Title: Evolution and Development of the Anuran skeleton
Description: This dissertation investigates skeletal development and evolution in frogs (Order Anura) by analyzing gene function, gene expression, and developmental morphology, and through the generation of transgenic tadpoles. Each chapter addresses specific controversies or bridges gaps between different investigative methodologies within the field. Chapter 1 describes the expression and function of the runx2 gene in Xenopus laevis. Morpholino-based silencing of runx2 reveals an unexpected role for the protein product in the differentiation of larval chondrocytes. Runx2's role in cartilage formation is shared with zebrafish but not mouse or chicken. Additionally, in situ hybridizations with an anti-runx2 mRNA probe, along with anti-sox9 and col2a1 probes, reveal some of the earliest patterning of the craniofacial skeleton in Xenopus. Chapter 2 describes the mRNA distribution of three genes--runx2, sox9 and col2a1--in the direct-developing frog Eleutherodactylus coqui. The precocious development of adult head and limb skeletons in E. coqui is associated with the expression of these genes in the embryo. The expression of both sox9 and runx2 in the head characterizes several skeletal changes associated with direct development. The distribution of both genes in the limb reveals an unexpected early distal expression pattern, which differs from that in amniotes and contradicts current models of proximal-to-distal skeletal differentiation along the limb axis.

Chapter 3 investigates the convergence of skeletal development between Eleutherodactylus coqui and the Sri Lankan shrub frog Philautus silus, which has independently evolved terrestrial direct development. Several tadpole-specific features of the developing skeleton that are lost in E. coqui are retained in P. silus. This study reveals that the unique embryologic changes driven by the evolution of direct-development in E. coqui are not inevitably shared with other direct-developing lineages. Chapter 4 returns to Xenopus to describe regulation of the col2a1 gene in cartilage and provide a tool for transgene expression in cartilage-forming cells. A combination of bio-informatics and functional tests reveal a conserved region in the first intron that is capable of driving gene expression in cartilage when connected to a short promoter sequence. This cartilage-specific reporter construct provides a valuable tool for future research into anuran skeletal development and evolution.