The tetrapod limb is an excellent and well-studied model for investigating the development and evolution of morphological characters. We have come to understand many details of limb development mechanisms—the roles of individual genes and gene products in morphological patterning and how they interact with other genes and gene products. Most of these data are from the amniote model species mouse and chicken. It is unclear how general these mechanisms are among the tetrapods, and understanding the extent to which they are general or variable is of great importance (Stopper and Wagner,2005). Comparatively little is known about mechanisms of limb development in the other main branch of tetrapods, the amphibians, though we do know of many instances of variation in developmental mechanisms between the amniotes and amphibians, and within each of these groups (Stopper and Wagner,2005). Despite all that we know about the genetics of limb development in model organisms, and all that we know about variation in molecular mechanisms of limb development, cases for mechanistic changes that are causal in the evolution of morphology are rare. Further elucidation of developmental mechanisms in limbs with differing morphologies should help to shed light on how changes in mechanisms of development cause evolutionary changes in morphology.
One major difference in limb development that exists among tetrapods is that of the order of digit development. Amniotes and anurans develop their digits in relative synchrony; digit cartilage condensation and bone chondrification usually start with the penultimate posterior digit, which is quickly followed by development of the most posterior digit and the more anterior digits developing in a posterior-to-anterior sequence (Shubin and Alberch,1986). Urodeles, however, develop their most anterior two digits first, followed by the posterior digits in an anterior-to-posterior sequence (Shubin and Alberch,1986; Wake and Shubin,1998). Considering the current understanding of tetrapod relationships and the phylogenetic distribution of these modes of development, it is likely that the shared amniote and anuran pattern is ancestral, and the urodele pattern is derived (Shubin,1995; Vorobyeva and Hinchliffe,1996; Wagner et al.,1999; Stopper and Wagner,2005). There are several hypotheses that have been posed to explain the novel order of digit development in urodeles. One hypothesis states that this difference can be explained by a polyphyletic origin of lissamphibians (Holmgren,1933; Jarvik,1980; Hanken,1986). In this case, anurans are thought to be more closely related to amniotes than they are to urodeles (Fig. 1A). This hypothesis, however, is refuted by the vast majority of morphological and molecular studies; most evidence supports monophyly of lissamphibians (Fig. 1B), with anurans and urodeles being equally closely related to amniotes, and more closely related to each other than either is to amniotes (Milner,1988; Hedges et al.,1990; Trueb and Cloutier,1991; Marshall and Schultze,1992; Cannatella and Hillis,1993; Eernisse and Kluge,1993; Larson and Dimmick,1993; Ahlberg and Milner,1994; Duellman and Trueb,1994; Zardoya and Meyer,2001). Another hypothesis suggests that urodeles evolved the heterochronic shift to earlier anterior digit development as an adaptation for aquatic larvae to interact with substrate (Wake and Marks,1993). A third hypothesis posits that an ancestor of all extant urodeles had limbs reduced to two digits—those homologous to digits 3 and 4 of ancestral tetrapods—and then subsequently evolved novel, later-developing posterior digits that have no homologues in amniotes or anurans (Wagner et al.,1999). Here we investigate one major aspect of anterior-posterior patterning, the Hedgehog signaling pathway, and ask what role Shh plays in the derived mode of digit development in urodeles.
The patterning of tetrapod limbs is largely directed by signaling across the three major axes: proximal-distal, dorsal-ventral, and anterior-posterior. Signaling along the anterior-posterior axis is mediated by the zone of polarizing activity (ZPA). The ZPA lies at the posterior margin of the limb bud, near the distal end. The Sonic Hedgehog (Shh) protein is expressed in, and secreted from, the ZPA. While the exact nature of the ZPA's activity is not fully understood, one model poses that Shh diffusion from this region forms a posterior-to-anterior gradient in its concentration, with the highest levels at the posterior (Saunders and Gasseling,1968; Riddle et al.,1993). Expansion along the anterior-posterior axis is directed by Shh and the anterior-posterior identity of any region of the limb is thought to be specified by the amount of Shh received by that region. Multiple lines of evidence suggest that the shh expression domain and the general anterior-posterior patterning function of Shh in limbs has been conserved across tetrapods (Riddle et al.,1993; Chang et al.,1994; Endo et al.,1997; Imokawa and Yoshizato,1997; Torok et al.,1999; Hanken et al.,2001). In urodeles, including Ambystoma mexicanum, although the limb shh domain appears to be slightly smaller and more proximal than in non-urodele tetrapods, shh expression is very similar in timing and location to that of non-urodele tetrapods (Imokawa and Yoshizato,1997; Torok et al.,1999).
To better understand the functions of the members of the Hedgehog family during limb development in urodele amphibians, and to analyze the fine-scale temporal dynamics of the patterning properties of Hedgehog gene products, we use cyclopamine to inhibit Hedgehog signaling during five different time windows of limb development in axolotl (A. mexicanum). It was previously shown that, when administered during limb regeneration in A. mexicanum, cyclopamine causes varying reductions in digit number based on its concentration (Roy and Gardiner,2002). In our experiments, cyclopamine exposure generally causes reductions in the anterior-posterior extent of limb development, including digit loss, and failures of limb element separation. Cyclopamine exposure causes digit reduction in the exact opposite order of their development (i.e., the posterior digits are affected first). This pattern mimics the pattern of natural variation in urodele digit reduction.