Roles and expression of Fgf/RTK signaling modulators, Sproutys and Sulfs, in Xenopus limb development and regeneration

Abstract

The amphibian model, Xenopus laevis, is capable of perfect epimorphic limb regeneration prior to metamorphosis. It had been indicated that the amphibians’ abilities to regenerate lost structures may be attributed to certain intrinsic factors, such as unique patterns of gene expression following trauma. Transcriptome studies and more recent molecular analyses have shown that members of the fibroblast growth factor (fgf) families are re-expressed in regenerating Xenopus limb. The expression of certain fgf members, such as fgf-8 and -10, plays positive roles in successful limb regeneration in Xenopus. Therefore, the functions of this group of morphogens during regeneration are of particular interest to understanding the secrets to regenerative success in regenerative taxa, such as the clawed frogs. Members of the Fgf are morphogens that direct cell differentiation and functions during developments. Their biological activities are regulated by a variety regulators, such as the intracellular inhibitor, Sproutys (Spry), and the extracellular heparan sulfates (Sulf). These modulators act on the Fgf-triggered mitogen-activated protein kinase (MAPK) signaling cascade at various points, influencing the eventual biological response. We hypothesised that, similar to the reports in other vertebrate models, Xenopus spry and sulf also play important roles in regulating Fgf activities in limb development, and possibly regeneration. This was indicated by in situ hybridisation data described in Chapter 3 and 4, showing that the expression profiles of three of the Xenopus spry and two sulf genes either complemented or overlapped with known region of the fgfs expression during development and regeneration. The developmental expression patterns of the Xenopus spry and sulf genes were distinct from their avian and murine homologues. They also demonstrated varying levels of expression in either the proliferating blastema or the apical epidermal cap in regenerating limb. These data indicate unique functions of the two sets of genes compared to their avian and mammalian homologues. Based on these observations, we speculate that fine manipulation of the Fgf- activated MAPK pathways may be achieved by controlling the expression of spry and/or sulf. To test the last hypothesis, we attempted functional studies by generating a heat shock inducible spry-1a over-expressing X. laevis line. Transgenic frogs carrying a novel heat shock inducible DNA-directed RNAi transgene system were also created, in hope to allow for sequence-specific gene knock-down/-out experiments in Xenopus. Regrettably, these experiments did not result in any usable data. In conclusion, this study provides the first detailed expression profiles of the Xenopus spry and sulf during limb development and regeneration. The dynamic and distinct expressions of these genes in Xenopus compared to the other model systems suggest unique functions of these genes in the amphibian model that may also be related their more superior regenerative ability. Therefore, further functional study targeting the effects of these Fgf regulators in limb regeneration is warranted. Finally, although the functional experiments using transgenics described in Chapter 3 and 5 did not yield any usable data, we believe that the detailed procedures discussed here will be useful to future studies.