Project Summary/Abstract Cerebellar hypoplasia may contribute to syndrome-specific cognitive and adaptive deficits in Down syndrome, which affects more than a quarter million individuals in the United States. The Ts65Dn mouse model of Down syndrome reflects several important cerebellar phenotypes observed in individuals with Down syndrome, including reduced total volume and reduced granule cell density. During development of the cerebellar cortex, granule cell precursors proliferate in response to the mitogen Sonic hedgehog. Granule cell precursors isolated from Ts65Dn mice proliferate less than control cells when treated with Sonic hedgehog, and a single dose of the Sonic hedgehog agonist, SAG, on the first day after birth rescues cerebellar morphology and some learning deficits in Ts65Dn mice. However, it is unknown which trisomic genes contribute to inhibition of Sonic hedgehog-dependent proliferation. Understanding how trisomy causes cerebellar hypoplasia and other developmental phenotypes is critical to establishing tractable drug targets for ameliorating Down syndrome- associated cognitive deficits. Therefore, we hypothesize that the overexpression of specific trisomic genes negatively regulates the endogenous Sonic hedgehog pathway during cerebellar development. We will address this hypothesis by overexpressing human chromosome 21 genes in a series of in vitro screens to identify candidates that inhibit Sonic hedgehog signaling. Candidates that are identified in these screens and are expressed in the developing cerebellum will be overexpressed in primary granule cell precursors. We will also optimize a technique for CRISPR/Cas9-mediated chromosome elimination in aneuploid cell lines derived from a mouse model of Down syndrome. Inducible chromosome elimination in a mouse model of Down syndrome would provide a method for detecting the developmental origins of phenotypes caused by trisomy. Together, successful completion of this project will yield new insights into how overexpression of human chromosome 21 genes dampen response to Sonic hedgehog during cerebellar development and will produce a powerful tool for identifying how dosage imbalance acts to produce phenotypes in Down syndrome.