CONTENTS

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.
Proximo-distal

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.
Anterior-posterior

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.

Limb Muscle

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, 1994).

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., 1996).

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., 1994).

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 bud.

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 by thalidomide.

Learning Objectives

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?

Digging Deeper:

Dr. Carthew's lecture on limb development.

Recent Literature

Altabef, M., Clarke, J. D. W. and Tickle, C. 1997. Dorso-ventral ectodermal compartments and origin of apical ectodermal ridge in developing chick limb.

Carpenter, E. M., Goddard, J. M., Davis, A. P., Nguyen, T. P. and Capecchi, M. R. 1997. Targeted disruption of Hoxd-10 affects mouse hindlimb development.

Cygan, J. A., Johnson, R. L. and McMahon, A. P. 1997. Novel regulatory interactions revealed by studies of murine limb pattern in Wnt-7a and En-1 mutants.

Dealy, C.N. 1997. Hensen's node provides an endogenous limb-forming signal. Develop. Biol. 188: 216-223.

Dealy, C.N., Seghatoleslami, M.R., Ferrari, D. and Kosher, R.A. 1997. FGF-stimulated outgrowth and proliferation of limb mesoderm is dependent upon syndecan-3. Develop. Biol. 184: 343-350.

Deng, C., Bedford, M., Li, C., Xu, X., Yang, X. Dunmore, J. and Leder, P. 1997. Fibroblast growth factor receptor-1 (FGFR-1) is essential for normal neural tube and limb development. Develop. Biol. 185: 42-54.

Hérault, Y., Fraudeau, N., Zákány, J. and Duboule, D. 1997. Ulnaless (Ul), a regulatory mutation inducing both loss-of-function and gain-of-function of posterior Hoxd genes. Development 124: 3493-3500.

Knezevic, V., De Santo, R., Schughart, K., Huffstadt, U., Chiang, C., Mahon, K. A. and Mackem, S. 1997. Hoxd-12 differentially affects preaxial and postaxial chondrogenic branches in the limb and regulates Sonic hedgehog in a positive feedback loop.

Lu, H.-C., Revelli, J.-P., Goering, L., Thaller, C. and G. Eichele. 1997. Retinoid signaling is required for the establishment of a ZPA and for the expression of Hoxb-8, a mediator of ZPA formation. Development 124: 1643-1651.

Michaud, J.L., Lapointe, F. and Le Douarin. 1997. The dorsoventral polarity of the presumptive limb is determined by signals produced by the somites and by the lateral somatopleure. Development 124: 1453-1463.

Ohsugi, K., Gardiner, D.M. and Bryant, S.V. 1997. Cell cycle length affects gene expression and pattern formation in limbs. Develop. Biol. 189: 13-21.

Ohuchi, H., et al. 1997. The mesenchymal factor, FGF10, initiates and maintains the outgrowth of the chick limb bud through interaction with FGF8, an apical ectodermal factor. Development 124: 2235-2244.

Peichel, C.L., Prabhakaran, B. and Vogt, T.F. 1997. The mouse Ulnaless mutation deregulates posterior HoxD gene expression and alters appendicular patterning. Development 124: 3481-3492.

Stratford, T. H., Kostakopoulou, K. and Maden, M. 1997. Hoxb-8 has a role in establishing early anterior-posterior polarity in chick forelimb but not hindlimb.

Tamura, K., Yokouchi, Y., Kuroiwa, A. and Ide, H. 1997. Retinoic acid changes the proximodistal developmental competence and affinity of distal cells in the developing chick limb bud. Develop. Biol. 188: 224-234.

Vargesson, N., Clarke, J.D.W., Vincent, K., Coles, C., Wolpert, L. and Tickle, C. 1997. Cell fate in the chick limb bud and relationship to gene expression. Development 124: 1909-1918.

Yang, Y., Drossopoulou, G., Chuang, P.-T., Duprez, D., Marti, E., Bumcrot, D., Vargesson, N., Clarke, J., Niswander, L., McMahon, A. and Tickle, C. 1997. Relationship between dose, distance and time in Sonic Hedgehog-mediated regulation of anteroposterior polarity in the chick limb.

Zeller, R. and Duboule. 1997. Dorso-ventral limb polarity and origin of the ridge: on the fringe of independence? BioEssays 19: 541-546.

References

Browder, L.W., C. A. Erickson and W.R. Jeffery. 1991. Developmental Biology. Third Edition. Saunders College Publishing. Philadelphia.

Cohn, M.J., J.C. Izpisúa-Belmonte, H. Abud, J.K. Heath and C. Tickle. 1995. Fibroblast growth factors induce additional limb development from the flank of chick embryos. Cell 80: 739-746.

Crossley, P.H., G. Minowaqda, C.A. MacArthur and G.R. Martin. 1996. Roles for FGF8 in the induction, initiation, and maintenance of chick limb development. Cell 84: 127-136.

Daston, G., E. Lamar, M. Olivier and M. Goulding. 1996. Pax-3 is necessary for migration but not differentiation of limb muscle precursors in the mouse. Development 122: 1017-1027.

Florini, J.R. A.B. Roberts, D.Z. Ewton, S.L. Falen et al. 1986. Transforming growth factor ß: a very potent inhibitor of myoblast differentiation, identical to the differentiation inhibitor secreted by buffalo rat liver cells. J. Biol. Chem. 261: 16509-16513.

Geduspan, J.S. and M. Solursh. 1992. A growth-promoting influence from the mesonephros during limb outgrowth. Dev. Biol. 151: 242-250.

Goulding, M., A. Lumsden and A.J. Paquette. 1994. Regulation of Pax-3 expression in the dermomyotome and its role in muscle development. Development 120: 957-971.

Itoh, N., T. Mima and T. Mikawa. 1996. Loss of fibroblast growth factor receptors is necessary for terminal differentiation of embryonic limb muscle. Development 122: 291-300.

Konigsberg, I. R. 1971. Diffusion-mediated control of myoblast fusion. Dev. Biol. 26: 133-152.

Kardami, E. D. Spector and R.C. Strohman. 1985. Myogenic growth factor present in skeletal muscle is purified by heparin-affinity chromatography. Proc. Natl. Acad. Sci. USA 82: 8044-8047.

Lash, J.W. and L. Saxén 1972. Human teratogenesis: in vitro studies of thalidomide-inhibited chondrogenesis. Dev. Biol. 28: 61-70.

Lathrop, B. E. Olson and L. Glaser. 1985. Control by fibroblast growth factor of differentiation in the BC3H1 muscle cell line. J. Cell Biol. 100: 1540-1547.

Massagué, J., S. Cheifetz, T. Endo and B. Nadal-Ginard. 1986. Type ß transforming growth factor is an inhibitor of myogenic differentiation. Proc. Natl. Acad. Sci. USA 83: 8206-8210.

Niswander, L., S. Jeffrey, G.R. Martin and C. Tickle. 1994. A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371: 609-612.

Olson, E.N., E. Sternberg, J.S. Hu, G. Spizz and C. Wilcox. 1986. Regulation of myogenic differentiation by type ß transforming growth factor. J. Cell Biol. 103: 1799-1805.

Riddle, R.D., R.L. Johnson, E. Laufer and C. Tabin. 1993. Sonic hedgehog mediates the polarizing activity of the ZPA. Cell 75: 1401-1416.

Sassoon, D., G. Lyons, W.E. Wright et al. 1989. Expression of two myogenic regulatory factors myogenin and MyoD1 during mouse embryogenesis. Nature 341: 303-307.

Tajbakhsh, S. and M.E. Buckingham. 1994. Mouse limb muscle is determined in the absence of the earliest myogenic factor myf-5. Proc Natl Acad USA 91: 747-751.

Williams, B.A. and C.P. Ordahl. 1994. Pax-3 expression in segmental mesoderm marks early stages in myogenic cell specification. Development 120: 785-796.

Yaffe, D. 1971. Developmental changes preceding cell fusion during muscle differentiation in vitro. Exp. Cell Res. 66: 33-48.

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Last revised Tuesday, July 21, 1998