Roles of Zic2 and Zic3 in early development

Abstract

The Hedgehog (Hh) signaling pathway plays a crucial role in modulating embryonic development. Malfunctions in the vertebrate Hh pathway involving Sonic Hedgehog Homolog (Shh) have been linked to cancers including basal cell carcinoma and developmental disorders like holoprosencephaly. Zic3, a zinc-finger transcription factor, is hypothesized to activate Shh- mediated Hh signaling. This is based on data demonstrating ZIC3 often binds to GLI consensus motif, and Zic3-depleted embryos express lower levels of Shh. To test this hypothesis, a line of zebrafish embryos carrying a nonsense mutation of Zic3 was examined for morphology of the forebrain and retina. Unexpectedly, no visible defects were found in embryos homozygous for the mutant allele through five days of development, and an expression assay of three Hh pathway genes (Shh, Hhip, and Gli2a) via in situ hybridization in Zic3 mutants showed normal patterns of Hh target expression. These data suggest Zic3 has redundant functions or gene compensation is occurring, in comparison to Zic3-depleted embryos. In a parallel approach, we are investigating Alxl, a candidate target of Zic2. Alxl is strongly implicated in the development of neural crest (NC) cells, as is the Zic gene family. Zic2 and Alxl promote specification of NC cells. Pericytes, a mural cell type important for vascular development, are one of many cell lineages derived from NC cells. Zic2 mutants exhibit hemorrhage indicative of aberrant vasculogenesis, which may be due to aberrant pericyte formation. To test this hypothesis, immunohistochemistry was used to assay expression of Pdgfrb, a gene expressed in pericytes, in Alxl morpholinoinjected embryos. Analysis of the dorso-medial midrain-hindbrain boundary found smaller quantities of pericytes within Alxl- depleted embryos than in controls, suggesting Alxl has an impact on migration, differentiation, or a combination of both factors on pericyte development. This study has provided potential insight into Zic gene family function in early development and may enhance understanding of diseases associated with it.