastida MF, Fernandez-Teran M, Ros MA.
The developing limb and the control of the number of digits.
Clin Genet 2005: 67: 143–153. # Blackwell Munksgaard, 2005
Congenital malformations of the limbs are among the most
frequent congenital anomalies found in humans, and they preferentially
affect the distal part – the hand or foot. The presence of extradigits, a
condition called polydactyly, is the most common limb deformity of the
human hand and is the consequence of disturbances in the normal
program of limb development. However, despite the extensive use of the
developing limb as a classical developmental model, the cellular and
genetic mechanisms that control the number and identity of the digits
are not completely understood. The aim of this review is to introduce the
reader to the current state of knowledge in limb development and to
provide the necessary background for an understanding of how
deviations from the normal developmental program may lead to
polydactyly.
The morphology of the human hand is of extraordinary
importance not only because it is an
excellent tool to manipulate the environment
but also because it is at the root of the evolutionary
progress of our species. The design of the
human limb follows the basic plan of the pentadactylous
tetrapod limb. Three segments can be
readily differentiated: (i) the proximal segment
called the stylopodium, which contains a single
skeletal element (humerus/femur), (ii) the intermediate
segment called the zeugopodium, which
contains two skeletal elements (radius–ulna/
tibia–fibula), and (iii) the distal segment called
the autopodium, containing the numerous skeletal
elements of the hand or foot (Fig. 2b).
Between the zeugopod and the autopod, another
segment can be distinguished that corresponds
to the carpus/tarsus and is called the mesopodium.
In the autopodium, the digits are numbered
according to their position, from anterior
to posterior, so that the most anterior digit, the
thumb in the human hand, is called digit 1 and
A Talamillo, Mf Bastida,
M Fernandez-Teran and Ma Ros
Departamento de Anatomía y Biología
Celular, Universidad de Cantabria,
Santander, Spain
Key words: digit morphogenesis –
limb development – limb malformation –
polydactyly
Corresponding author: María A. Ros,
Departamento de Anatomía y Biología
Celular, Universidad de Cantabria, 39011
Santander, Spain.
Tel.: þ34 942 201933;
fax: þ34 942 201903;
e-mail: rosm@unican.es
Received 16 November 2004,
revised and accepted for publication
22 November 2004
the most posterior digit, the little finger, is called
digit 5. What distinguishes the morphology of
the human hand is the presence of an opposed
thumb that makes it a magnificent multifunctional
tool.
Malformations affecting the limb and particularly
the number of digits are among the most
frequent congenital malformations in humans
(1, 2). Polydactyly implies the occurrence of
supernumerary fingers or toes, whereas oligodactyly
indicates that the number of digits is less
than five. The anomalies in the number of digits
can appear as an isolated condition or be part
of a systemic syndrome (3, 4). Interestingly, the
number of digits has traditionally aroused human
curiosity. In the era before ultrasound, the
first question asked on the birth of a child
inquired whether the number of fingers or toes
was correct. This curiosity seems to correspond
to an interest in the general health of the newborn
rather than in a possible functional alteration
of the hand. It thus reflects the recognized
143

Talamillo et al.
fact that polydactyly may be associated with
other more general syndromes. Isolated polydactyly
is frequently inherited as an autosomal dominant
trait (3).
Molecular and cellular bases of limb
development
The first morphological evidence of a limb
during embryonic development is the emergence
of a bulge at the appropriate level in the lateral
body wall (Fig. 1a). This bulge will rapidly form
a bud consisting of mesenchymal cells of mesodermal
origin that are covered by the ectoderm.
The main source of the limb mesoderm is the
somatopleura, but other contributions come
from the migration of muscle precursors from
the somites and from the progressive invasion
of the limb bud by endothelial and nerve cells.
This apparently simple bud will develop into a
complete and patterned limb under the control of
a few well-identified signaling centers. Signaling
centers are specialized groups of cells that produce
(A) (B) A (C)
(E)
Fgf10
Wnt
(F)
P
D
Fgf8
ZPA
P
(G)
V
D
AER
(D)
(H)
Fig. 1. (a) Chick embryo after 2 days of incubation. The emerging
wing bud is indicated by the yellow bracket. (b) Schematic
diagram of a limb bud with the axes and major signaling centers
indicated. The apical ectodermal ridge (AER) is in blue and the
zone of polarizing activity in red. (c) Semithin section of the tip of a
chick limb bud showing the pseudostratified morphology of the
AER (arrows). (d) Semithin section of the tip of a mouse limb bud
showing the polystratified AER (arrows). (e) In situ hybridization
showing the expression of Fgf8 in the AER of a 3-day chick wing
bud. (f) In situ hybridization showing the expression of Fgf8 in the
AER of a 6.5-day chick leg bud. (g) Dorsal view of the flank of a
chick embryo immediately after the application of a microsphere
(arrow) loaded with Fgf8. (h) Supernumerary limb that resulted
from the experiment shown in (g). (i) Schematic diagram
indicating the process of AER induction.
144
and secrete molecules to direct the developmental
behavior of neighboring cells. The three main signaling
centers identified in the growing limb bud
are the apical ectodermal ridge (AER), the zone of
polarizing activity (ZPA), and the non-ridge ectoderm,
each one being primarily responsible for
directing growth and patterning along one of the
three orthogonal axes (Fig. 1b) (reviewed in 5–7).
The AER and fibroblast growth factors
Possibly, the main signaling center during limb
development is the AER (Fig. 1b). The AER is
the epithelial rim at the distal border of the limb.
It forms a specialized structure – a pseudostratified
epithelium in birds (Fig. 1c) and a squamous polystratified
epithelium in the mouse (Fig. 1d) – that
separates the dorsal from the ventral surfaces of
the limb bud. The importance of this structure is
clearly evidenced by the phenotype resulting when
it is removed experimentally. Removal of the
AER arrests further development and results in a
proximo-distal transverse truncated limb (8). Also,
several mutations, spontaneous in chick and targeted
in mice, are characterized by the inability to
form an AER, and consequently, they are amelic
(9–11). The earlier the stage at which the AER is
lost, the more proximal the level of truncation is
(8). This phenotype was taken to indicate that the
AER is necessary for the progressive specification
of distal identities (fates) in the sub-AER mesoderm
called the progress zone. The progress zone
model assumes that the cells become progressively
distalized in relation to the time that they spend in
the progress zone under the influence of the AER
(12). However, the AER is also necessary early on
for the survival, and later for the proliferation, of
the subjacent mesoderm. Therefore, the cell death
or reduced proliferation that follows its removal
can also account for the truncated phenotype (13).
The action of the AER is mediated by several
members of the family of fibroblast growth factors
(Fgfs), five of them (Fgf8, Fgf4, Fgf2, Fgf9, and
Fgf17) being secreted by the AER cells (14–17).
Of these, Fgf8 is expressed by all AER cells, from
their early specification to their disappearance at
late limb development, and is thus considered the
marker of the AER (Fig. 1e,f) (17). A source of
FGF can replace all AER functions, and furthermore
is sufficient to trigger the development of a
complete extra supernumerary limb if applied to
the flank (interlimb) region (Fig. 1g,h) (18).
The induction of the AER results from a classical
epithelial–mesenchymal interaction, whose molecular
bases are currently being unraveled. It has
recently been shown that the pre-limb ectoderm, in
birds and mammals, requires Wnt signaling to

form an AER (Fig. 1i) (9, 11, 19, 20). In the
absence of this Wnt signaling, the AER and,
consequently, the limb fail to develop (9, 11, 21).
In the chick, early Fgf10 expression in the pre-limb
mesoderm is responsible for the induction of Wnt
signaling in the ectoderm (Fig. 1i) (19, 20). This
Wnt expression is also regulated by bone morphogenetic
protein (BMP) signaling originating in the
ventral ectoderm (22, 23). Once the AER is
formed, it also requires maintenance from the subjacent
mesoderm, likely mediated by Fgf10 (19).
The ZPA and Sonic hedgehog
A second signaling center of crucial importance in
limb development is the ZPA, a group of mesodermal
cells located at the posterior border of the
limb bud (Fig. 1b) (24). The ZPA was discovered
in the course of certain transplantation experiments
when it was found that grafting of posterior
cells to the anterior border of a normal limb
Shh (A)
ZPA
?
stylopodium
RA
3
zeugopodium
4
4
3
autopodium
2
4
4
4
h
3
3
(B)
(C) (D)
(E)
Fig. 2. (a) In situ hybridization showing the expression of Shh in
the zone of polarizing activity (ZPA). (b) Whole-mount skeletal
preparation of a 10-day chick wing bud. The regions of the limb
and the number of digits are indicated. (c) Schematic diagram to
illustrate the grafting of ZPA or retinoic acid (RA; in a carrier
microsphere) at the anterior limb border. The endogenous ZPA is
also depicted in red. (d) Skeletal preparation showing a mirror
image duplication obtained after anterior application of RA. Note
the absence of the most anterior digit, digit 2 in the chick wing. (e)
Skeletal preparation showing a mirror image duplication obtained
after anterior application of Shh. Note the three elements in the
zeugopod. (f) Skeletal preparation of the forelimb of an Shh null
mouse. The humerus (h) is normal but the element present in the
zeugopod (z) cannot be identified, and no digits develop.
3
z
(F)
Polydactyly
bud led to a mirror image duplication of the limb
(24). The same kind of duplication was obtained
after applications of retinoic acid (Fig. 2c,d), which
is capable of inducing another ZPA (25–28). The
ZPA cells produce Sonic hedgehog (Shh) (29)
(Fig. 2a), a potent signaling molecule that has
critical functions during embryonic development
(30) and elicits the same properties as the ZPA
(Fig. 2e) (31, 32). Shh is thus considered to be the
signaling molecule emanating from the ZPA and
responsible for all its properties.
The duplicated limb obtained after the ectopic
anterior activation of Shh signaling (exogenous
applications of Shh or grafting of any tissue expressing
or inducing Shh) (29, 31–33) is polydactylous
but with the peculiarity that the extradigits develop
as a mirror image duplication of the normal digits
(Fig. 2d,e). In human pathology, there is a very
rare congenital abnormality called the mirror
hand that is equivalent to the phenotypes obtained
after the exogenous application of a strong source
of Shh at the anterior border of the limb (32). The
mirror hand is characterized by a symmetric duplication
of part of the limb. The number of digits is
excessive, but usually the thumb or great toe is
absent. Symmetric duplication of elements of the
forearm (two ulnas) can also occur in a phenotype
similar to that shown in Fig. 2e. Therefore, it is
accepted that the mirror hand represents an early
and strong erroneous activation of Shh signaling at
the anterior border of the limb and it is not usually
included in the classifications of polydactyly but
considered as a distinct clinical entity.
In the absence of Shh signaling, as in the Shh
mutation in the mouse and the spontaneous chick
mutation called oligozeugodactyly, the development
of the limb starts normally but progressively
collapses, resulting in a tapered limb lacking distal
structures (Fig. 2f) (34, 35, 36). Nevertheless, the
complete proximo-distal axis is realized in the hindlimb,
where a single digit (digit 1) is formed (34–36).
Shh is absolutely required to make a normal skeletal
pattern from the elbow/knee joint outwards. Besides
patterning defects, the considerable abnormal cell
death seen in the absence of Shh signaling at least
accounts for part of the phenotype.
The non-AER ectoderm and the interaction between
the signaling centers
Patterning in the dorso-ventral axis of the limbs
depends on the non-AER ectoderm (reviewed in
37). The pre-limb ectoderm shows specific
domains of gene expression. Thus, the prospective
ventral ectoderm expresses Engrailed-1 (En-1),
which controls ventral patterning by restricting
145

Talamillo et al.
Wnt7a expression to dorsal ectoderm (38, 39).
Wnt7a induces the expression of the homeobox
gene Lmx1, which is responsible for the dorsalization
of the mesoderm (40).
Finally, it is important to emphasize that the
limb signaling centers are interdependent. Their
coordinated function is required for the appropriate
development of the normal limb (41–44).
Morphogenesis of the digits
The digits are a novel evolutionary acquisition of
the tetrapod limb (45, 46). During embryonic
development, they arise as single chondrogenic
condensations that later segment and grow. In
the rounded digital plate (autopodium), the digits
soon become demarcated and form the digital
rays that are separated by the flattened interdigital
tissue (Fig. 3a–c). The fate of the cells in
these two regions is very different; while the digits
will form in the digital rays (Fig. 3d), the interdigital
tissue will disappear through apoptosis
(Fig. 3e). Interestingly, the cell death fate of the
interdigit can be experimentally diverted to form
a digit if the appropriate signals are provided
(Fig. 3f) (47, 48). Indeed, the simple generation
of a wound in the interdigital tissue is sufficient to
activate the chondrogenic phenotype resulting in
an extra digit (M.A.R., personal observations).
The cellular and molecular mechanisms controlling
the formation of the correct number of
(A) (B) (C)
11.5 dpc
(D)
Tgfβ
12.5 dpc 14.5 dpc
(E)
Fig. 3. (a–c) Pictures of mouse forelimbs of stages 11.5, 12.5, and
14.5 days post coitum (dpc) illustrating the evolution of the shape
of the autopodium and progressive morphogenesis of the digits.
(d) In situ hybridization of a 7-day chick leg bud showing
expression of transforming growth factor b2(Tgfb2) in the digital
rays. (e) Terminal deoxynucleotidyl transferase-mediated
deoxyuridinetriphosphate nick-end labeling assay in a 13.5 dpc
mouse forelimb showing interdigital cell death. (f) Skeletal
preparation of a chick leg bud with a supernumerary digit
(arrowhead) that developed in the third interdigital area after
application of a microsphere loaded with TGFb 1 day earlier.
146
(F)
digital rays are not completely understood but
correlate with the size of the digital plate and
can be modified at relatively late stages.
In the pentadactylous limb, each digit presents
a specific morphology according to its anterior–
posterior position in the autopodium and is therefore
distinguishable from the other digits. The
chick is a widely used model for studying limb
development and particularly useful for studies
on digit identity, because in the leg, each digit
has a different number of phalanges, which is
therefore taken as diagnostic for the identity of
the digit. Experimental studies with this model
have demonstrated that the identity of the digits
remains labile up to a relatively late stage in
development (49). At the time the phalangeal
condensations of the digits are first being laid
down, it is still possible to modify the identity of
the digits by interfering with the signals emanating
from the encompassing interdigits. BMP signaling,
regulated by the transcription factors
Tbx2 and Tbx3, is a firm candidate for this function
(49, 50). There is some controversy as to
whether interdigital signaling acts directly upon
the digital rudiments to control their identity or
whether there is indirect effect acting through the
AER to prolong its duration and therefore the
time for digit elongation (51).
Classification of polydactyly
Many efforts have been made to establish a comprehensive
classification of the different types of
polydactyly. The appearance of an extradigit in
the hand or foot may occur in a number of defects
ranging from an almost unnoticed nubbin to a
complete duplication of one or several digits. Presently,
the most accepted classification of polydactylies
follows a topographic criterion based on
the position of the extradigit in the hand or foot.
Polydactyly affecting the thumb or great toe is
classified as radial/tibial polydactyly (Fig. 4a,b).
When the affected digit is the little finger, the
polydactyly is referred to as ulnar/fibular
(Fig. 4c,d), and cases in which the three central
digits are affected are referred to as central polydactyly,
the least common of the three types. This
classification, which was introduced by Swanson
(52) and Buck-Gramcko (53), has replaced
previous classifications into pre- and postaxial
polydactylies. It has the advantage of avoiding
confusion between the central morphological axis
in the human hand, running along the third digit,
and the more posterior metapterygial axis.
Each type of polydactyly is subclassified mostly
according to the severity of the duplication

(A) (B)
(C)
defect. Several classifications for radial polydactyly
have been described (reviewed in 54, 55). The
most accepted classification, first developed by
Wassel (56), includes seven subtypes from type I
to type VII depending on the bifurcation level
and has since been extended by Wood (57) and
Tada et al. (58). A different system for classifying
radial polydactyly, along both the transverse axis
(involved ray) and the longitudinal axis (extent of
proximo-distal duplication), has been used by
Blauth and Olason (59). This classification was
extended by Buck-Gramcko and Behrens (60),
who include duplications occurring at joint as
well as at bone level; each bone segment is subclassified
as either partly or totally duplicated.
Temtamy and McKusick (3) classified ulnar
polydactyly into types A and B, based on the
extradigits being either well developed (type A)
or rudimentary (type B).
It is not uncommon for an extradigit to be fused
in some way to the neighboring digits (webbing
between the digits or osseous fusions), a condition
called syndactyly (Fig. 4b). The association
between polydactyly and syndactyly is known as
polysyndactyly and reflects interference common
to the mechanisms of digit specification and interdigital
cell death.
Etiology of polydactyly
(D)
Fig. 4. Human congenital polydactylies. (a) Radial polydactyly
with duplication of the thumb. (b) Polydactyly of the foot with
the duplication of first toe. (c) Ulnar polydactyly with an extra
little finger. (d) Polydactyly of the foot with an extra fifth toe.
As indicated above, a polydactylous limb
implies some kind of interference with normal
development. Because the control of digit morphogenesis
is complex and remains labile for a
considerable time, the possibilities for interference
are numerous. Despite the enormous
advance in our understanding of the processes
that control digit formation, it is not always
possible to provide a reasonable explanation for
each of the multitude of outcomes observed in
human limb pathology.
The number of digital rays that develop in the
autopodium depends on the amount of tissue
available. Thus, the control of the size of the
limb bud is of decisive importance. Three factors
strongly influence the size of the limb bud: (i) the
number of progenitors that start up the bud, (ii)
the rate of proliferation, and (iii) the amount of
cell death. A deregulation of these factors results
in a subsequent modification in the pattern of the
autopodium and the number of digits.
Involvement of Fgf signaling
Polydactyly
FGF signaling from the AER appears to function
at a very early stage in limb development to
ensure that enough progenitor cells are available
to form the normal limb skeleton (61). This conclusion
is reached because the double Fgf8;Fgf4
knockout limbs are smaller than normal from
their earliest emergence. Furthermore, the
increased cell death observed on these doublemutant
embryos at proximal level contributes to
their marked reduction in size by 10.5 days after
coitum and results in the total absence of skeletal
elements (61). In the case of the conditional single
Fgf8 knockout, the limb is hypoplastic at the
three proximo-distal levels with the absence of
the first digit (61–63) and abnormal areas of
cell death. This indicates that a sufficient amount
of FGF signaling is required for the appropriate
survival of the cells and consequently to produce
a normal digit number (61–64).
Conversely, mutations that cause increased Fgf
signaling from the ectoderm result in polydactyly.
For example, doubleridge is a transgene-induced
mutation in the mouse characterized by thickening
of the AER and increased Fgf signaling in the
subjacent mesoderm that results in forelimb ulnar
polydactyly (65). The transgene insertion in
doubleridge causes a notable decrease in the
expression of Dkk1, a secreted Wnt antagonist
that modulates Wnt signaling in the ectoderm
(66) and that has also been shown to mediate
BMP control of apoptosis (67). Likewise, other
mutations that cause expansion of the AER, such
as the genetic disruption of En-1 that interferes
with the normal formation and maturation of the
AER, result in polydactyly (39). An increase in
147

Talamillo et al.
FGF signaling, rather than changes in the morphology
of the AER, is responsible for the
increased proliferation and cell survival of the
underlying mesenchyme, which leads to the syndactyly
and polydactyly phenotype.
Recently, the genetic modifications performed
to elevate Fgf signaling through the introduction
of a hypermorphic mutant form of the receptor 1
of FGF (FGFR1) (68) lead to the appearance of
radial polydactyly by a mechanism involving
extended AER function and reduced cell death.
Interestingly, the topographic restriction of polydactyly
to digit 1 suggests a specific FGFR1dependent
regulation of the pool of cells destined
to give rise to this specific digit.
Involvement of Shh signaling
Shh signaling has also been shown to promote
proliferation through the activation of Cyclin
D1 and Cyclin D2 (69). The ectopic application
of Shh to the anterior limb bud results in a duplicated
limb bud that reflects a dramatic increase in
Gli3 (A) Alx4 (B) Hoxd13 (C) Hand2 (D)
WT
(E)
FL HL
FL
148
Xt/Xt (G) Xt/Xt
(H)
(I)
HL
2
2
4
WT
(J)
3
the amount of tissue. Conversely, in the Shh
mutant limb, there is a general decrease in proliferation
(34) and a dramatic increase in cell death
that accounts for the deficient phenotype. Therefore,
it is very likely that the cooperation of
proliferating signals from the AER and from the
ZPA is required to obtain a normal amount of
tissue to permit normal development.
Shh signaling is mediated by the Gli family of
Zn-finger transcription factors. In vertebrates,
three members of the family have been identified,
Gli1, Gli2, and Gli3 (70, 71). Of these, Gli3,
whose expression is complementary to that of
Shh (Figs 5a and 2a), is the only member with
mutations giving a limb phenotype, and hence is
the most relevant for limb development (72). Gli3
is a complex factor that can function as a weak
activator or as a strong transcriptional repressor
when processed to a short truncated form, Gli3R,
in the absence of Shh signaling (73). Extratoes
(Xt) is a spontaneous polydactylous mutation in
the mouse (Fig. 5g,h) representing the null allele
of Gli3 (72). Interestingly, in humans, mutations
Hoxd
Hand2
Gli3R
Shh
(F)
(K)
FGF
Fig. 5. Pattern of the expression of Gli3
(a), Alx4 (b), Hoxd13 (c), and Hand2 (d) in
3-day chick limb buds. Skeletal preparation
of the wild-type mouse forelimb
(e) and hindlimb (f). Skeletal preparation
of the Xt/Xt forelimb (g) and hindlimb (h)
exhibiting polydactyly. (i) In situ hybridization
of an Xt/Xt forelimb of 11.5 days
after coitum showing ectopic anterior
activation of Shh (arrowhead). (j) Duplication
of the second digit (most anterior
digit in the chick wing) obtained after
overexpression of Hand2. (k) Schematic
diagram showing the genetic interactions
that govern limb development.

in Gli3 are responsible for at least three polydactylous
syndromes, the Pallister–Hall, the
Greig cephalopolysyndactyly, and the postaxial
polydactyly type A. This variety of limb phenotypes
reflects the complexity of this transcription
factor as well as its extraordinary importance in
controlling the number of digits.
Besides Xt, other spontaneous or induced
mutant mice exhibit polydactyly. These mice are
excellent models for understanding the morphogenesis
of the digits and the genetic basis of
human polydactyly. These models include
strong’s luxoid (lst), luxate (lx), X-linked polydactyly
(Xpl), recombination-induced mutant 4
(Rim4), hemimelic extra toes (Hx), and sasquatch
(Ssq) mutants. The gene disrupted in the lst
mutant is Aristaless-like 4 (Alx4), a paired-type
homeobox transcription factor expressed in the
anterior mesenchyme of the limb bud (Fig. 5b)
(74–76). Although the genetic bases of lx, Xpl,
and Rim4 mutants remain unknown, it has been
shown that Hx and Ssq mutations (77, 78) affect
a long-range cis-acting regulatory element controlling
Shh expression (79, 80). The approximate
location of this regulator is within or near intron
5oflimb region-1 (Lmbr1) gene, about 1 Mb away
from the Shh gene (79, 80). Interestingly, in
humans, a major locus for dominant radial polydactyly
has been mapped to 7q36, a chromosomal
region syntenic to Lmbr1 region of mouse chromosome
5. It has been shown recently that point
mutations in this long-range regulatory element
segregate with polydactyly in unrelated families
affected with radial polydactyly and in the Hx
mouse mutant (80). However, the mutation of
the Lmbr1 gene results in Acheiropodia, a severe
human congenital abnormality that presents loss
of all bones of hands and feet (81). All the radial
polydactylies mentioned above are characterized
by some ectopic activation of Shh in the anterior
limb bud mesenchyme (Fig. 5i for Xt/Xt) (75, 78,
82, 83). Curiously, doublefoot (84), which is
another spontaneous mouse mutant with polydactyly,
shows ectopic activation of Indian
Hedgehog, a close relative of Shh (85).
Of remarkable interest was the finding that the
limb phenotype of the double Shh;Gli3 mutant
was identical to that of the Gli3 null mice
(86, 87), indicating that one of the main functions
of Shh signaling in the posterior mesenchyme is
to prevent processing of Gli3 to its repressor form
(73, 86, 87). Therefore, the proposal that Shh
signaling initially primes the limb mesoderm
as territory to produce digits (88) may now be
reinterpreted as defining a Gli3R-free territory.
This finding also indicates that the ectopic anterior
activation of Shh in the Gli3 mutant limb is
irrelevant for the phenotype, whereas this is not
the case for the double Alx4;Shh mutant. Based
on these observations, a classification of polydactyly
into two groups, according to their dependence
or not on anterior ectopic activation of
Shh, has been proposed. Group 1 polydactyly
would include those polydactylies, such as in the
Alx4/Alx4 mutant, that require anterior activation
of Shh signaling and develop mirror image duplications
with normal digit identity. The mirror hand
would thus be included in this group. The difference
between a simple radial polydactyly and the
mirror hand (with the absence of the thumb) would
reside in the strength and time of activation of the
ectopic anterior Shh signaling. Group 2 polydactyly
is independent of anterior activation of
Shh signaling. This group is represented by the
Xt/Xt mutant and is characterized by the lack of
mirror image digit duplications and altered digit
identity (86, 87).
The observation that the limb bud of the Gli3
mutant is broader than normal suggests that Gli3R
may be implicated in the control of the limb bud
size. In the chick, the spontaneous mutations
talpid 2 and talpid 3 , whose genetics are presently
unknown, show broader limbs and polydactyly.
talpid 2 has been shown to have a constitutive
activation of Shh signaling and very low levels of
Gli3R (73, 89), supporting the implication of
Gli3R in the control of the size of the limb bud.
Involvement of 5 0 Hoxd genes and Hand2
Polydactyly
The most 5 0 Hoxd genes are expressed in posterodistal
nested domains during limb development
(Fig. 5c for Hoxd13) and have been shown to
control digit number and pattern downstream of
Shh as well as regulating Shh expression
(reviewed in 46). Mutations in the Hoxd13 gene
are responsible for the human synpolydactyly
syndrome (90), a rare dominantly inherited limb
malformation, which results from different-sized
expansions of a 15-residue amino-terminal polyalanine
tract in this gene (90–92).
Hand2 is a bHLH transcription factor, with an
expression pattern very similar to that of Hoxd12
in the limb bud (Fig. 5d) (93, 94). It has been
implicated in the establishment of the limb
anterior–posterior polarity previous to Shh by
reciprocal genetic antagonism with Gli3 (93–95).
Hand2 also controls Shh activation of expression
(93, 94).
The activation of the most 5 0 Hoxd and Hand2
genes does not require Shh (34, 35, 93, 94),
and furthermore, the forced expression of either
of them throughout the limb bud results in
duplications of anterior digits as well as ectopic
149

Talamillo et al.
activation of Shh at the anterior level (Fig. 5j) (93,
94, 96), indicating that these transcription factors
are capable of triggering Shh expression (93, 96,
97). However, once Shh expression is activated in
the ZPA, the expressions of Hoxd11–13 and
Hand2 require Shh signaling as indicated by the
strong downregulation observed in the Shh
mutant limb (34, 35). These observations suggest
the existence of a positive feedback loop between
5 0 Hoxd and Hand2 and Shh (Fig. 5k).
In addition, it has been shown that the Hoxd
genesarerequiredforpropergrowthofthelimb,
as suggested by the reduction of the initial size of the
limb bud formed in their absence (97). Of particular
interest is the polydactylous phenotype obtained
after upregulation of Hoxd11 expression induced
by the genetic removal of Hoxd12 and Hoxd13 (98).
Finally, a direct biochemical interaction between
Hoxd12 and Gli3 proteins has been demonstrated
recently (99). The association between these two
transcription factors converts Gli3R from a transcriptional
repressor to a transcriptional activator
(99). These results suggest a model in which the
ratio of Gli3 to total 5 0 Hoxd protein across the
limb bud regulates digit pattern and identity.
Concluding remarks
Human limb malformations, like other congenital
anomalies, are frequently associated with defects
in other organs, thus forming part of a systemic
syndrome (100). This is because they are caused
by mutations in genes that affect important signaling
pathways repetitively used during embryonic
development at different times and locations.
Therefore, the alteration of one of these signaling
pathways may result in pleiotropic effects affecting
multiple organ systems in the developing
embryo.
Much work remains to be done to define the
precise role of all the above-mentioned factors in
the control of the number of digits. The development
of appropriate animal models is of great help,
because they permit the analysis of the underlying
molecular mechanisms when this is not possible in
humans. However, the phenotypes obtained in
mice and in human are not always the same. In
part, this is because the mutations in mouse are
engineered to result in a complete loss of function,
whereas naturally occurring mutations in man may
yield products of unknown function.
Acknowledgements
We are very grateful to Dr G.-Ullate Vergara del Servicio de
Neonatologı´ a del Hospital Universitario ‘Marque´ s de Valdecilla’
for the pictures of human polydactyly. We thank I. A.
150
Williams for his careful revision of the English in this manuscript.
A.T. is the recipient of a postdoctoral fellowship from the
Fundacio´ n Marque´ s de Valdecilla. Work in M.A.R. laboratory
is supported by grant FP12002-02946 from the Spanish
Ministery of Education and Science and grant API 04/03
from the Instituto de Formacio´ n e Investigacio´ n ‘Marque´ sde
Valdecilla’.
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