(P368.05) A Genetically Encoded Fluorescent Sensor for monitoring Sonic Hedgehog (Shh) Signaling

Society for Neuroscience Global Connectome, 2021

Sonia L. Bernal, Santiago Uribe-Cano, and Andreas H. Kottmann

Shh signaling is critical for the development, maintenance and function of the central nervous system. Shh acts in a concentration dependent manner and 1.8 fold changes in Shh signaling strength can result in vastly different biological outcomes as evidenced by Shh dependent cell fate determination during development. Shh signaling is spatially highly restricted and affects cellular physiology within seconds to hours. For example, we have shown previously that the precise spatial and temporal integration of multiple sources of Shh in the pMN domain of the developing spinal cord is critical for the switch from neurogenesis to oligogenesis (Starikov et al., bioRXiv; doi: https://doi.org/10.1101/534750 ). Further, we have shown that mesencephalic dopamine neurons (DAN) in the adult brain engage in neuronal co transmission using Shh. Here, activity dependent release from of Shh from DAN impinge on cholinergic neurons of the striatum and affect the rate of reinforcement learning in the normal brain and the formation of dyskinesia in parkinson’s models (Malave et al.; bioRXiv: https://doi.org/10.1101/2020.03.09.983759). To determine how moment to moment changes in Shh signaling strength are interpreted by Shh receiving cells and integrated with other extra cellular signals, tools need to be generated that allow quantifying Shh signaling strengths with subcellular spatial and millisecond temporal resolution. We have developed an activation-based biosensor for Smoothened, the G-protein coupled receptor (GPCR) downstream effector of Shh signaling in target cells. Guided by the prior design of GPCR-Activation Based (GRAB) sensors and analysis of their sequence homology, we inserted a circularly permutated (cp) GFP module into the third intracellular loop of Smoothened via HiFi assembly. The sensor was characterized in HEK293 cells with Smoothened pharmacology, using a vector that produces a Smoothened receptor with a non-fused GFP as a control. Our results demonstrate the sensor achieves physiologically relevant affinity and specificity, with sub-second kinetics. My poster will present the biophysical characterization of this novel recombinant SmoGRAB sensor and validation of its ability to detect physiological relevant dynamics of Shh signaling in vitro.