Normal brain function relies on the correct assembly of neural circuits during development. This process starts with the patterning of neural progenitors along the dorsal-ventral and anterior-posterior axes to give rise to distinct subtypes of neurons. A number of key transcription factors (TFs) control the process of neuronal subtype specification. Work in the mouse has shown that the homeodomain (HD) TF Gsx2 plays essential roles in the patterning and differentiation of neuronal cell types that arise from progenitors in the lateral ganglionic eminence (LGE) of the embryonic mouse telencephalon. These progenitors give rise to cell types that include the striatal projection neurons of the basal ganglia and interneurons in the olfactory bulb, both of which are severely reduced in mouse Gsx2 mutants. Accordingly, human patient studies identified 2 pathological GSX2 variant alleles in children with serious neurological symptoms, including dystonia and intellectual disabilities. Consistent with these symptoms, MRI imaging revealed severe basal ganglia agenesis. One GSX2 variant results in a null allele, however, the other is a missense variant (Q251R) that alters a key amino acid in the DNA binding HD. We generated a mouse model of this human variant and our initial studies suggest that the Q>R variant leads to a strong embryonic LGE and basal ganglia phenotype that is morphologically similar to embryos with Gsx2 null alleles. Furthermore, our preliminary data indicate that this human HD variant alters Gsx2 DNA binding specificity, and thereby may account for the observed phenotypes. Moreover, we recently determined that Gsx2 binds and regulates target genes via two mechanisms; as a monomer Gsx2 represses gene expression whereas on a subset of DNA sites cooperative Gsx2 binding to dimer sites appears to facilitate gene expression. Intriguingly, the Dlx HD TFs, which lie downstream of Gsx2 during LGE progenitor maturation, also bind monomer sites but instead of repressing they activate gene expression. In this application, we propose to determine how Gsx2 and the Dlx TFs regulate LGE gene expression during basal ganglia development. To achieve this goal, we will test the following hypotheses in 3 independent specific aims: 1) To test the hypothesis that Gsx2 controls basal ganglia development by mediating distinct gene regulatory outcomes in a DNA binding site dependent manner. 2) To test the hypotheses that Gsx2 and Dlx TFs regulate a common set of LGE genes though direct competition for shared enhancer elements. 3) To test the hypothesis that the GSX2Q251R human variant causes altered DNA binding specificity, and thereby results in the mis-regulation of LGE gene expression and ultimately basal ganglia agenesis. Our approach will combine the use of mouse genetics and human forebrain neural stem cell cultures with molecular, biochemical, and genomic approaches to study transcriptional control of neuronal specification in the developing basal ganglia. ...