Mechanism and Evolution of Mammalian Hedgehog Signaling

The Hedgehog (Hh) signaling pathway is an evolutionarily conserved cascade important for embryonic development and postnatal regeneration. Mutations in components of the Hh pathway in humans result in a number of congenital birth defects, such as holoprosencephaly, as well as numerous cancers. In the fruit fly Drosophila melanogaster, transduction of the Hh signal from the positive membrane effector Smoothened (Smo) to the Ci/Gli transcription factors is mediated by a large cytoplasmic complex. This complex is scaffolded by the kinesin Costal-2, and contains the key regulator Fused (Fu), a putative serine-threonine kinase, and Suppressor of Fused (Sufu), a regulator of Ci/Gli proteins. The role of Fu and Sufu in mammalian Hh signaling and development are less well-characterized. In this dissertation, we show that mouse Fu is dispensable for mammalian Hh signaling, but instead participates in construction of the central pair apparatus of motile cilia. Strikingly, zebrafish Fu participates in both motile ciliogenesis and Hh signaling. We link Fu and a Costal-2 ortholog, Kif27 in mouse and Kif7 in zebrafish, to motile ciliogenesis, and advance hypotheses as to how these proteins evolved functions in diverse processes. We also show that mouse Sufu plays an evolutionarily conserved role in promoting the stabilization of Gli2 and Gli3. In part, Sufu opposes the activity of Spop, an adaptor protein for E3 ubiquitin ligases. The increased Hh pathway activity observed in Sufu mutant mice and embryonic fibroblasts can be attributed in part to elevated Gli1 activity. Finally, we investigate the trafficking of mouse Smo to the primary cilium, an organelle required for its activity in Hh transduction. We demonstrate that translocation of Smo to the cilium is necessary but not sufficient for pathway activation, as some classes of Smo antagonists can stimulate movement to the cilium. This result suggests a multi-step model for Smo activation in mammalian Hh transduction. Taken together, the data presented in this dissertation shed light on the evolution of the Hh pathway from invertebrates to mammals, and provide a deeper mechanistic understanding of mammalian Hh transduction that will be useful in the future design of rational therapies