| Protein kinase B (PKB), also known as Akt, is a serine/threonine protein kinase that regulates key enents in metabolism, proliferation, cell survival, and differentiation. As an important downstream mediator of IGF1 signaling pathway, it implicates critical role in insulin action and growth regulation. Im mammalian, there are three isoform of PKB, PKBα/Akt1, PKBβ7Akt2, PKBγ/Akt3. They are highly conserved whereas encoded by different genes. They also have distinct function, in vivo. Mice deleted AktIdisplay increased prenatal mortality and a reduction in body size; Akt2 deletion cause type 2 diabetes-like syndrome; and Akt3 deletion present small size of brain. Although we have abundant progresses of Akt function research, its efftcts during early developmental stages are still unclear due to the in uetro developmental model of mouse. In our study, we use zebrafish embryo as model to investigate the function of PKB in early developmental stages.Aktl is ubiquitous in organism which is thought to be the critical PKB isoform for survival. In zebrafish, only akt2 and akt2like are cloned. So we expresses consistent activated mouse Aktl (myr-Aktl) in zebrafish embryos, by which we have observed a selective increase in relative brain length and height.In contrast,inhibiting PI3K by treating those embryos with LY294002, the enlargement of brain size recovered to normal. Then we test cell proliferation, cell fate and apoptosis in brain regions after myr-Aktl injection. The results demonstrate that Aktl could reduce the cell number of apoptosis but do not affect cell proliferation and fate in embryo brain. Moreover, by checking the apoptosis cell from UV-induced and heatshock-induced, we demonstrate the neuroprotection of Aktl in zebrafish embyo.Then, we isolated and identified a pkby/akt3 cDNA in Zebrafish, Danio rerio, by reverse transcription polymerase chain reaction and rapid amplification of cDNA ends (RACE) methods. The full length cDNA of Zebrafish akt3 comprises 2874 base pairs (bp) with an open reading frame (ORF) of 1440 bp, encoding 479 amino acids. Analysis of the deduced amino acid sequences revealed that zebrafish Akt3 contain all three domains and two phosphorylation sites conserved among Akt family members, and showed high similarity with known sequence from human AKT3 (95.8%), rat AKT3 (94.7%), mouse AKT3 (95.4%).Analyzing embryos of different development stages by RT-PCR displayed that akt3 highly expressed at 0-4hpf (hours past fertilization) and fall below the detective sensitivity at 6-12hpf. After 16hpf, its expression began to elevation and maintained at relative high level from 60hpf to 96hpf. Whole mount in situ hybridization analysis showed that zebrafish akt3 express all over the embryo and have no special expression pattern. In adult fish, RT-PCR analysis of tissue distribution demonstrated that akt3 expressed in all tested tissue sample, except gill, and highly expresses in brain and ovary. Additionally, we fundamentally addressed the role of akt3 in zebrafish embryonic development by microinjecting overactivated myr-akt3 in early stage embryo. We found that overexpressing akt3 leads to growth retardation in embryos and causes tail deformities. More important, it increases the embryonic brain thickness and brain length at 24hpf which indicated that akt3 is essential for normal embryonic brain development.These results demonstrated that PKB/Akt might play an important role in zebrafish embryogenesis and neurogenesis.
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