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.

In mammalian ES cells, the transcription factors Klf4 and Klf2 contribute to maintenance of pluripotency and self-renewal and are regulated by Pou5f1/Oct4. In the early zebrafish embryo Pou5f1/Oct4 is necessary for expression of three Klf2/4 family members, klf2a, klf2b and klf17 (previously klf4b), similar to the regulation reported for mammalian ES cells. In this study, we analyzed blastula and gastrula stage Klf regulatory networks and their influence on zebrafish embryonic patterning. We show that Pou5f1 acts in combination with region-specific factors to activate klf2a, klf2b, and klf17 in the superficial cell layer of the embryo. In addition, Pou5f1 acts together with the BMP signaling pathway to activate and maintain expression of klf2a and klf2b in a ventral ectodermal domain. We used microarray expression profiles of klf2a, klf2b and klf17 knockdown and overexpression embryos to identify Klf target genes, which reveals that Klfs participate in specification of the extraembryonic enveloping layer (EVL). We discuss mechanistic implications of simultaneous activation of transcriptional targets by ubiquitous, like Pou5f1, and region-specific inducers, emerging as a common regulatory motif in early development.

Early patterning of the vertebrate neural plate involves a complex hierarchy of inductive interactions orchestrated by signalling molecules and their antagonists. The morphogen retinoic acid, together with the Cyp26 enzymes which degrade it, play a central role in this process. The cyp26a1 gene expressed in the anterior neural plate thus contributes to the fine modulation of the rostrocaudal retinoic acid gradient. Despite this important role of cyp26a1 in early brain formation, the mechanisms that control its expression in the anterior neural plate are totally unknown. Here, we present the isolation of a 310-base-pair DNA element adjacent to cyp26a1 promoter, displaying enhancer activity restricted to the anterior neural plate of the zebrafish gastrula. We show that unlike that of cyp26a1, expression driven by this cyp26a1 anterior neural plate element (cANE) is independent of retinoic acid. Through deletion analysis, we identify a 12-nucleotide motif essential for cANE activity. A consensus bipartite binding site for SoxB:Oct transcription factors overlaps with this motif. Mutational analysis suggests that SoxB binding is essential for its activity. We discuss the contribution of this study to the elucidation of the regulatory hierarchy involved in early neural plate patterning.