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Abstract
The importance of understanding the molecular mechanisms driving cell fate specification lies in the fact that loss of cell identity due to dysregulation of the common developmental processes in adult tissues can lead to cancer. One example that is indicated in both tumorigenesis and embryonic development is a dual-specificity transcription factor Max gene-associated protein (MGA). MGA is known to be a tumor suppressor gene for its ability to antagonize MYC dependent cell proliferation and promoting differentiation in cell culture. It is also required in promoting dorsal-ventral patterning by positively regulating BMP signaling in zebrafish development. Here we identified a novel role of MGA in neural crest specification during zebrafish early embryonic development utilizing both antisense oligonucleotides and CRISPR/Cas9 system. The depletion of Mga from 1-cell stage embryos using antisense oligonucleotides resulted in defects in various lineages of the neural crest cell population. Furthermore, a marked decrease in early neural crest markers was observed during gastrulation. Mga depleted embryos show a marked decrease in Bmp signaling activity and an increase in Myc activity, as revealed by analysis of a Myc responsive transgene and endogenous Myc target gene expression profile. Overexpression of Mxd3 mRNA, a known Myc antagonist, in Mga depleted embryos fails to rescue neural crest defects. This is the first demonstration that Mga acts as a Myc antagonist in vivo, and the neural crest defect seen in Mga depleted embryos is due to altered Bmp signaling activity which is independent of Myc. We successfully generated five potentially null Mgaa mutant alleles using the CRISPR/Cas9 system. These mutants fail to show a morphologically distinct phenotype when compared to wild type. However, further molecular analysis of Myc and Bmp pathways in Mgaa mutants show similar but milder alterations are seen in knockdown embryos. Furthermore, we identified Mgaa paralog, Mgab, using the bioinformatic approach and confirmed its expression at various developmental stages. The mild changes in our Mgaa mutants may be explained by genetic compensation resulting from non-sense mediated decay of mgaa mRNA inducing increased expression of mgab. Therefore, the generation of mgaa and mgab double mutants is required to characterize Mga function in zebrafish embryonic development.