Tissue-Specific Roles for Hedgehog Co-Receptors During Vertebrate Embryogenesis

Abstract

The Hedgehog (HH) signaling pathway is essential for the proper patterning of multiple tissues during vertebrate embryogenesis, including the spinal cord, craniofacial structures, and limbs. At the cell surface, secreted HH ligands signal through the canonical receptor Patched 1 (PTCH1) and three co-receptors– GAS1, CDON and BOC. Interestingly, these proteins share similar expression patterns throughout development, bind to HH ligands, and interact with PTCH1. Together, these HH co-receptors are essential to transduce HH signaling in multiple tissues during embryogenesis.Recent studies suggest that, in addition to their combined, essential role in HH signal transduction, these co-receptors also play individual, tissue-specific roles in the regulation of HH signaling. However, the tissue-specific contributions of these co-receptors remain largely unexplored during vertebrate embryogenesis. The goals of this dissertation are to: 1) investigate novel tissue-specific roles for the HH co-receptors and 2) elucidate the mechanism of HH co-receptor function during vertebrate embryogenesis. During craniofacial development, the individual deletion of HH co-receptors results in variable craniofacial defects, in which the severity of the phenotype is dependent on the genetic background. To investigate the individual and combined contributions of the HH co-receptors to craniofacial development, I analyzed Gas1, Cdon and Boc mutant embryos on a congenic C57BL/6J background. The deletion of Gas1 and Cdon result in variable degrees of holoprosencephaly; a birth defect characterized by the complete or partial failure of the forebrain and the midface structures. In contrast, Boc deletion results in midface widening. Increasing severity of the craniofacial defects in Gas1 and Cdon mutants correlated with decreased levels of HH pathway activity, while Boc mutants display increased levels of HH pathway function. These data suggest that the HH co-receptors differentially contribute to craniofacial development. Further, Boc deletion in a Gas1 null background partially ameliorates the phenotypes observed in Gas1 single mutants. However, the rescue of the craniofacial defects observed in Gas1;Boc mutants is restricted to the craniofacial structures, while other tissues patterned by HH are more severely affected. Mechanistically, BOC can also selectively restrict proliferation in the cranial neural crest-derived mesenchyme. These data suggest that Boc functions as a tissue-specific HH pathway antagonist during craniofacial development.In this thesis, I also investigated the contribution of HH co-receptors to digit specification and limb patterning. Notably, previous studies have demonstrated roles for both Gas1 and Boc in HH-dependent digit specification, but not for Cdon. However, the known redundant functions of these HH co-receptors, combined with the early (embryonic day 9.5) lethality of Gas1;Cdon;Boc triple mutants has hindered efforts to definitively examine a potential contribution of Cdon to limb development. To dissect the function of CDON during HH-dependent digit specification, I performed limb-specific deletion of Cdon in a Gas1;Boc null background. Embryos lacking Gas1, Cdon and Boc display both severe digit specification defects in the radius, ulna, tibia and fibula. These experiments demonstrate that CDON does indeed contribute to digit specification and limb patterning. Overall, the data presented in this dissertation demonstrate that HH co-receptors contribute in both a redundant and tissue-specific fashion to HH signal transduction, and that these co-receptors are multi-functional regulators of HH signaling during vertebrate embryogenesis.