Signaling centers in early limb development

Several signaling centers have been identified in patterning of the developing limb bud. The zone of polarizing activity (ZPA) located in the distal posterior limb is a source of the secreted factor Sonic hedgehog (Shh) responsible for anterior-posterior patterning of the limb. Another signaling center, the apical ectodermal ridge (AER) is located at the distal tip of the limb and is a source of fibroblast growth factors (Fgfs) required for proper limb outgrowth. This dissertation examines components of a positive feedback loop between Shh in the ZPA and Fgfs in the AER. The bone morphogenetic protein (Bmp) antagonist Gremlin is an intermediate in the Shh-Fgf feedback loop, and termination of the loop occurs by expansion of a region in the posterior limb that is refractory to Gremlin expression. In this dissertation, we demonstrate that Shh acts indirectly via Bmp activity to upregulate Gremlin. Bmp activity is necessary and sufficient for Gremlin upregulation in a concentration-dependent manner, and Shh activity is not independently required. We present evidence that breakdown of the Shh-Fgf loop results from failure of Bmp activity to upregulate Gremlin in the posterior. Finally, we show that Shh activity is required for exclusion of Gremlin in the posterior. These findings contribute to our understanding of how the Shh-Fgf loop is maintained and terminated, as well as to the role of Bmp activity in early limb development.;The second focus of this dissertation is a mechanism for localization of the ZPA. Given its importance in patterning the limb, how Shh expression is tightly restricted to the posterior margin has been a major question in limb development. In this dissertation, we identify the non-AER dorsal-ventral ectoderm border overlying the anterior and posterior limb margins as a new signaling center that regulates the restricted expression of Shh. Expression of the transcription factor Tbx2, restricted to the anterior and posterior limb margins and known to regulate Shh, requires proximity to this signaling center. Moreover, grafts of this signaling center are sufficient to induce ectopic Tbx2 and to expand Shh anteriorly. These findings support a model explaining the restriction of Shh to the posterior margin throughout limb outgrowth as well as the restriction of ectopic Shh to the anterior margin that occurs when other mechanisms regulating Shh expression are disrupted.