Transcriptome Analysis Suggested Striking Transition Around the End of Epiboly in the Gene Regulatory Network Downstream of the Oct4-Type POU Gene in Zebrafish Embryos

Abstract

Zebrafish pou5f3 encodes a Class V POU transcription factor, Pou5f3, which regulates various developmental processes, including neurogenesis and brain formation. In the current study, we attempted to comprehensively identify the Pou5f3 downstream genes around the end of epiboly, when the competence of the mid-hindbrain region to Pou5f3 suppression changes drastically, by the microarray method and a heat-inducible dominant-interference pou5f3 gene (en-pou5f3) that functionally suppresses pou5f3. At late epiboly and early somitogenesis stages, we identified genes whose expression was altered in en-pou5f3-induced embryos, revealing numerous genes regulated differently by Pou5f3 at the two stages. The validity of the microarray data was confirmed by whole mount in situ hybridization and quantitative RT-PCR. Many of the downstream genes were implicated by the Gene ontology (GO) analyses in transcriptional regulation and neural development and were enriched with sox genes and bHLH genes such as her genes. Interestingly, we noticed a tendency that Notch-dependent her genes were activated, whereas Notch-independent her genes were downregulated by Pou5f3 suppression. Among the Notch-independent her genes, her3, which is orthologous to mammalian Hes3, was suggested to be strongly activated endogenously by Pou5f3. In the upstream DNA of this gene, we found two noncoding conserved sequences (NCRs), which harbored consensus binding sites for Pou5f3, Sox, and Nanog. We further showed in reporter assays that the transcriptional regulatory activity of the her3 upstream DNA was strongly enhanced by SoxB1, and this SoxB1-mediated activation was weakened by Pou5f3. Deletion experiments showed that both upstream NCRs were involved in transcriptional repression.