Signaling receptor turnover by endocytic pathways in the patterning of the vertebrate nervous system

The patterning of the vertebrate nervous system is a key event in the generation of cell diversity and the wiring of the brain. It is thought that Sonic Hedgehog (Shh) signaling acts as a “morphogen” gradient specifying distinct cell types based on the concentration threshold of Shh signaling along the dorso-ventral axis. The default model states that Shh is a signaling molecule expressed in a gradient of high to low in the ventral nervous system. It binds to a receptor called Patched1 (Ptch1) at the cell surface in initiation an intracellular series of events that ultimately leads to transcriptional activation and cell fate acquisition. Normally, in the absence of ligand, Ptch1 prevents the activation of a co-receptor called Smoothened, which in turn is required for the activation of Shh pathway through the Gli transcription factors. Ptch1 therefore acts as a negative regulator for Shh pathway activation and the binding of Shh to Ptch1 relieves this inhibition. However, it is unclear how a graded response to Shh signaling is achieved in growing tissues such as the developing neural tube. What remains to be determined is how Ptch1 helps create a graded response to the Shh morphogen field in the developing vertebrate nervous system. The binding of Hedgehog ligands to Ptch1 triggers their internalization through the endocytic pathway and ultimately to the lysosome, where Ptch1 is degraded. Thus, one way to set the output range of Shh signaling is to control the steady-state levels of Ptch1 through endocytosis. In Drosophila and mammalian cells, it is known that ubiquitin ligases target Ptch1 to degradation and allow for the activation of Shh signaling. However, it is not known how Ptch1 internalization and targeting to endocytic and lysosomal vesicles is regulated. In a screen for novel Ptch1-interacting proteins, we identified proteins implicated in endocytic shuttling that may regulate the targeting of Ptch1 to intracellular vesicles. This is expected to promote Ptch1 degradation and enhance Shh-dependent ventral patterning in the developing nervous system. Our objectives are: (1) validate the role of these putative Ptch1-interacting proteins in endocyctic processing using assays in cell culture model systems; and (2) evaluate whether the over-expression and knockdown of these proteins during neural development can alter ventral patterning in response to Shh signaling. The proposed research addresses a fundamental question in neurobiology: how is the graded response to morphogen signaling controlled? We use a combination of in vitro and in vivo approaches in tractable model systems to address this question, providing an engaging training opportunity in basic cellular and developmental biology. Altogether, this research will significantly advance our understanding of the Shh signaling and lead to fundamental insights into how pattern is generated in the developing vertebrate nervous system.