Role of vertebrate GAGA associated factor (vGAF) in early development of zebrafish.

Homeotic genes and their genomic organization show remarkable conservation across bilaterians. Consequently, the regulatory mechanisms, which control hox gene expression, are also highly conserved. The crucial presence of conserved GA rich motifs between Hox genes has been previously observed but what factor binds to these is still unknown. Previously we have reported that the vertebrate homologue of Drosophila Trl-GAF preferentially binds to GA rich regions in Evx2-hoxd13 intergenic region of vertebrate HoxD cluster. In this study, we show that the vertebrate-GAF (v-GAF) binds at known cis-regulatory elements in the HoxD complex of zebrafish and mouse. We further used morpholino based knockdown and CRISPR-cas9 knockout technique to deplete the v-GAF in zebrafish. We checked expression of the HoxD genes and found gain of the HoxD4 gene in GAF knockout embryos. Further, we partially rescued the morphological phenotypes in GAF depleted embryos by providing GAF mRNA. Our results show that GAF binds at intergenic regions of the HoxD complex and is important for maintaining the spatial domains of HoxD4 expression during embryonic development.