Morphogenetic signaling from the cell surface to the nucleus during vertebrate eye development - Hedgehog Signal Transduction from the Cell Surface to the Nucleus during Vertebrate Eye Development The Hedgehog (Hh) pathway is essential for eye development, controlling a diverse array of fundamental patterning, morphogenesis, and differentiation events during oculogenesis. Misregulated Hh signaling instigates a spectrum of human developmental eye disorders, including holoprosencephaly, cyclopia, uveal coloboma and retinal dystrophy. This signaling pathway is therefore a prime therapeutic target for eye development disorders and for regenerative medicine efforts. During Hh signal transduction, the cell surface atypical G protein-coupled receptor (GPCR) SMOOTHENED (SMO) communicates with GLI transcription factors in the nucleus, via a mechanism that has remained enigmatic for decades. We recently found that SMO directly binds to the catalytic subunit of protein kinase A (PKAcat) and physically blocks its enzymatic activity. As a result, PKAcat cannot phosphorylate and inhibit GLI, leading to GLI activation. Thus, our work highlights two potential routes – traditional G protein coupling and our new direct PKAcat inhibition pathway – for Hh signal transduction from the cell surface to the nucleus during oculogenesis. How these two pathways regulate different stages of eye development, and how to target each of them optimally for therapeutic purposes, remains largely unknown. We hypothesize that SMO controls eye development by deploying the G protein coupling and PKA-binding pathways in a spatiotemporally distinct manner. Here we propose to combine organoid, and animal models, along with protein structure approaches, to uncover how SMO orchestrates eye development. We will: (1) learn how the SMO G protein coupling and PKA-binding pathways controls retinal development, using human retinal organoid development and retinal ganglion cell (RGC) differentiation as outputs for Hh pathway-dependent signaling processes in the human retina; (2) assess the impact of each SMO-dependent pathway on ocular Hh signaling in a whole animal context, using zebrafish to define the spatiotemporal contributions of each SMO-dependent pathway to optic fissure formation and RGC differentiation in vivo; (3) solve the structure of a vertebrate SMO / PKAcat complex using X-ray crystallography and cryo-electron microscopy, to learn how SMO engages PKAcat at the atomic level, enabling precise manipulation of this interaction for therapeutic purposes. To carry out the proposed studies, we have assembled a team of experts in biochemical / cell biological mechanisms of Hh signal transduction (Myers), structure / biophysics of PKA (Taylor), human cellular and organoid retina models (Wahlin), and zebrafish eye development (Kwan). The knowledge gleaned from our work will be vital in the development of therapeutics for a range of debilitating eye disorders, as well as for production of specific ocular cell types in regenerative medicine. It will also foster the development of drugs that selectively modulate either the SMO-G protein or SMO / PKAcat signaling pathways, enabling more precise generation of desired ocular cell types for regenerative medicine and more effective treatments for ocular conditions in the future.
