An evolutionarily conserved kernel of gata5, gata6, otx2 and prdm1a operates in the formation of endoderm in zebrafish
An evolutionarily conserved subcircuit (kernel) dedicated to a specific developmental function is found at the top of the gene regulatory networks (GRNs) hierarchy. Here we comprehensively demonstrate that a pandeuterostome endoderm specification kernel exists in zebrafish. We analyzed interactions among gata5, gata6, otx2 and prdm1a using specific morpholino knockdowns and measured the gene expression profiles by quantitative real-time RT-PCR and in situ hybridization. The mRNA rescue
... t validated the specificity of the morpholino knockdown. We found that the interactions among gata5, gata6, otx2 and prdm1a determine the initial specification of the zebrafish endoderm. Although otx2 can activate both gata5 and gata6, and the prdm1a/krox homologue also activates some endoderm transcription factors, a feedback loop from Gata to otx2 and prdm1a is missing. Furthermore, we found the positive regulation between gata5 and gata6 to further lock-on the mesendoderm specification by the Gata family. Chromatin immunoprecipitation was used to further validate the recruitment of Otx2 to the gata5 and gata6 loci. Functional assays revealed that module B of gata6 and the basal promoter of gata5 drive the gene at the mesendoderm, and mutational analysis demonstrated that Otx2 and Gata5/6 contribute to reporter gene activation. This is the first direct evidence for evolutionarily conserved endoderm specification across echinoderms and vertebrates. the posterior pharyngeal arch and nerve in zebrafish (Olesnicky et al., 2010; Birkholz et al., 2009; Rossi et al., 2009) . In Xenopus, Prdm1/ Blimp1 is hypothesized to induce anterior endomesoderm and promote head formation (de Souza et al., 1999) . The roles of otx2 and prdm1a in the endoderm have received little attention in zebrafish. Previously, we constructed zebrafish GRNs (Chan et al., 2009b) (http://www.zebrafishworld.org) by integrating published evidence for interactions with the spatial/temporal expression pattern extracted from the Zebrafish Information Network (ZFIN) (http://zfin.org). In this study, we establish the interrelationship among four evolutionarily conserved transcription factors, gata5, gata6, otx2 and prdm1a in early endoderm specification by using morpholino (MO) perturbation coupled with quantitative RT-PCR (Q-PCR) and in situ hybridization. We demonstrate their structural and functional relationships in the genomic code of gata5 and gata6 for interaction and activation from Otx2 and Gata5/6. With this new insight, we suggest that the kernel of these four evolutionarily conserved transcription factors is also important, however with some degree of modification, for activating endodermal transcription factors during early zebrafish development. Results The spatial and temporal expression profiles of otx2, gata5, gata6 and prdm1a Key conserved transcription factors operating a subcircuit (kernel) are essential for determining the endoderm in early embryogenesis. To investigate the roles of gata5, gata6, otx2 and prdm1a in mesendoderm specification in zebrafish, we first examined the temporal expression profiles of these genes. We extracted total RNA from wild-type embryos at 0.5, 2.5, 3.5, 4.5, 5.0, 5.5, 6.5 and 8 hpf (hours post-fertilization) and performed real-time RT-PCR. As shown in Fig. 1A , all four transcription factors were undetectable at 1.5 hpf and started to be expressed around 4 hpf. The expression of prdm1a before 5 hpf was undetectable (not shown). The expression of gata5, gata6 and otx2 increased until it peaked at around 5 hpf. The expression peaked again at 11 hpf and then declined from 16 to 24 hpf (data not shown). The time of the four genes' expression correlated with mesendoderm specification in zebrafish.