Conserved function of Caenorhabditis elegans UNC-30 and mouse Pitx2 in controlling GABAergic neuron differentiation

We are taking a cross-species approach to identify genes that are required for mammalian GABAergic neuron differentiation. On the basis of homeodomain similarity, the vertebrate Pitx genes appear to be orthologs of unc-30, a Caenorhabditis elegans gene necessary for differentiation of the GABAergic phenotype of type D neurons. One of the Pitx genes, Pitx2, is expressed in regions of GABAergic neurogenesis in the mammalian brain. These observation s led us to test the functional conservation of the mouse Pitx2 and worm un c-30 genes using a rescue assay. Pitx2 rescues the GABAergic differentiatio n defect and partially rescues the axon guidance and behavioral phenotypes of unc-30 mutants, indicating a high degree of functional conservation betw een these evolutionarily related genes. Previous studies show that UNC-30 d irectly regulates the unc-25/glutamate decarboxylase gene that encodes the enzyme for GABA synthesis. We find that the promoter regions of the mouse a nd human genes coding for the 67 kDa glutamate decarboxylase (Gad1) also co ntain binding sites matching the UNC-30/Pitx2 consensus binding site sequen ce. We show that these sites specifically bind to Pitx2 protein in vitro an d that in transfected neuroblastoma cells, the Pitx2 binding sites contribu te to the basal activity of the Gad1 promoter. Furthermore, in cotransfecti on experiments, we find that Pitx2 strongly activates the Gad1 promoter. Th ese results indicate that Pitx2 may regulate Gad1 expression in mammals, su ggesting a new role for this key developmental transcription factor as a re gulator of GABAergic differentiation during mammalian neural development. O ur results suggest that some of the mechanisms regulating GABAergic differe ntiation are evolutionarily conserved.