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Development 1994 Supplement, 181-186 (1994)
Printed in Great Britain @ The Company of Biologists Limited 1994
181
Hox genes and growth: early and late roles tn limb bud morphogenesis
Bruce A. Morgan1,2 and CIiff Tabin3
lCutaneous Biology Research Center, MGH East, Charlestown MA 02129, USA
2Department of Dermatology, Harvard Medical School, Boston MA 02115, USA
sDepartment of Genetics, Harvard Medical School, Boston MA 02115, USA
SUMMARY
In recent years, molecular analysis has led to the identification of some of the key genes that control the morphogenesis of the developing embryo. Detailed functional
analysis of these genes is rapidly leading to a new level of
understanding of how embryonic form is regulated. Understanding the roles that these genes play in development can
additionally provide insights into the evolution of morphology.
The 5' genes of the vertebrate Hox clusters are expressed
in complex patterns during limb morphogenesis. Various
models suggest that the Hoxd genes specify positional
identity along the anteroposterior (A-P) axis of the limb.
Close examination of the pattern of Hoxd gene expression
suggests that a distinct combination of Hoxd
gene expressed in different digit primordia is unlikely to
specify each digit independently. The effects of altering the
pattern of expression of the Hoxd-I1 gene at different times
during limb development indicate that the Hoxd genes have
separable early and late roles in limb morphogenesis. In
their early role, the Hoxd genes are involved in regulating
the growth of the undifferentiated limb
mesenchyme.
Restriction of the expression of successive 5' Hoxd genes to
progressively more posterior regions of the bud results in
the asymmetric outgrowth of the limb mesenchyme. Later
in limb development, Hoxd genes also regulate the maturation of the nascent skeletal elements. The degree of
overlap in function between different Hoxd genes may be
different in these early and late roles. The combined action
of many Hox genes on distinct developmental processes
contribute to pattern asymmetry along the A-P axis.
in the limb
Comparative molecular analysis suggests that the genes that
gave rise to the modern Hox clusters have been specifying
regional differences in the animal body for possibly more than
one billion years (Kappen and Ruddle, 1993; Shubert et a1.,
1993). Sequence comparisons suggest that, at the time of the
origin of the chordate, nematode and arthropod lineages, there
were between 5 and 7 members of the Hox complex (Shubert
et al. , 1993). Since that time, these complexes have undergone
expansion and duplication independently in different lineages
ultimately generating, for example, 38 members in the four
clusters of Hox genes observed in gnathostome vertebrates.
During the course of this expansion, these genes retained their
original functions in patterning the anterior-posterior (A-P)
body axis and also acquired new functions in regulating other
aspects of morphogenesis. When novel developmental innovations arise in evolution, modifying preexisting body plans,
the derived embryonic steps tend to occur late in ontogeny
("the general precedes the spectalized" in von Baer's formulation; Gould, 1977). In performing patterning roles that are phylogenetically old, and therefore occur developmentally early,
there may be a high degree of overlap in function between paralogous genes in different Hox clusters, and even between
sequentially arranged genes within a cluster (see Fig. 1 for
nomenclature). Continued function in these early roles constrains their divergence (as discussed by Holland et al.,1992).
Key words: Hox gene, limb bud morphogenesis, axis specification,
anteroposterior determination
However, in roles that arose later in evolution and are frequently observed later in development of the embryo, the differences in function between paralogue groups and between
different genes in a paralogue group are more pronounced,
taking advantage of divergence in regions not required for
primitive function. This concept is generally useful in attempting to decipher the role of the Hox genes in morphogenesis and
is illustrated in our consideration of the role of the Hox genes
in limb development.
The limb arises from an accumulation of cells in the lateral
plate mesenchyme. These cells induce a specialized structure
in the overlying ectoderm, the apical ectodermal ridge (AER).
Subsequent proliferation and outgrowth of the limb mesenchyme is dependent on signals from the AER; in turn the
AER is dependent on the underlying mesenchyme for its maintenance. Mesenchymal cells in the region subjacent to the AER
(the so-called progress zone) remain in a highly proliferative
and undifferentiated state. As the limb bud grows, cells are
continuously displaced from this zone. Displaced cells
decrease their rate of proliferation and subsequently begin to
differentiate, leading to a proximal-to-distal wave of differentiation. Cells that will participate in the formation of distal
structures are still being generated when cells participating in
the formation of proximal structures are beginning to differentiate.
182
B. A. Morgan and C. Tabin
The Hox genes are expressed in intriguing patterns during
morphogenesis of the wing and leg. These patterns evolve in
complex ways as development proceeds. In earlier work in the
mouse limb, these changing patterns of expression were
described as the continuing evolution of a single expression
domain (Dolle
et al.,
1989). Examination
of Hox gene
expression in the chick limb bud reveals that the evolving
pattern of gene expression described for each gene actually
represents temporal and spatial overlap of several distinctly
regulated expression domains.
Members of the Hoxd cluster are sequentially expressed
during the development of the chick limb bud (Fig. 2). In the
early phases of limb bud development, transcripts of the Hoxd-
9 and Hoxd- I0 genes are expressed across the A-P extent of
the nascent wing and leg bud. Activation of the nine paralogues
is synchronous with the initial outgrowth of the bud. It is
during this phase of development (stages 17 -19) that the presumptive stylopod is displaced from the progress zone
(Saunders, 1948).
The next phase of limb outgrowth involves the activation of
the Hoxd- I I and Hoxd- 12 genes in progressively restricted
domains in the posterior half of the bud. This occurs at a stage
when the presumptive zeugopod is being displaced from the
progress zone (stage 19 through 21122). Finally, Hoxd-|3 is
activated in the posterior distal region of the limb bud at stage
20. As development proceeds, expression of Hoxd-10, HoxdI I , Hoxd- 12 and Hoxd- I 3 come to occupy very similar
domains in the distal segment (autopod) and remain strongly
expressed in this region while the nested domains of expression
in the proximal segments of the limb are fading. For most of
these genes, a clear separation
of
proximal and distal
13 is only weakly
expression domains is observed. Hoxdexpressed in the posterior zeugopod at
this stage, but is strongly expressed in
the autopod in a domain that extends
slightly anterior to that of the other
Hoxd genes.
The apparent separation
n
-,/\\.
Hoxd
morphogenesis.
The early pattern of Hoxd gene expression in the chick limb
correlates
I
chronous with initial limb outgrowth and at this stage bud
outgrowth is symmetric along the A-P axis. Growth of the bud
becomes markedly asymmetric along the A-P axis with a
distinct posterior bias when the Hoxd- I I and Hoxd- 12 genes
are activated in the posterior half of the bud. This bias in
outgrowth is observed during stages 19 to 23, a period when
the presumptive sylopod and zeugopod constituents are being
displaced from the progress zone. Subsequent growth of the
bud becomes less asymmetric as the distal domains of Hoxd
gene expression spread to the anterior regions of the bud.
This correlation between the timing and position of early
rl_
-t..- ET ETE
i
-ll
,'
l/ i
876
54
321
-.'
:. --.
13-t 12 11 10
with the outgrowth of the bud. The onset of
I0 throughout the bud is syn-
expression of Hoxd-9 and Hoxd-
I.-J.
/
of
regulated in proximal and distal regions of the mouse limb bud.
The degree of overlap between domains prevents their observation as distinct entities in the wild-type animal. However,
transgenic mice bearing different segments of the Hoxd- I I
fused to a p-galactosidase reporter construct demonstrate that
expression of Hoxd-I I may be independently regulated in the
proximal (stylopod), central (zeugopod), and distal (autopod)
segments of the limb (Gerard et al., 1993). In particular,
expression in the distal segment of the limb is separable from
expression in the central segment.
As development proceeds, Hoxd expression fades in the
mesenchyme with the exception of the perichondrial regions.
The maintenance of Hoxd gene expression in the perichondrial regions correlates with the expression of a second vertebrate
hedgehog homologue, Indian hedgehog. This late pattern of
expression may represent an independently regulated phase of
Hox gene transcription indicative of a separable role in limb
9
lnsecU
Vertebrate
Ancestor
i
Paralogue
gene expression into several phases is
confirmed by recent identification of
Sonic hedgehog as a gene involved in
the regulation of the Hoxd gene
in distal mesenchyme.
expression
of Hoxd genes is
independent of Sonic regulation. Hoxd-
Early expression
9 and Hoxd-L0 appear in proximal
regions before Sonic is expressed
(Laufer et al., unpublished data). The
posterior distal expression of Hoxd- I I ,
Hoxd-
12 and Hoxd- I 3 in the limb
to be regulated by Sonic;
appears
ectopic expression of Sonic in the
anterior region of the bud is sufficient
to elicit ectopic expression of these
in the anterior distal region
(Riddle et &1., 1993; Laufer et ol.,
genes
unpublished data).
Analysis of the sequences required
to drive expression of the mouse HoxdI I gene suggests that Hox gene
expression
is also
independently
Fig. 1. Vertebrate Hox genes. There are four clusters of vertebrate Hox genes. Numbers in the
boxes represent previous nomenclature. Currently, genes are referred to by their cluster letter
(right) and paralogue number (l-13 listed above). At the time of insect/vertebrate divergence,
there were between 5 and 7 Hox genes (diagrammed above the vertebrate clusters). The four
modern vertebrate clusters apparently arose by serial duplication of a 13 member cluster with
subsequent deletions. As a result, the homologous genes in different clusters (referred to as
paralogues) are more similar to each other than they are to adjacent genes in the same cluster.
Paralogous genes differ by a few amino acids in the homeobox region and are therefore expected
to have similar DNA-binding characteristics. Most divergence between paralogues is observed
in the N-terminal half of the protein. The N-terminal region is presumed to interact with other
proteins or otherwise modulate activity of the protein, but direct evidence of its function in
vertebrates has not yet been obtained.
Role of Hox genes in limb bud morphogenesis
183
Stage
19
)
I
D-9
D-9 and D-l
20
)
I
1
il
ilt
D-9, D-l 1 and D-l 3
tv
D-9 and D-13
22
23
25
Fig. 2. Hoxd gene expression in the chick leg bud. The chick leg bud
is diagrammed at various stages of development (Hamburger and
Hamilton, 1951, stages listed at left). These sketches represent a
view of the dorsal surface of the right limb bud (anterior is towards
the top of the page). The expression of the Hoxd-9, Hoxd-I I and
Hoxd-L3 genes are diagrammed. To simplify the diagram, Hoxd-LO
and Hoxd-12 are not shown. At early stages (19-22) Hoxd-10
expression resembles that of Hoxd-9. During stages22 and23,
Hoxd- I0 expression fades in the anterior regions and its expression
domain approximates that shown in yellow for Hoxd-9, Hoxd-I I
plus Hoxd-L3. By stage 25 Hoxd-9 expression is negligible and the
color codes shown represent the designated gene combinations
without Hoxd-9. The Hoxd- 10 expression domain at later stages
resembles that of Hoxd-l I. The expression of Hoxd-|2 is very
similar to that of Hoxd-l/ throughout development, although it is not
found in the anterior most regions of the Hoxd- I / expression domain
through stage 23.
Hoxd gene expression and the growth of the undifferentiated
timb mesenchyme could reflect the response of both Hox gene
expression and cell proliferation to a common inducer, or even
a requirement for additional cell divisions to achieve the
sequential activation of increasingly 5' Hox genes. Alternatively, one role of the 5' Hoxd genes early in limb bud development may be to mediate the asymmetric growth of the bud.
a case, the discrete regulation of these genes in the three
segments of the limb may reflect a fundamental role of these
genes in the evolution of the appendage; the sequential appearance of elements capable of activating these genes would correspond to the addition of segments along the proximal distal
axis.
The effects of ectopic expression of Hoxd- I I suggest that
regulation of the growth of undifferentiated mesenchyme is an
In such
Fig. 3. Hoxd-I
I
At either
2l
expression compared to a fate map of the leg bud.
or stage 26, domains of Hoxd-I expression in the
leg bud (blue) are restricted to the regions posterior to the primordia
of digit I. At early stages, Hoxd- I / expression encompasses the
primordia of the fibula (f), the posterior metatarsals (m) and digits II
through IV. The position of the primordia of distal skeletal element
can only be approximated at this stage. As development proceeds,
expression of Hoxd- I / remains strong in digits II, ilI and IV as well
as the posterior metatarsals and the perichondrium of the fibula.
Skeletal elements whose growth is inhibited by ectopic expression of
Hoxd-I
stage
I
I are shown in violet.
of the Hoxd genes in the limb. These ectopic
expression experiments were performed using a replication
competent retroviral vector to express the mouse Hoxd-I I
protein specifically in the developing chick limb bud (Morgan
et al., 1992) Hoxd-II is normally expressed in the posterior
regions of the bud in a domain that encompasses the primordia
of the fibula, posterior metatarsals and digits II, III and IV (see
Fig. 3). The retrovirus was used to infect the entire limb bud
and therefore expand that domain of expression into anterior
regions. These infections led to complex and variable phenotypes which reflects the mechanistic constraint on virusmediated ectopic expression (see Fig. 4 legend).
Perhaps the most striking phenotype associated with the
mis-expression of Hoxd- I I in regions anterior to its normal
domain of expression in the leg bud was the appearance of an
additional phalange in digit I leading to a morphology similar
to that of digit II (Fig. 4A,B). Anterior digits containing either
an additional phalange or a single elongated phalange were
observed, while no effect was observed on the bones of the
posterior digits II, III and IV, which arise from the region in
which Hoxd- I / is norrnally expressed.
A conceptually similar phenotype is observed in Hoxd-I I-
early role
184
B. A. Morgan and C. Tabin
injected wings. Cells in the anterior region of the chick wing
which do not express Hoxd- I I do not normally give rise to
skeletal elements. Ectopic expression of Hoxd-l I in the
anterior region of the wing results in the formation of an additional digit at the anterior edge of the wing which resembles
digit II in structure.
The apparent homeotic change was observed in roughly 307o
of the infected legs showing any phenotype. This level of penetrance indicates that the digit-affecting action of Hoxd- I I
occurs early in limb development, when only 307o of injected
limb buds arc abeady fully infected by the spreading virus.
Both the appearance of an additional phalange in digit I in
the leg and an additional anterior digit in the wing can be
ascribed to the proposed effect of the Hoxd genes on outgrowth
Fig. 4. Phenotypic consequences of ectopic Hoxd-l I expression. A
retrovirus encoding the mouse Hoxd- 11 cDNA was used to express
ectopically this protein in the chick leg bud (Morgan et al., 1992).
This wild-type chick foot is shown in A. Note that the first metatarsal
(m) is a deltoid bone arising distally. Excluding the terminal claw,
digit I has one phalange (p) while digits II, III and IV have 2,3 and 4
phalanges respectively. In a foot infected with the Hoxd-L1virus (B),
the anterior two digits have a similar structure which includes the
two phalanges normally found in the second digit. The anterior
metatarsal now arises proximally and has a structure similar to that
of the posterior tarsometatarsals. The first and second metatarsals are
approximately half the length of a normal second metatarsal. Digits
III, IV and the posterior metatarsals are relatively normal. The
curvature of the posterior metatarsals and digits may be caused by
the lack of growth of the anterior metatarsals and failure of
interdigital cell death. (C) In a similar fashion, the tibia (t) and fibula
(0 of a wild-type leg at day 1 1 of incubation (left) or day 5 of
incubation (center) are shown. At day 5 of incubation, the primordia
of the tibia and fibula are approximately equal in length. The tibia
and fibula of an infected embryo are very similar to that of an
uninfected embryo at this stage. However, as development proceeds
the tibia of an infected embryo fails to elongate normally and the
tibia and fibula remain the same length at day 1 1 of incubation
(right). To achieve a domain of contiguous infected cells roughly
encompassing the entire limb bud, several focal infections are
induced by microinjection of virus in the lateral plate mesenchyme
early in development. As development proceeds these infections
spread to adjacent cells and coalesce to encompass the entire limb
bud. However, the precise position of the initial infections is
somewhat variable, as is the time when infection has spread
sufficiently to emcompass the entire bud. For some buds, this
process is complete by stage 2I, whrle for others it may be as late as
stage 24 or 25. Effects on distal skeletal elements cannot be reliably
assayed before day 1 1, making it impossible to directly relate the
degree of infection at an early stage with a particular phenotype in an
affected embryo. Therefore, population approaches must be
employed, correlating the degree of infection of early harvested
specimens with the range of phenotypes later in development. The
continuous spread of the virus during the course of incubation
increases the penetrance of phenotypes which result from Hox gene
activity later in development. Because there is a proximal to distal
progression of differentiation in the limb, this will be observed in
two ways. The effects of altered Hox expression on a given
developmental process (e.g. cartilage condensation) will be evident
more frequently in distal structures where this process occurs later,
allowing more time for viral spread. Furthermore, at a given level
along the proximal distal axis, phenotypes resulting from influences
on later developmental events will also be observed more frequently
than those reflecting earlier activity.
or survival of undifferentiated limb mesenchyme. We propose
that procedures that increase the proliferation or survival of
limb bud mesenchyme lead to the formation of additional cartilaginous condensations. We suggest that, when these condensations are sufficiently separated, they give rise to additional bones. Insufficiently separated condensations fuse to
form a single enlarged bone. Hence the effects on distal
elements of ectopic Hoxd- 11 expression in the anterior region
of the wing bud resemble those observed when elevated levels
of bFGF are achieved either by implantation of an FGF-soaked
bead or by implantation of FGF-secreting cells in the anterior
wing bud (Riley et al., 1993). In both cases, increased proliferation or survival of undifferentiated limb mesenchyme leads
to the formation of additional bones. Indeed, earlier activation
of Hoxd- I I in the anterior region of the hind limb bud can have
a more pronounced effect on limb growth leading to the
formation of an additional digit anterior to the normal digit I.
A role for the Hox genes in stimulating growth of limb precursors is sufficient to explain the appearance of an additional
bone in the anterior digit after ectopic expression of Hoxd- I I
in the anterior region. However, this postulated function is not
sufficient to explain other skeletal phenotypes observed in
response to ectopic Hoxd- 11. Most of these reflect a failure of
specific bones to mature properly after apparently normal
initial development (Fig. 4).Ectopic Hoxd-[1 expression in the
anterior region leads to an abnormally short second tarsometatarsal, while the third and fourth tarsometatarsals ate
comparatively normal. In a similar fashion, the tibia is reduced
C.
<-$-,il
rf
Role of Hox genes in limb bud
to half its normal length and is now very similar in length to
the fibula. This phenotype was observed in more than half of
the affected specimens as compared to the one third that
showed an additional phalange in digit I. The higher penetrance suggests that these phenotypes result from a later effect
of altered Hoxd-4 expression on the limb bud. (When the
injected virus has had time to spread completely in a higher
percentage of limbs.)
If the digital phenotypic effect were more prevalent than the
proximal effect, one might ascribe the difference in penetrance
to the fact that there is a proximal-to-distal wave of differentiation in the limb bud. Thus the Hox genes could act at a single
developmental stage (e.g. cartilage condensation); and when
that stage occutred proximally (early) there would naturally be
less frequent complete infection than when that same stage
occurred in distal regions (late). However this is not the case:
the proximal phenotype is more frequent, and hence occurs
later in time. This strongly implies that phenotypes in regions
proximal to the digits are not due to the effect on early proliferation (that gives rise to the digit phenotypes), but rather that
they result from a distinct later effect of altered Hoxd
expression on the developing limb. Consistent with this
hypothesis is the observation that at early stages of development, the initial condensations that will become the tibia and
fibula appear normal in infected limb buds. Through stage 26,
there is no obvious difference between the zeugopod of the
infected and uninfected contra-lateral limbs. At this stage, the
cafirlaginous condensations that will contribute to the tibia and
fibula are approximately equal in length. The norrnal disparity
between the size of these bones at later stages arises as a consequence of greater subsequent growth of the tibia compared
to the fibula. Hoxd- I I expression in anterior regions prevents
this preferential growth leading to a phenotype in which both
bones are similar lengths. In a similar fashion, the second
matatarsal also fails to undergo normal elongation and is
reduced to roughly half the size of the norrnal bone. Even when
the anterior metatarsal shows a complete phenotypic conversion to resemble the posterior metatarsal, both are half the
length of a normal metatarsal. (Fig. 4B).
These effects could be due to persistent expression of the
Hoxd- I I gene in differentiating cells where it is normally
turned off. However, such an explanation would predict that
all bones in the limb would be affected equally since Hoxd
genes are nonnally down regulated in all but the perichondrial
regions within the diagrammed domain of expression. The fact
that this inhibitory effect on bone elongation is observed only
in bones from the regions where Hoxd- I I is not normally
expressed suggests that it is more specific. Some other aspect
of differential gene expression such as the expression of the
endogenous gene or Hoxd-L2 in the normal expression domain
of Hoxd- I I prevents the inhibitory action of the exogenous
gene in this region. Alternatively the exogenous Hoxd-11 interferes with the activity of factors that arc only responsible for
stimulating the preferential growth of the tibia and anterior
metatarsal. Other Hox genes are likely targets for this interaction; protein-protein interactions between different Hoxd gene
products which inhibit transcriptional activity have
been
described (Zappavigna et a1., 1994). This late effect on bone
growth has also been observed in gene ablation experiments
with the mouse Hoxd-L3 gene. Absence of the Hoxd-L3 gene
product leads to delayed and incomplete ossification of the
morphogenesis
185
proximal phalanges in the anterior and posterior digits (Dolle
et al., 1993).
It has been proposed that the combination of Hoxd genes
expressed in a digit primordium specifies the unique identity
of each digit in a combinatorial code and mediates its characteristic morphogenesis (Tabin, 1992). The apparent homeotic
nature of the morphological changes in the anterior digit and
metatarsal resulting from ectopic expression of Hoxd- I I can
be interpreted to support such a model. The separable early and
late roles of Hox genes that emerge from the analysis presented
here do not in themselves contradict this view. Rather they
provide a two-phase mechanistic basis for the effect of Hox
genes on limb (including digit) morphology.
However, the simple Hox-code model is excluded when one
also takes into account the extremely dynamic expression
patterns of the multiple domains of each Hox gene within the
limb (Nelson, Morgan and Tabin, unpublished data). The Hoxd
genes do appear to act early when there are nested expression
patterns of the Hoxd genes along the anterior-posterior limb
axis. However, they also clearly function later as well, when
their relative domains are very different, and not at all aligned
with unique digit primordia. Indeed, at this later time all of the
Hoxd genes are expressed across the entire distal limb bud and
may play partially redundant roles in this domain. This is consistent with the finding that deletion of the Hoxd- 13 gene
affects all the digits (not just the most posterior one), but results
in a fairly subtle effect in mice whose other Hoxd genes are
intact (Dolle et al. , 1993). Thus, while the expression of Hox
genes in the limb bud appear to regulate digit morphology, they
do not encode digit identity by a simple combinatoral code.
An attempt was recently made to apply the Hox code model
to the problem of understanding the origin of pentadactyly (five
digits) in modern tetrapods (Tabin,1992). A two part argument
was proposed.
(
1)
It is know that the regulation of the number of digits
produced in a limb field is independent of the regulation of the
morphology of the digits. If there were a constraint on the
number of distinct morphological types of digits that could be
specified in a given limb field such that only five 'different'
digit types were permissible, then more than five digits might
never be able to evolve with a selective advantage in a population. Evidence presented in support of this hypothesis
included the fact that polydactylous individuals arising spontaneously in many species do not typically have a novel extra
digit but rather have a morphological duplicate of either the
most preaxial
or postaxial digit. Similarly,
polydactylous
Acanthostega (8 total digits) have
only five or fewer morphological types of digits. Finally, when
extra distal 'digits' arise in tetrapod evolution with unique morphologies, they are invariably produced by modification of a
wrist bone and are not true extra digits (e.g. Panda Bears, some
moles, some frogs). Thus, there does indeed appear to be some
constraint, limiting number of potential digit 'types' to five.
(2) It was further argued that the Hox code could directly
produce such a constraint since only five combinatorial codes
are possible from the Hoxd genes expressed in the limb.
However, as we have seen, the Hoxd genes do not act in a
simple combinatorial code for 'digit identity'. While they do
contribute to the regulation of digit morphology, our current
understanding of their action does not provide an indication of
a constraint on potential morphologies. Either such a constraint
primitive tetrapods such
as
186
B. A. Morgan and C. Tabin
remains to be discovered in the subtler aspects of Hox gene
action or else one will have to look elsewhere for it.
In summary, rather than an alteration in Hoxd code
producing the apparent homeotic effect of Hoxd- I I misexpression, we suggest that the effect on the anterior digit results
from an early stimulatory effect on limb outgrowth which
generates sufficient additional mesenchyme to generate an
additional chondrification center. At this stage of development,
all of the 5' Hoxd genes may have qualitatively similar roles.
Appropriately timed ectopic expression of another 5' Hoxd
gene might well achieve a similar result. Later effects on
skeletal morphogenesis may refle ct paralogue specific roles in
regulating bone growth. The fact that these genes are having
an effe ct at a stage when the expression domains of the HoxdI and Hoxd-12 genes are very similar in the autopod
10, Hoxd-I
renders the hypothesis that different combinations of Hoxd
genes specify digit identity improbable. Rather, the combined
early action on the accumulation
of
undifferentiated mes-
enchyme and later action on skeletal growth lead to the characteristic pattern of the chick limb skeleton.
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