Identification of Gli3 target genes during corpus callosum development

Abstract

The corpus callosum (CC) is the largest white matter fibre tract in the mammalian telencephalon responsible for the transfer and coordination of information between the two cerebral hemispheres. The development of this structure is tightly regulated by a complex interactions between signalling molecules and transcription factors. Gli3 is a zinc finger transcription factor best known for its effects in mediating sonic hedgehog (Shh) signalling. It has previously been shown that conditional inactivation of Gli3 from cortical progenitor cells leads to patterning defects resulting in an agenesis of corpus callosum (AgCC) phenotype. However, the exact molecular mechanisms by which Gli3 controls this patterning In this project I take two approaches to identify downstream targets of Gli3 through which it controls CC development using the Gli3 conditional knockout mouse Emx1Cre;Gli3fl/fl. First, I look at the interaction between Gli3 and the Hippo pathway, with the hypothesis that Gli3 is an upstream regulator of the pathway. In order to do this I analyse the Hippo pathway target Yap and compare the protein levels of its phosphorylated and non-phosphorylated state using quantitative western blot analyses at E14.5 and E16.5. However, no significant difference is observed in the protein levels between mutant and control embryos at either age. The second approach uses data from a previously performed RNA-Seq experiments to identify potential directly regulated targets of Gli3. In situ hybridisation was used to analyse the expression profiles of eight genes identified from the RNA-seq. From this expression analysis two genes were chosen for further study: Foxb1 and Pappa. Following the expression analysis, promoter sequences and predicted enhancer regions for Foxb1 and Pappa were subjected to bioinformatics analysis to identify potential Gli3 binding sites. This analysis revealed two enhancer regions with evolutionarily conserved Gli3 candidate binding sites for Pappa while one conserved binding site was identified in the Foxb1 promoter. The in vitro and in vivo functionality of these sites were then tested using a protein-DNA binding assay and in utero electroporation respectively. These analyses showed that one of the putative Pappa enhancers is significantly repressed by Gli3 in the septum