The role and regulation of TGF-beta signaling during Xenopus regeneration and development

Signaling by TGF-β ligands is essential for regulating multiple processes in embryonic development and adult tissue homeostasis, including early mesendoderm formation, patterning of body axes, tissue growth, and wound healing. Many of these processes are recapitulated during epimorphic regeneration of lost tissues. In this dissertation, I explore the roles of TGF-β signals in embryonic development and epimorphic regeneration, primarily using the frog Xenopus laevis as a model system. SB-431542 is a chemical inhibitor that blocks signaling by TGF-β ligands. I have characterized the effects and specificity of SB-431542 in vivo using Xenopus and zebrafish embryos. I have also generated mutant type I TGF-β receptors that are resistant to inhibition by SB-431542, and have used these receptors both to test inhibitor specificity and to identify specific ligand-receptor pairs involved in TGF-β-dependent processes during development. The Xenopus tail can fully regenerate following experimental amputation. I examined the localization of p-Smad2, the direct intracellular transducer of TGF-β signaling, and found that it is upregulated throughout regeneration in a dynamic and highly regulated pattern. Inhibition of TGF-β signals during regeneration with SB-431542 resulted in failure to regenerate at multiple points. More specifically, TGF-β signaling plays several distinct roles in regeneration: (1) initial formation of a regenerative wound epithelium, (2) early establishment of the regeneration bud, and (3) later stimulation of cellular proliferation in the regenerating tail. I have also examined the role of TGF-β signals in post-gastrulation development. Both p-Smad2 and the TGF-β ligand GDF11 are expressed in similar patterns during tailbud stages in Xenopus . Morpholino knockdown of GDF11 results in defects in axial elongation of the tail and trunk and the complete elimination of p-Smad2, indicating that GDF11 generates the endogenous p-Smad2 pattern in the tailbud stage embryo in order to control axial patterning. I also show that cleavage by BMP-1/Tolloid family proteases is important to activate GDF11 signaling in Xenopus embryos, indicating that this regulatory mechanism is important in vivo . In summary, the work presented in this dissertation identifies novel and essential roles and regulatory mechanisms for TGF-β signaling in development and regeneration.