Important Kernel for endoderm development in zebrafish: subcircuits between gata5, gata6 and otx2

Wen-Fang Tseng¹, Te-Hsuan Jang, Chang-Ben Huang¹ and Chiou-Hwa Yuh^{1,2, 3}

曾文芳¹，讓德選¹, 黄昶斌¹，喻秋華^{1, 2, 3}

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

In the whole gene regulatory networks, the most important subcircuits also called “Kernel” which is highly conserved during evolution. From sea urchin and star fish, the endoderm Kernel involves gata, otx, and forkhead transcription factors. In zebrafish, many evidences have shown that otx2, and gata5/6 play crucial roles during early embryogenesis. However, previous study has focusing on the role of otx2 in the head and gata5/6 on the mesodermal tissue, few of them have discussed about function of them on endoderm formation. Moreover, the interrelations between otx2 and gata5/6 are rarely defined. In this study, we analyze otx2, gata5 and gata6 gene regulatory subcircuits in zebrafish. With this information, we may fully understand the gene regulatory networks that otx2, gata5 and gata6 operates in zebrafish endoderm development.

We established the subcircuits of otx2, gata5 and gata6 using specific morpholino against them, and then measured certain gene expression profiles by quantitative real time RT-PCR, finally validated by in-situ hybridization. We chose 35 genes that express in hypoblast at 5 hpf in zebrafish embryos as our candidate genes. After we have identified genes positively or negatively regulated by otx2, gata5 and gata6, we analyzed these data and built up the subcircuits between those genes by BioTapestry software. Form this subcircuits, we found at 5hpf , otx2 positively regulates gata5 and gata6, and gata5 and gata6 can positively regulate each other and form a positive feed-back loop. Form the result, we propose that otx2 transmits signals to gata5 and gata6, and then these two genes provide a stable out put by forming a positive feed back loop. With this out put, genes for endoderm development including sox32, foxa2, and sox17 at later stages were activated.

Chromatin- immunoprecipitation using antibodies against Otx2, Gata5, and Gata6 was carried out to search for the in-vivo binding site in the embryo. To validate the chromatin-immunoprecipitation result, each module will be linked to basal promoter and EGFP reporters constructs and test their function in-vivo in live embryos, co-injection of morpholino and site-directed mutations will also be carried out to find the direct target sites. Eventually, we hope our study in subcircuits of otx2, gata5 and gata6 will serve as a valuable resource for the establishment of a complete endoderm gene regulatory network, and delineate how those important transcription factors work together and determine the cell fate during the zebrafish development.