Molecular Mechanism of the Murine Homeobox Gene-GSH-1 (Abstract)

Abstract

Evidence from drosophila to human indicates that the homeobox genes are developmental important. These genes encode transcription factors capable of initiating genetic cascades that determine cell type specificity. The sequence of Gsh-1 cDNA (2.1 kb) revealed an open reading frame of 261 amino acids, including a homeodomain that closely resembles that encoded by Antennapedia. Expression of mRNA for Gsh-1 has been localized to the neuroepithelial tissues of the developing central nervous system. Two symmetrical stripes of expression extend from the posterior neural tube to the hindbrain region where they spread out into a complex restricted pattern in the developing midbrain and forebrain regions. To study the in vivo function of Gsh-1, we knocked out the gene via homologous recombination in embryonic stem cells. Homozygous mutants exhibit extreme dwarfism, sexual infantilism and significant perinatal mortality. The mutant pituitary is small in size and hypocellular, with severely reduced number of growth hormone and prolactin producing cells. The Gsh-1 gene is shown to be essential for growth hormone releasing factor (GRF) gene expression in the arcuate nucleus of the hypothalamus. The mutant phenotype indicates a critical role for Gsh-1 in the genetic hierarchy of the formation and function of the hypothalamic-pituitary axis. The next vertical extension of our understanding of Gsh-1 function will base on the isolation of Gsh-1 downstream target genes. To this end, we have generated transgenic mice carrying the SV40 T-antigen under the control of Gsh-1 5’-flanking sequences. All mice expressed the transgene developed brain tumors. Cell lines from 4 transgenic mice were established and exhibited characteristics similar to brain tumors. Furthermore, we established several Gsh-1 null (-/-) cell lines by crossing the Gsh-1/SV40 T-antigen and Gsh-1 knock out mice. These immortalized cells should provide a valuable resource for the identification of Gsh-1 downstream target genes. In order to accomplish this aim, an inducible Gsh-1 cDNA construct was transfected into these immortalized Gsh-1 null cell lines, RNA differential display and DNA microarray were then used to fish out the Gsh-1 downstream target genes. More than 100 differential expressed clones have been isolated. Northern blot and in situ hybridization are performing to further confirm that they are truly differential expressed clones.