Project description:Pax6 is a transcription factor with key functional roles in embryonic development. In order to identify downstream effectors of Pax6 in the developing cerebral cortex we performed microarray analysis. We compared gene expression profiles of cortical tissues isolated from wild type and Pax6-/- mouse embryos. In order to identify Pax6 downstream targets we carried out microarray analysis of Pax6-/- mutant mice. Pax6 is highly expressed in the mouse cerebral cortex at embryonic day E14.5, therefore we selected this tissue in order to compare gene expression profiles between wild type and Pax6-/- homozygous cortici. RNA samples were isolated from three mutant and three wild type embryos.
Project description:Pax6 is a highly conserved transcription factor among vertebrates and is important in various aspects of the central nervous system (CNS) development. However, the gene regulatory circuitry of Pax6 underlying these functions remains elusive. We find that, following expression in neural progenitor cells, Pax6 targets many promoters embedded in an active chromatin environment. Intriguingly, many of these sites are also bound by another progenitor factor, Sox2, which cooperates with Pax6 in gene regulation. A combinatorial analysis of Pax6 binding dataset with transcriptome changes in Pax6-deficient neural progenitors reveals a dual role for Pax6, in which it activates the neuronal (ectodermal) genes while concurrently represses the mesodermal and endodermal genes thereby ensuring the unidirectionality of lineage commitment towards glutamatergic neuronal differentiation. Furthermore, Pax6 is critical for inducing activity of transcription factors that elicit neurogenesis and repress others that promote non-neuronal lineages. In addition to many established downstream effectors, Pax6 directly binds and activates a number of genes that are specifically expressed in neural progenitors but have not been previously implicated in neurogenesis. The in utero knockdown of one such gene, Ift74, during brain development impairs polarity and migration of new-born neurons. These findings demonstrate new aspects of the gene regulatory circuitry of Pax6, revealing how it functions to control neuronal development at multiple levels to ensure unidirectionality and proper execution of the neurogenic program.
Project description:Novel target genes for the two transcription factor isoforms Pax6 and Pax6(5a) were identified by generating mouse embryonic fibroblast cell lines stably expressing either Pax6 or Pax6(5a), and comparing their gene expression pattern with the original mouse embryonic fibroblast cell line. Flp-In-3T3 cell lines were generated according to the manufacturers instruction (Invitrogen), using the mouse Pax6 and Pax6(5a) cDNA as insert. Correct inserts were verified by PCR, sequencing and Westernblot.
