Structural And Functional Study Of Two Important Genes In The IGF Signaling Pathway In Teleost Fish

The insulin-like growth factor (IGF) signaling pathway is a conserved pathway that plays fundamental roles in regulating animal embryonic growth and development as well as in adult physiology and aging。In mammals, it contains two IGF ligands (IGF-1 and IGF-2), two IGF receptors (IGF-1R, IGF-2R), and six IGF binding proteins (IGFBPs). Despite many advances in our understanding of the fish IGF pathway in teleost, many questions remain unanswered. For example, the full-length structure of IGFBP-4 has not been reported in any teleost species and its function in fish is unknown. Likewise, there are no reports on the post-transcriptional regulation of IGF genes in any teleost fish. In this study, we use two teleost fish models-fugu (Takifugu rubripes) and zebrafish (Danio rerio), to investigate the function of fish IGFBP-4 and IGF-2 mRNA binding protein 2 (IGF2BP2).In the first part of this thesis, I report the characterization of the fugu IGFBP-4 gene. I found that its chromosomal location, gene, and protein structure are similar to its mammalian orthologs. Fugu IGFBP-4 mRNA is easily detectable in all adult tissues examined with the exception of spleen. Older animals tend to have higher levels of IGFBP-4 mRNA in the muscle and eyes compared with younger animals. Starvation results in significant increases in IGFBP-4 mRNA abundance in the muscle, liver, gallbladder, and brain. Overexpression of fugu and human IGFBP-4 in zebrafish embryos results in a significant decrease in body size and somite number, suggesting that fugu IGFBP-4 inhibits growth and development, possibly by binding to IGFs and inhibiting their binding to the IGF receptors. These results provide new information about the structural and functional conservation, expression patterns, and physiological regulation of the IGFBP-4 gene in a teleost fish.Next, I report my findings on two IGF2BP2 genes, igf2bp2a and igf2bp2b, in zebrafish. Zebrafish igf2bp2a and ig/2bp2b are located on Chr9 and Chrl, respectively. Phylogenetic and synteny analyses indicated that they are co-orthologs of the human 1GF2BP2 gene. The zebrafish igf2bp2a has 4 mRNA transcripts due to alternative splicing (igf2bp2a-A, igf2bp2a-B, igf2bp2a-C and igf2bp2a-D). Zebrafish IGF2BP2a-A contains 2 RNA recognition motifs (RRM) in the N-terminal region, and 4 hnRNP K homology (KH) domains in the C-terminal region. During early embryogenesis, igf2bp2a-A mRNA is detected in 0-4 hpf embryos, but becomes undetectable between 6 to 20 hpf; igf2bp2a-B and igf2bp2a-C is undetectable; igf2bp2a-D is only expressed in 0-6 hpf embryos. These data suggest that igf2bp2a-A and igf2bp2a-D are maternally deposited. In adult stages, the 4 igf2bp2a alternative splicing transcripts mRNA are primarily expressed in the ovary. The expression of igf2bp2a mRNA is found exclusively in oocytes in all stages of zebrafish oogenesis. Overexpression of the four IGF2BP2a alternative splicing isoforms and target knockdown IGF2BP2a have no obvious effect on zebrafish muscle, brain and notochord development and cell patterns. However, target knockdown of IGF2BP2a markerly reduced the numbers of primodrail germ cells (PGCs) during zebrafish development.The zebrafish igf2bp2b has 2 mRNA transcripts due to alternative splicing (ig/2bp2b-A and igf2bp2b-B). The 2 mRNA transcripts encode the same ORF which contains four KH domains but lacks any RRM domains. Zebrafish igf2bp2b mRNA is ubiquitously expressed during zebrafish embryogenesis and in adult tissues. Target knockdown of IGF2BP2b inhibited zebrafish embryos growth and development, Moreover, target knockdown of IGF2BP2b disrupted the notochord of the tail. However, IGF2BP2b knockdown morphants had no major effect on brain, eye, muscle or vascular patterning during zebrafish development. Furthermore, IGF2BP2b knockdown results in an increase in igf2a, igf2b and mycb mRNA levels. These data provide new information about the structural and expression patterns of IGF2BP2. Furthermore, these findings suggest that IGF2BP2b may play an important role in zebrafish embryogenesis.