The function of Gas1 in Hedgehog signaling

Cellular signaling initiated by Hedgehog (HH) binding to Patched1 has profound importance in mammalian embryogenesis, genetic disease, and cancer. HH acts as a morphogen to specify distinctive cell fates using different concentration thresholds but our knowledge of how the concentration gradient is interpreted into the activity gradient is incomplete. The membrane protein Growth Arrest Specific Gene 1 (GAS1) was thought to be a negative regulator of the HH concentration gradient. Here, I present unexpected genetic evidence that Gas1 positively regulates HH signaling in multiple developmental contexts, an effect particularly noticeable at regions where HH acts at low concentration. Using a combination of in vitro cell culture and in ovo electroporation assays, I demonstrate that GAS1 enhances HH signaling activity in a cell-autonomous manner. These results support a model in which GAS1 helps transform the HH protein gradient into the observed activity gradient. I propose that Gas1 is an evolutionarily novel, vertebrate-specific HH pathway regulator.;Holoprosencephaly (HPE) is a common birth defect predominantly affecting the forebrain and face and has been linked to mutations in the SHH gene. Using cell surface binding assays, in vitro activity assays, and a limb bud explant culture assay, I provide evidence that SHH contains a unique binding surface for its interaction with GAS1 which is also important for maximal signaling activity. Within this surface, the N115 residue of human SHH has been documented to associate with HPE when mutated to lysine (N115K). I provide evidence that HPE associated with this mutation can be mechanistically explained by a severely reduced binding of SHH to GAS1, and predict a similar result if a mutation were to occur at Y80. Our data support the hypothesis that GAS1 is a candidate modifier loci for HPE.;To elucidate the biochemical/cellular mechanism of GAS1's enhancement of HH signaling, I performed live cell imaging of fluorescently labeled GAS1, HH, and Patched1 proteins. I discovered evidence supporting that GAS1 acts a HH transporter, delivering HH to the intracellular location where Patched1 is located.