Down syndrome (DS), caused by trisomy for human chromosome 21 (Hsa21), is the most common genetically defined cause of intellectual disability. Structural abnormalities that are thought to contribute to learning problems are seen in the cerebellum and hippocampus of people with the condition. Treatments to correct these parts of the brain would tangibly improve the lives of children and young adults with DS and would allow them to better integrate into society. Relevance to NICHD: We recently showed that that a single injection on the day of birth of Sonic Hedgehog (Shh) pathway agonist, SAG, permanently normalizes the size and gross anatomy of the cerebellum, leads to better adult spatial problem solving, and restores electrophysiological correlates of learning in the CA1- hippocampal subfield of the Ts65Dn mouse model of DS. A number of questions remain before this finding can be translated to therapy for people. We will determine how region-specific expression of Shh in the Ts65Dn brain affects hippocampal function using a conditional temporal-spatial genetics approach. This will allow us to increase Shh expression around the time of birth only within the Purkinje cells of the cerebellum, or within the hippocampus, to determine where Shh stimulation is necessary and/or sufficient to correct the behavior and physiology of adult Ts65Dn mice in hippocampal tests (Specific Aim 1). Another restraint on advancing Shh treatments to the clinic is the very wide range of effects of this potent growth factor, making systemic application in people problematic. A more confined method of drug delivery might limit side effects. Another approach is to understand which Hsa21 genes act to down-regulate the Shh pathway response in cerebellum, which might suggest more druggable targets. We identified several candidate genes in a screen of over 100 Hsa21 cDNAs in two Shh reporter cell lines and in zebrafish embryos. Genes that significantly decrease Shh pathway activation in the LIGHT2 assay will be evaluated in additional Shh-sensitive assays to assure that they generalize across different biological systems and their specificity determined using genetic models (Specific Aim 2). Finally, Davisson's Ts65Dn mouse transformed research in DS but after 20 years, limitations in this and in fact in all mouse models have emerged. We will open a new era of investigation with a better model for behavioral and pharmacological testing by making a rat with a stably integrated copy of Hsa21 (Specific Aim 3).