The downstream genes regulated by six3 and transcriptional regulation analysis of six3 in Zebrafish Embryo

Chung-Hao Chao¹ and Chiou-Hwa Yuh^1,2,3

趙崇豪^1,、喻秋華^1,2,3

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

The overall goal of our Lab is to establish the gene regulatory networks underlying the mesoderm development in zebrafish. In this study, I focus on six3a and six3b which belong to Six family of homeobox-containing genes and are expressed in the most anterior neural plate at the beginning of neurulation in various vertebrates. The function of six3 has been widely investigated as a crucial regulator of eye and forebrain development in vertebrate. However, rarely papers regard the transcription regulation of six3.

Previous studies demonstrate that six3b expression is activated from 8 hours post fertilization (hpf) in axial mesoderm, and subsequently, in the overlying anterior neural plate from which the optic vesicles and forebrain will develop. After knockdown of six3b expression by Morpholino anti-sense oligonucleotide which blocks the protein translation, some have developmental defects in eyes and brain and almost all the embryos had curled down tails. From my Q-PCR data, the expression of otx1, mybbp1a, otx2, foxh1, gbx1, pax2a, gbx2, and og9x were decreased at early stage (5 ~ 11 hpf) after removal of six3b. The expression of gbx1, pax2a, otx1, pou1 were decreased at later stages (16~ 24 hpf). Those genes were under positive regulation by six3b. In addition, the expression of mybbp1a (8hpf), etv5 were increased in six3b morphants at early stage, prdm1, six3b, tbx16, brn1.2, foxa3, bon, gata6, hey1, irx3a, tbx24, sox4 were increased in six3b morphants, indicating those genes were under negative regulation by six3b. Other researchers in our lab also found that six3b expression is increased in sox17, otx2, six3b, and gbx2 morphants, indicating that sox17, otx2, gbx2 and six3b itself negative regulate six3b. Based on six3b perturbation experiment, I have built GRN during 5hr to 24hr of zebrafish development.

To elucidate the transcription regulation of six3a, we perform the in vivo reporter assay. Alignment of a 21kb region surrounding zebrafish six3a with several species identified a cluster of non-coding conserved modules. We generated different GFP reporter constructs containing different regions of zebrafish six3a conserved modules and each linearlized construct was injected into zebrafish one-cell stage embryos and GFP expression images were taken at different stages. Three modules were identified that regulate GFP reporter express in respective six3a expression region forebrain and eyes, module A and module C drive GFP expression in forebrain and eyes at late stage whether module J drives GFP expression in forebrain and eyes from early stage. From our study, we hope to have a comprehensive view of how the brain and eye are formed.