A subtractive screen for Pax6 target genes in *P19 embryonal carcinoma cells.

The highly conserved transcription factor Pax6 is an early, crucial regulator of eye and nervous system morphogenesis throughout the Metazoa. Haploinsufficiency causes development of reduced or abnormal eyes in mice and humans. Homozygous loss of Pax6 function results in severe brain defects and the complete absence of eyes and olfactory structures in mice, rats, humans, and Drosophila , and neurosensory defects in C. elegans. Misexpression promotes the growth of ectopic eyes or eye tissue in Drosophila and Xenopus, suggesting that Pax6 functions near the top of a regulatory hierarchy controlling eye formation. However, relatively few direct gene targets of Pax6 regulation are known. In order to identify direct target genes of Pax6 transactivation, I employed suppression subtractive hybridization to enrich a complex cDNA population for Pax6-upregulated messages. A multipotent murine P19 embryonal carcinoma cell line carrying a tetracycline-repressible PAX6 expression cassette produced Pax6-induced and uninduced cDNA pools for PCR based subtraction. Potentially upregulated cDNAs were cloned into a plasmid vector after forward enrichment and hybridized to forward-subtracted and reverse-subtracted probes to assess differential abundance in the two cDNA pools. Ninety-eight clones, representing 76 unique sequences, were selected for further examination via northern blot analysis. Among these, the thymosin beta4 gene (Tmsb4x ), is consistently upregulated 3- to 4-fold in P19 cells following Pax6 induction. This potential Pax6 target encodes a conserved actin-binding protein that is strongly expressed in the developing nervous system, including retinal ganglion cells. Thymosin beta4 facilitates normal actin filament turnover and is likely to have a role in axon pathfinding and neuron migration---processes which are also disrupted in Pax6 homozygous mutants. Sequence analysis of the mouse Tmsb4x locus revealed three consensus binding sites for the Pax6 paired domain, at -6.3 kb, -221 bp, and within intron 1, which could mediate transcriptional regulation. In cotransfection experiments in NIH-3T3 cells, Pax6 appeared to activate expression of a luciferase reporter containing the Tmsb4x promoter, 5' proximal region and first intron (-7.8 to +1.2 kb). My work thus establishes a potential link between Pax6 and a protein with a central function in cytoskeletal dynamics.Ph.D.Biological SciencesGeneticsMolecular biologyUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/124037/2/3121909.pd