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Vertebrate Limb Development
Hey, Bud, what do you want to be when you grow up?
The vertebrate limb is an outgrowth of the embryonic body wall, consisting
of mesenchyme derived from the somites and the somatic portion of the lateral
plate mesoderm, surrounded by an ectodermal jacket. In the fully-developed
limb, the muscle, cartilage, connective tissue, skin and skin derivatives
(to say nothing of the nerves and circulatory elements) are integrated into
a complex functional structure.
You should review in your textbook formation of the limb bud, limb morphogenesis
and the reciprocal interactions between the ectoderm and mesoderm that are
necessary to form the limb. The following points are salient:
Limb bud formation
- The distal tip of the limb bud is covered with a transient structure
known as the apical ectodermal ridge (AER). Limb outgrowth is dependent
upon interactions between the AER and the underlying mesoderm.
- If the AER is removed, the mesoderm stops dividing.
- If a supernumerary AER is grafted adjacent to a developing limb, a
supernumerary limb results.
- Prospective limb mesoderm grafted under flank ectoderm will promote
formation of an AER and a supernumerary limb.
- If limb bud mesoderm is removed from an early limb bud, the AER regresses
and the mesoderm ceases proliferation.
- Therefore, limb outgrowth involves reciprocal interactions between
the AER and underlying mesoderm.
What Controls Establishment of Limb Axes?
- The limb had three axes: Proximo-distal; Anterior-posterior and Dorsal-ventral.
Removal of the AER at progressively later stages results in a truncated
limb with progressively more distal elements. Thus, these elements are
laid down in a proximal-to-distal direction.
The AER does not regulate P-D polarity. Fates of cells are determined by
the length of time they spend in the progress zone. Those residing
there the longest become the most distal elements.
This axis is determined by the zone of polarizing activity (ZPA),
which is located at the junction between the limb bud and the body wall.
Grafts of the ZPA to the anterior margin of a host limb bud causes duplication
of digits in mirror-image symmetry.
A diffusible morphogen (retinoic acid?) is released from the ZPA.
We are now going to elaborate on two topics: limb muscle determination
and differentiation and determination of the limb bud itself.
As we have previously discussed, the
cells that will produce the limb muscles in the vertebrate embryo migrate
away from the lateral region of the somites and develop into muscle within
the limbs. Myogenic regulatory factors (MRFs) are required for their differentiation,
but the expression of MRFs is delayed until the cells have migrated into
the developing limbs and become established there (Figs. 2 and 3, Table
1, Sassoon et al., 1989; Figs. 2 and 3, Tajbakhsh and Buckingham,
What distinguishes the migratory limb myogenic precursors from the other
muscle precursors derived from the somites, and what triggers their delayed
terminal differentiation? Differentiation of myogenic cells in culture is
arrested by growth factors, such as FGF and TGF-ß (Konigsberg, 1971;
Yaffe, 1971; Kardami et al., 1985; Lathrop et al., 1985; Olson
et al., 1986; Massagué et al., 1986; Florini et
al., 1986). These results have led to the hypothesis that differentiation
of myogenic precursors occurs in response to low growth factor levels (Konigsberg,
1971; Yaffe, 1971).
A corollary of this hypothesis would be that growth factor signaling is
responsible for preventing myogenic differentiation within the somites and
allowing migration of myoblasts to the limb buds. Upon their arrival in
the limb buds, a down-regulation of growth factor signaling would allow
their differentiation. This hypothesis has been tested by perturbing FGF
signaling (Itoh et al., 1996). The results of these experiments suggests
that FGF prevents differentiation of myogenic precursors within the somites;
instead, these cells migrate to the limb buds, where they differentiate,
presumably because of reduced growth factor signaling. A decline in growth
factor signaling can be controlled either by a decline in the ligand, a
decline in the receptor or both.
A feature of the limb precursor cells is their expression
of Pax-3, which is a transcription factor of the paired-box type (Figs.
1, 2 and 7, Goulding et al., 1994; Fig. 6, Williams and Ordahl, 1994).
A role for Pax-3 in limb development is further suggested by observations
on the mouse mutant splotch. Embryos homozygous for splotch
lack a functional Pax-3 gene and develop into mice without skeletal
limb muscle but having normal trunk musculature (Figs. 8 and 9, Goulding
et al., 1994). These observations suggest that Pax-3 is essential
for the proliferation, specification and migration of the limb myogenic
precursors. Recent evidence suggests that Pax-3 is necessary for migration,
rather than differentiation, of these cells (Daston et al., 1966).
The Roles of the FGF Family in Limb Bud Induction and Development
The limb bud itself is derived from the lateral plate mesoderm and its
overlying ectoderm. The signals involved in limb bud induction come from
the intermediate mesoderm, which lies between the somite and lateral plate
mesoderm and forms the mesonephros. The involvement of the mesonephros in
limb induction is shown by retardation of limb development caused by mesonephros
ablation (Fig. 3, Geduspan and Solursh, 1992) and enhancement of limb development
in vitro by co-culture of limb bud and mesonephros (Fig. 7, Geduspan
and Solursh, 1992).
We previously discussed the role of FGFs in migration of myogenic precursors
to the limb bud and their terminal differentiation in the limb. There is
evidence that members of the FGF family are also involved in induction of
the limb bud itself. Cohn et al. (1995) have shown that beads soaked
in FGFs1, 2 or 4 will induce ectopic wings when implanted in flank mesoderm.
Once induced, FGF family members may also play a role in subsequent development
of the limb. For example, FGF2 and 4 can substitute for the apical ectodermal
ridge (AER) at the tip of the limb bud to maintain limb outgrowth and pattern
the developing limb (Niswander et al., 1993; Fallon et al.,
We shall now examine the putative role of one of the FGFs (FGF8) in induction
and formation of the chick limb. The possibility that FGF8 might be at least
one of the endogenous inducers produced by the intermediate mesoderm was
investigated by determining the expression of the Fgf8 gene in that
tissue in the chick embryo and its effects on limb development (Crossley
et al., 1996).
As shown in Figure 2 A-E, Fgf8 is expressed in the intermediate mesoderm
at the level of both the prospective forelimb and hindlimb when normal limb
induction is occurring. To determine whether FGF8 is capable of inducing
a limb, beads soaked in the protein were inserted into the lateral plate
mesoderm in the interlimb region of chick embryos. As shown in Figs. 2G
and H, the beads were capable of inducing ectopic limbs.
These results indicate that FGF8 is produced at the right time and place
and has the functional capacity to induce limbs.
Just before the limb bud protrusion becomes detectable, expression of Fgf8
becomes detectable in the ectoderm within the limb territory, and expression
becomes enhanced and persists in the limb ectoderm as limb outgrowth occurs
(Fig. 3). Expression of Fgf8 is not seen elsewhere in the ectoderm.
One of the functions of the AER is to maintain the zone of polarizing activity
(ZPA), a region of mesoderm in the posterior margin of the limb bud that
plays an important role in determining the anterior-posterior axis of the
limb (see Fig. 15.12, Browder et al., 1991). The functional properties
of the ZPA are mediated by Sonic hedgehog (Shh), which is
expressed in the ZPA (Fig. 4, Riddle et al., 1993). Application of
retinoic acid (RA) to the anterior margin of a limb bud will initiate an
ectopic ZPA. The ectopic ZPA expresses Shh, but only if
either an intact AER or exogenous FGF is present (Riddle et al.,
1993; Niswander et al., 1994).
This result suggests that a member of the FGF family produced by the
AER may be required for induction of Shh activity.
Is FGF8 one of the endogenous signals for Shh expression? Shortly
after the initial expression of Fgf8 in the ectoderm, Shh
expression is initiated underlying the posterior end of the domain of those
Fgf8-expressing cells (Figs. 3E and F, Crossley et al., 1996).
As development proceeds, the number of Shh-expressing cells increases
and the domain of expression extends proximal to that of the posterior margin
of the Fgf8-expression domain (Figs. 3G-I). These results are suggestive
that FGF8 protein plays a role in induction of Shh expression.
To test this hypothesis, anterior limb mesoderm was exposed to a bead soaked
in FGF8 and a bead exposed to RA. To eliminate endogenous FGF, the AER was
removed. Shh expression was detected in the mesodermal cells adjacent
to the FGF8 bead (Fig. 3J). Neither FGF8 nor RA alone could induce Shh
expression (Fig. 3K).
As previously discussed, FGF2 and 4 can perform the essential functions
of the AER. To determine whether FGF8 can substitute for the AER in maintaining
expression of Shh in the ZPA, the AER was removed from a limb bud
and an FGF8 bead was placed adjacent to posterior limb bud mesoderm; Shh
expression was maintained 24 hours later (Fig. 3L). In the absence of FGF8
beads, no Shh expression could be detected.
On the basis of the results described thus far, Crossley et al. (1996)
have proposed that FGF8 produced in the intermediate mesoderm initiates
Fgf8 expression in the surface ectoderm in the limb field, which
- in turn - promotes outgrowth of the underlying lateral plate mesoderm
and expression of Shhin the posterior margin of the emerging limb
To test this hypothesis, they implanted an FGF8 bead in the mesoderm in
the interlimb region (Fig. 4A). The implant triggered an outgrowth of the
lateral plate mesoderm and expression of Fgf8 in the overlying ectoderm
(Fig. 4B). A similar result was obtained using a bead soaked in FGF4; i.e.,
FGF4 also triggers lateral plate mesoderm outgrowth and Fgf8 expression
(Figs. 4C, D and E). Expression of Fgf4 was also detected, but at
a later time; the chronology of gene expression in the ectopic limbs is:
Fgf8, followed by Shh, followed by Fgf4.
This suggests that Fgf8 expression in the ectoderm initiates limb
bud outgrowth and Shh expression and that both Fgf4 and
Fgf8 (and possibly other FGF family members) are expressed in the AER
to sustain limb bud outgrowth and maintain Shh expression in the
ZPA. A model based upon these results is shown in Figure 5.
The involvement of the intermediate mesoderm in the induction of limb development
may provide an explanation for the effects of the drug thalidomide on limb
development. This drug, which was withdrawn from the market in 1961, caused
limb deformities if taken by the mother at critical times during pregnancy.
Lash and Saxén (1972) observed that thalidomide prevented the human
mesonephros from promoting cartilage growth in vitro. Furthermore,
radioactive thalidomide was found to bind to the mesonephros. These observations
suggest that the FGF8 signaling that normally originates in the mesonephros
to trigger limb bud development may be prevented by thalidomide. It will
be interesting to learn whether the FGF8 signaling mechanism is inhibited
- What is the AER, and what role does it play in limb outgrowth?
- Oh, yeah? What's your evidence.
- What are the three axes of the limb, and what controls their establishment?
- Discuss experiments that tell us how the ZPA functions and what morphogen
might be released from it.
- What is the proposed relationship between growth factor signaling and
differentiation of limb muscles?
- What is the proposed role of Pax-3 in limb myogenesis?
- Where does the limb bud originate?
- Which tissue induces limb bud formation?
- What's the evidence?
- What factors signal limb bud formation?
- What's the evidence?
- What is the presumed role of sonic hedgehog in limb formation, and
what are the interactions between it and FGFs?
- What might we conclude about thalidomide action in perturbing limb
development from in vitro experiments?
- How might thalidomide interact with FGF8?
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