Expression and function of FIH in zebrafish embryo

• Main Authors: Chian-Yi Chen, 陳倩旖
• Other Authors: Chin-Hwa Hu
• Format: Others
• Language: zh-TW
• Published: 2011
• Online Access: http://ndltd.ncl.edu.tw/handle/40804663857893321452

碩士 === 國立臺灣海洋大學 === 生物科技研究所 === 99 === bHLH-PAS proteins act important functions in vertebrate embryos. Hypoxia inducible factors (HIFs) are required for oxygen homeostasis. In normoxia conditions, HIF alpha subunits are targeted for proteosomal degradation by prolyl hydroxylase (PHD) and the von Hippel-Lindau (VHL) E3 ligase complex. Furthermore, the aspartic residues in the C-terminus of un-degraded HIF1α are subjected by factors-inhibit-HIF (FIH) to block its transactivation activity. When cells are subjected to hypoxia, HIF alpha subunits are stabilized and in turn associate with HIF beta subunit (ARNT) in the nucleus and activate target genes. Nevertheless, how the HIF alpha subunits are stabilized and sustain their transcriptional activities in embryos still remains to be elucidated. Here we have investigated the function of FIH in zebrafish embryos. A FIH-specific morpholino oligonucleotide was applied into embryos at 1-cell-stage to block FIH translation. Morphological changes with small head size, curved tail were observed in FIH morphants. Massive apoptosis and suppressed erythrocytosis and angiogenesis was also detected in FIH morphants. The specificity of FIH morpholino was tested by CMV:FIH-EGFP fusion gene in which the DNA fragment with 5’UTR sequence and the first three codons of FIH was fused to EGFP ORF fragment. It appears that the FIH morpholino successfully repressed the fluorescent protein expression. Synthetic FIH mRNA with mutated 5’UTR sequence could partially rescue impairs observed in FIH morphants, suggesting that FIH morpholino acts in a sequence-specific way. Apparently FIH acts critical roles in zebrafish embryo. The function of aspartic hydroxylation of HIF1α in zebrafish embryo needs further examination.