The Gene Regulatory Networks of Sox32 and Sox17 in the Zebrafish Embryo Development

Tzu-Min Chan^{1, 2}, Chung-Hao Chao^{1, 2}, Horng-Dar Wang², Yen-Ju Yu¹, and Chiou-Hwa Yuh^1,3,4

詹子民^{1, 2}, 趙崇豪^1,2, 汪宏達², 余彥儒¹, 喻秋華^1,3,4

¹Division of Molecular and Genomic Medicine, National Health Research Institute, ²College of Life Science and Institute of Biotechnology, National Tsing-Hua University, ³College of Life Science and Institute of Bioinformatics and Structural Biology, National Tsing-Hua University⁴, Department of Biological Science & Technology National Chiao Tung University

In vertebrate development, the process of gastrulation leads to three germ layers: ectoderm, endoderm, and mesoderm. The endoderm originates from the most marginal blastomeres of blastula stage embryos in zebrafish. Nodal signals transduced by its receptors lead to the activation of gata5, bon and og9x, which then activates sox32 to promote the expression of sox17 and activates the endoderm differentiation. Here, we established the subcircuits of sox32 and sox17 using morpholino against sox32 and sox17, and measured certain gene expression profiles by quantitative real time RT-PCR and validation by using in-situ hybridization. We identified several interesting functional motifs that are important building blocks in zebrafish developmental Gene Regulatory Networks (GRNs). At early stage, sox32 and sox17 autoregulatory lock on the endoderm fate. Early activation turns to late repression for sox32 to sox32 itself and otx2 to sox17. Late transcription factors repress early activator by gata5 activates sox17 and then sox17 represses gata5; gbx1 activates sox32 and then sox32 represses gbx1. We also found sox32 and sox17 repress many early transcription factors such as foxH1, and sox17 represses itself at later stage.

Furthermore, we uncovered the relevant sox17 cis-regulatory elements, and examined the specific input predictions of the GRNs. We discovered three conserved modules: A, B, and C, with a synergistic effect among them. It was revealed that Pou5f1 binding element on B module and Sox32 binding element on C module works synergistically. An evolutionary non-conserved R module exhibits a repressive effect on both the ventral and dorsal side. Together, we demonstrated directly the structural and functional relationships of the genomic code at this key node of the endoderm GRNs in zebrafish development. This information provides a new insight to the complexity of endoderm formation and serves as a valuable resource for the establishment of a complete endoderm gene regulatory network.