Mechanistic characterization of ZIC2 function during brain patterning

Abstract

Zinc finger of the cerebellum (ZIC) proteins constitute a family of transcription factors (TFs) with crucial roles during embryogenesis, particularly during neural development. Defects on the genes encoding these TFs cause a broad range of developmental disorders. In particular, Zic2 defects lead to holoprosencephaly, a congenital brain malformation resulting from the defective cleavage of cerebral hemispheres manifested with variable expressivity and incomplete penetrance. However, the target genes and mechanism of action of ZIC2 during brain development are largely unknown. Consequently, the molecular etiology of ZIC2-associated holoprosencephaly remains poorly characterized. To elucidate the molecular mechanisms by which ZIC2 contributes to proper brain development, I first analyzed how the loss of ZIC2 function affects the differentiation of embryonic stem cells into anterior neural progenitor cells (AntNPCs). Notably, I found that the knockout of Zic2 led to a drastic downregulation of dorsal brain genes, including major roof plate markers such as Lmx1a and Lmx1b. Next, one major objective in this project is to determine if ZIC2 directly activates these dorsal genes during AntNPC differentiation or if, alternatively, it represses ventral regulators which themselves antagonize brain dorsal identity. To achieve this, it is necessary to generate ZIC2 binding profiles genome-wide in AntNPC. Due to the lack of a specific antibody against ZIC2, I used CRISPR-Cas9 technology to generate a mouse embryonic stem cell line (mESC) in which the endogenous Zic2 was tagged with a C-terminal Flag-HA epitope. After demonstrating that Zic2 is expressed in this cell line at the same levels as in WT cells both at mRNA and protein level, I also showed that this cell line can be used to identify ZIC2 binding sites by chromatin immunoprecipitation (ChIP). This Zic2-Flag-HA mESC line will now allow us to perform chromatin immunoprecipitation and sequencing (ChIP-seq) and immunoprecipitation coupled to mass spectrometry (IP-MS) experiments to elucidate ZIC2 genomic binding sites and its possible interacting partners, which should provide major insights into the regulatory networks and mechanisms whereby ZIC2 contributes to brain development and human holoprosencephaly.