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 observations led us to test the functional conservation of the mouse Pitx2 and worm unc-30 genes using a rescue assay. Pitx2 rescues the GABAergic differentiation defect and partially rescues the axon guidance and behavioral phenotypes of unc-30 mutants, indicating a high degree of functional conservation between these evolutionarily related genes. Previous studies show that UNC-30 directly regulates the unc-25/glutamate decarboxylase gene that encodes the enzyme for GABA synthesis. We find that the promoter regions of the mouse and human genes coding for the 67 kDa glutamate decarboxylase (Gad1) also contain binding sites matching the UNC-30/Pitx2 consensus binding site sequence. We show that these sites specifically bind to Pitx2 protein in vitro and that in transfected neuroblastoma cells, the Pitx2 binding sites contribute to the basal activity of the Gad1 promoter. Furthermore, in cotransfection experiments, we find that Pitx2 strongly activates the Gad1 promoter. These results indicate that Pitx2 may regulate Gad1 expression in mammals, suggesting a new role for this key developmental transcription factor as a regulator of GABAergic differentiation during mammalian neural development. Our results suggest that some of the mechanisms regulating GABAergic differentiation are evolutionarily conserved.
Westmoreland
J J
JJ
Institute of Molecular Medicine and Genetics and Departments of Medicine and Cellular Biology and Anatomy, Medical College of Georgia, Augusta, Georgia 30912, USA.
McEwen
J
J
Moore
B A
BA
Jin
Y
Y
Condie
B G
BG
D000818:Animals; D001369:Axons / Q000187:drug effects / Q000502:physiology; D001665:Binding Sites / Q000502:physiology; D017173:Caenorhabditis elegans; D029742:Caenorhabditis elegans Proteins; D002454:Cell Differentiation / Q000187:drug effects / Q000502:physiology*; D002460:Cell Line; D017124:Conserved Sequence / Q000502:physiology; D015870:Gene Expression / Q000187:drug effects; D017930:Genes, Reporter; D005968:Glutamate Decarboxylase / Q000235:genetics / Q000378:metabolism; D015801:Helminth Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D018398:Homeodomain Proteins / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D007527:Isoenzymes / Q000235:genetics / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D009447:Neuroblastoma / Q000378:metabolism; D009474:Neurons / Q000166:cytology / Q000187:drug effects / Q000378:metabolism*; D009687:Nuclear Proteins; D010641:Phenotype; D011401:Promoter Regions, Genetic / Q000502:physiology; D017434:Protein Structure, Tertiary / Q000502:physiology; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism / Q000494:pharmacology; D012689:Sequence Homology, Nucleic Acid; D013379:Substrate Specificity / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism* / Q000494:pharmacology; D014162:Transfection; D019076:Transgenes; D005680:gamma-Aminobutyric Acid / Q000378:metabolism*; D000097577:Homeobox Protein PITX2
D029742:Caenorhabditis elegans Proteins; D005968:Glutamate Decarboxylase; D015801:Helminth Proteins; D018398:Homeodomain Proteins; D007527:Isoenzymes; D009687:Nuclear Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D005680:gamma-Aminobutyric Acid; D000097577:Homeobox Protein PITX2; C090946:unc-30 protein, C elegans; C402565:glutamate decarboxylase 1