Limb development in a "nonmodel" vertebrate, the direct-developing frog Eleutherodactylus coqui.

Mechanisms that mediate limb development are regarded as highly conserved among vertebrates, especially tetrapods. Yet, this assumption is based on the study of relatively few species, and virtually none of those that display any of a large number of specialized life-history or reproductive modes, which might be expected to affect developmental pattern or process. Direct development is an alternative life history found in many anuran amphibians. Many adult features that form after hatching in metamorphic frogs, such as limbs, appear during embryogenesis in direct-developing species. Limb development in the direct-developing frog Eleutherodactylus coqui presents a mosaic of apparently conserved and novel features. The former include the basic sequence and pattern of limb chondrogenesis, which are typical of anurans generally and appear largely unaffected by the gross shift in developmental timing; expression of Distal-less protein (Dlx) in the distal ectoderm; expression of the gene Sonic hedgehog (Shh) in the zone of polarizing activity (ZPA); and the ability of the ZPA to induce supernumerary digits when transplanted to the anterior region of an early host limb bud. Novel features include the absence of a morphologically distinct apical ectodermal ridge, the ability of the limb to continue distal outgrowth and differentiation following removal of the distal ectoderm, and earlier cessation of the inductive ability of the ZPA. Attempts to represent tetrapod limb development as a developmental "module" must allow for this kind of evolutionary variation among species.

Inhibition of neural crest migration in Xenopus using antisense slug RNA.

Based primarily on studies in the chick, it has been assumed that the zinc finger transcription factor Slug is required for neural crest migration. In the mouse, however, Slug is not expressed in the premigratory neural crest, which forms normally in Slug -/- animals. To study the role of Slug in Xenopus laevis, we used the injection of XSlug antisense RNA and tissue transplantation. Injection of Slug antisense RNA did not suppress the early expression of the related gene XSnail, but led to reduced expression of both XSlug and XSnail in later stage embryos, whereas the expression of another neural crest marker, XTwist, was not affected. Down-regulation of XSlug and XSnail was associated with the inhibition of neural crest cell migration and the reduction or loss of many neural crest derivatives. In particular, the formation of rostral cartilages was often highly aberrant, whereas the posterior cartilages were less frequently affected. The effects of Slug antisense RNA on neural crest migration and cartilage formation were rescued by the injection of either XSlug or XSnail mRNA. These studies indicate that XSlug is required for neural crest migration, that XSlug and XSnail may be functionally redundant, and that both genes are required to maintain each other's expression in the neural crest development of xenopus laevis.

Limb development and evolution: a frog embryo with no apical ectodermal ridge (AER).

The treefrog Eleutherodactylus coqui is a direct developer--it has no tadpole stage. The limb buds develop earlier than in metamorphosing species (indirect developers, such as Xenopus laevis). Previous molecular studies suggest that at least some mechanisms of limb development in E. coqui are similar to those of other vertebrates and we wished to see how limb morphogenesis in this species compares with that in other vertebrates. We found that the hind limb buds are larger and more advanced than the forelimbs at all stages examined, thus differing from the typical amniote pattern. The limb buds were also small compared to those in the chick. Scanning and transmission electron microscopy showed that although the apical ectoderm is thickened, there was no apical ectodermal ridge (AER). In addition, the limb buds lacked the dorsoventral flattening seen in many amniotes. These findings could suggest a mechanical function for the AER in maintaining dorsoventral flattening, although not all data are consistent with this view. Removal of distal ectoderm from E. coqui hindlimb buds does not stop outgrowth, although it does produce anterior defects in the skeletal pattern. The defects are less severe when the excisions are performed earlier. These results contrast with the chick, in which AER excision leads to loss of distal structures. We suggest that an AER was present in the common ancestor of anurans and amniotes and has been lost in at least some direct developers including E. coqui.