Abstract
Myc proteins control cell proliferation, cell cycle progression, and apoptosis, and play important roles in cancer as well in establishment of pluripotency. Here we investigated the control of myc gene expression by the Pou5f1/Oct4 pluripotency factor in the early zebrafish embryo. We analyzed the expression of all known zebrafish Myc family members, myca, mycb, mych, mycl1a, mycl1b, and mycn, by whole mount in situ hybridization during blastula and gastrula stages in wildtype and maternal plus zygotic pou5f1 mutant (MZspg) embryos, as well as by quantitative PCR and in time series microarray data. We found that the broad blastula and gastrula stage mych expression, as well as late gastrula stage mycl1b expression, both depend on Pou5f1 activity. We analyzed ChIP-Seq data and found that both Pou5f1 and Sox2 bind to mych and mycl1b control regions. The regulation of mych by Pou5f1 appears to be direct transcriptional activation, as overexpression of a Pou5f1 activator fusion protein in MZspg embryos induced strong mych expression even when translation of zygotically expressed mRNAs was suppressed. We further showed that MZspg embryos develop enhanced apoptosis already during early gastrula stages, when apoptosis was not be detected in wildtype embryos. However, Mych knockdown alone did not induce early apoptosis, suggesting potentially redundant action of several early expressed myc genes, or combination of several pathways affected in MZspg. Experimental mych overexpression in MZspg embryos did significantly, but not completely suppress the apoptosis phenotype. Similarly, p53 knockdown only partially suppressed apoptosis in MZspg gastrula embryos. However, combined knockdown of p53 and overexpression of Mych completely rescued the MZspg apoptosis phenotype. These results reveal that Mych has anti-apoptotic activity in the early zebrafish embryo, and that p53-dependent and Myc pathways are likely to act in parallel to control apoptosis at these stages.
Highlights
Apoptosis plays a crucial role during development and maintenance of homeostasis in multicellular organisms by eliminating damaged or unneeded cells [1,2]
For the zebrafish c-myc homologues myca and mycb, in MZspg embryos compared to WT, the mRNA levels derived from maternal expression have been found to be reduced before onset of zygotic transcription, but elevated at post-MBT stages (Figure S3A and C). mycl1a expression was not altered in Pou5f1 mutants compared to WT (Figure S3E). mycl1b expression in MZspg has been found to be normal during blastula and early gastrula stages, but downregulated during late gastrulation (Figure S3G)
We found that early zygotic mych expression as well as late gastrula stage mycl1b expression both directly depend on Pou5f1 activity
Summary
Apoptosis plays a crucial role during development and maintenance of homeostasis in multicellular organisms by eliminating damaged or unneeded cells [1,2]. Programmed cell death is intensely studied in model organisms, because its deregulation is involved in many diseases including cancer, Alzheimer’s disease, or immune deficiencies [3,4]. Myc proteins are transcription factors involved in regulation of various cellular functions including cell-cycle progression [14], differentiation [15,16], cell growth [17], and apoptosis [18]. During normal mouse embryonic development c-myc is expressed in many different cell types [31,32,33,34]. The importance of c-Myc in promoting early development was shown by the fact that c-myc homozygous knock-out mice are embryonic lethal [36], and its role in stem cell pluripotency when mouse fibroblasts were reprogrammed by overexpressing c-Myc together with Oct, Sox and Klf4 [37]. Myc makes complex contributions to pluripotency [38,39,40]
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