The contribution of chicken embryology to the understanding of vertebrate limb development

The chicken is an excellent model organism for studying vertebrate limb development, mainly because of the ease of manipulating the developing limb in vivo. Classical chicken embryology has provided fate maps and elucidated the cell-cell interactions that specify limb pattern. The first defined chemical that can mimic one of these interactions was discovered by experiments on developing chick limbs and, over the last 15 years or so, the role of an increasing number of developmentally important genes has been uncovered. The principles that underlie limb development in chickens are applicable to other vertebrates and there are growing links with clinical genetics. The sequence of the chicken genome, together with other recently assembled chicken genomic resources, will present new opportunities for exploiting the ease of manipulating the limb.     

Targeted misexpression of constitutively active BMP receptor-IB causes bifurcation, duplication, and posterior transformation of digit in mouse limb

Members of bone morphogenetic proteins (BMPs) play important roles in many aspects of vertebrate embryogenesis. In developing limbs, BMPs have been implicated in control of anterior-posterior patterning, outgrowth, chondrogenesis, and apoptosis. These diverse roles of BMPs in limb development are apparently mediated by different BMP receptors (BMPR). To identify the developmental processes in mouse limb possibly contributed by BMP receptor-IB (BMPR-IB), we generated transgenic mice misexpressing a constitutively active Bmpr-IB (caBmpr-IB). The transgene driven by the mouse Hoxb-6 promoter was ectopically expressed in the posterior mesenchyme of the forelimb bud, the lateral plate mesoderm, and the whole mesenchyme of the hindlimb bud. While the forelimbs appeared normal, the transgenic hindlimbs exhibited several phenotypes, including bifurcation, preaxial polydactyly, and posterior transformation of the anterior digit. However, the size of bones in the transgenic limbs seemed unaltered. Defects in sternum and ribs were also found. The bifurcation in the transgenic hindlimb occurred early in the limb development (E10.5) and was associated with extensive cell death in the mesenchyme and occasionally in the apical ectodermal ridge (AER). Sonic hedgehog (Shh) and Patched (Ptc) expression appeared unaffected in the transgenic limb buds, suggesting that the BMPR-IB mediated signaling pathway is downstream from Shh. However, ectopic Fgf4 expression was found in the anterior AER, which may account for the duplication of the anterior digit. An ectopic expression of Gremlin found in the transgenic limb bud would be responsible for the ectopic Fgf4 expression. The observations that Hoxd-12 and Hoxd-13 expression patterns were extended anteriorly provide a molecular basis for the posterior transformation of the anterior digit. Together these results suggest that BMPR-IB is the endogenous receptor to mediate the role of BMPs in anterior-posterior patterning and apoptosis in mouse developing limb. In addition, BMPR-IB may represent a critical component in the Shh/FGF4 feedback loop by regulating Gremlin expression.     

Cellular analysis of limb development in the mouse mutant Hypodactyly

The limb defect in the mouse Hypodoctyly (Hd) affects only the distal structures. Heterozygotes (Hd/+) lack all or part of the distal phalanx and the terminal claw of digit 1 on the hindlimbs; mice homozygous (Hd/Hd) for the mutation have just one digit on each of the four limbs. Early limb development in the mutant appears normal and a change in morphology can only be detected later. Limb buds of Hd/+ and Hd/Hd embryos become reduced in width, with Hd/Hd buds becoming very pointed instead of rounded. This change in bud shape is correlated with an increase in cell death anteriorly in Hd/+ hindlimbs and both anteriorly and posteriorly in Hd/Hd fore- and hindlimb buds. The apical ectodermal ridge is very pronounced in pointed Hd/Hd limb buds. Mesenchyme cells from the Hd/Hd mutant in culture show a cell-autonomous change in behaviour and less cartilage differentiates. © 1996 Wiley-Liss, Inc.     

Inhibition of Polarizing Activity in the Anterior Limb Bud Is Regulated by Extracellular Factors

Anterior-posterior patterning of the developing limb is largely viewed as a function of polarizing activity. Recent evidence in polydactylous mutants, however, indicates that development of proper pattern also requires the involvement of inhibitory pathways in the anterior limb that prevent secondary polarizing zone formation, thus limiting the number of digits produced. We report the novel finding that grafts of extracellular matrix from the Mouse Posterior Limb Bud-4 cell line can induce supernumerary digits, including digits with posterior phenotype, from anterior chick limb mesenchyme. Unlike previously described mechanisms of pattern specification during limb development, it is shown that the extracellular matrix effect is not associated with release of an active signal. Rather, evidence is presented suggesting that heparan sulfate moieties in extracellular matrix grafts bind an endogenous, extracellular factor involved in inhibition of anterior polarizing activity, leading to derepression of the anterior limb and induction of polarizing zone marker genes including Sonic hedgehog and Bone morphogenetic protein-2.     

A tissue culture analysis of the steps in limb chondrogenesis

Summary Tissue-culture methods can be used to test the developmental capacity of embryonic cells. In micro-mass cultures, derived from wing cells of stages 21 through 24 chick embryos, aggregates of cells form and then differentiate into cartilage nodules, as judged by the presence of an Alcian blue staining extracellular matrix. Wing cells derived from embryos as young as stage 17 can form aggregates. However, unless they are treated with db cyclic AMP and theophylline, it is not until stage 20 that these aggregates can produce cartilage in culture. In clonal cell culture, cartilage colonies are not produced by primary cell suspensions of limb cells until stage 25 when overt cartilage differentiation is occurring in vivo. It is possible to obtain clonable cartilage cells from limb cells from embryos between stages 20 and 24 if the cells are either treated with db cyclic AMP and theophylline or maintained in suspension culture for 12 to 48 hr. On the basis of these in vitro results a multiple step model for the conversion of limb mesenchyme into cartilage cells is proposed. The model involves the appearance of cells with a predisposition to form aggregates, development of the capacity to form cartilage in response to elevated levels of cyclic AMP, the appearance of receptors that translate changes in either cell shape or cell cycle parameters into elevated levels of cyclic AMP, aggregation, elevated levels of cyclic AMP, cartilage cell determination, and differentiation. This model can serve as the basis for further tests. Presented in the Opening Symposium on Nutritional Factors and Differentiation at the 28th Annual Meeting of the Tissue Culture Association, New Orleans, Louisiana, June 6–9, 1977. This work was supported by USPHS Training Grant HD00152 from the National Institute of Child Health and Human Development, while P.B.A. was a postdoctoral trainee, and by NIH Grant HD05505 to M.S.     

Patterning in the vertebrate limb.

The past few years have seen the isolation and characterization of some of the genes involved in the control of limb pattern formation. Their possible role in this fundamental process is discussed in the light of recent data, and an attempt is made to superimpose this molecular approach to patterning on pre-existing conceptual views.     

Integration and dissociation of limb elements in flying vertebrates: a comparison of pterosaurs, birds and bats

Flapping flight has evolved independently in three vertebrate clades: pterosaurs, birds and bats. Each clade has a unique flight mechanism involving different elements of the forelimb. Here, patterns of limb integration are examined using partial correlation analysis within species and matrix correlation analysis across species to test whether the evolution of flapping flight has involved developmental dissociation of the serial homologues in the fore- and hind limb in each clade. Our sample included seven species of birds, six species of bats, and three species of pterosaurs for which sufficient sample sizes were available. Our results showed that, in contrast to results previously reported for quadrupedal mammals, none of the three clades demonstrated significant integration between serial homologues in the fore- and hind limb. Unexpectedly, there were few consistent patterns of within-forelimb correlations across each clade, suggesting that wing integration is not strongly constrained by functional relationships. However, there was significant integration within the hind limbs of pterosaurs and birds, but not bats, possibly reflecting the differing functions of hind limbs (e.g. upright support vs. suspension) in these clades.     

Congenic method in the chick limb buds by electroporation

Electroporation is a powerful tool with which to study limb development. Limb development, however, remains an intricate series of events, requiring the precise dissection of developmental processes using relevant transgenes. In this review, we describe the anatomy of the limb field as the basis of targeted electroporation, and specific expression vectors are discussed. We share a useful protocol for electroporation of chick limb buds, and the expression pattern of enhanced green fluorescent protein in the limb buds is used to demonstrate relevant embryonic patterning. Finally, useful trouble-shooting techniques are described.     

Involvement of Wnt-5a in chondrogenic pattern formation in the chick limb bud

Members of the Wnt family are known to play diverse roles in the organogenesis of vertebrates. The full-coding sequences of chicken Wnt-5a were identified and the role it plays in limb development was examined by comparing its expression pattern with that of two other Wnt members, Wnt-4 and Wnt-11, and by misexpressing it with a retrovirus vector in the limb bud. Wnt-5a expression is detected in the limb-forming region at stage 14, and in the apical ectodermal ridge and distal mesenchyme of the limb bud. The signal was graded along the proximal-distal axis at stages 20-28 and also along the anterior-posterior axis during early stages. It disappeared in the cartilage-forming region after stage 26, and was restricted to the region surrounding the phalanges at stage 34. Wnt-4 and Wnt-11, other members of the Wnt-5a-subclass, were expressed with a distinct spatiotemporal pattern during the later phase. Wnt-4 was expressed in the articular structure and Wnt-11 was expressed in the dorsal and ventral mesenchyme adjacent to the ectoderm. Wnt-5a expression was partially reduced after apical ectodermal ridge removal, whereas Wnt-11 expression was down-regulated by dorsal ectoderm removal. Therefore, expression of these Wnt was differentially regulated by the ectodermal signal. Misexpression of Wnt-5a in the limb bud with the retrovirus resulted in truncation of long bones predominantly in the zeugopod because of retarded chondrogenic differentiation. Distal elements, such as the phalanges and metacarpals, were not significantly reduced in size. These results suggest that Wnt-5a is involved in pattern formation along the proximal-distal axis by regulation of chondrogenic differentiation.     

Making digit patterns in the vertebrate limb

The vertebrate limb has been a premier model for studying pattern formation - a striking digit pattern is formed in human hands, with a thumb forming at one edge and a little finger at the other. Classic embryological studies in different model organisms combined with new sophisticated techniques that integrate gene-expression patterns and cell behaviour have begun to shed light on the mechanisms that control digit patterning, and stimulate re-evaluation of the current models.