Expression And Function Of FoxO3 In Zebrafish Neural Development

Neural development in vertebrate is a rather complex event. To reveal the physiological effect of FoxO3a during neural development, we identified FoxO3 from the model animal zebrafish and undertook"loss-of-function"and"gain-of-function"experiments in zebrafish. This work provides a novel clue to explore the physiological function of FoxO3 in zebrafish.FoxO3 is a member of the FoxO subfamily of forkhead (fox) transcription factors and is known to control glucose metabolism, cellular proliferation, cellular differentiation, DNA repair, cell cycle arrest, longevity, oxidative stress, as well as cell apoptosis.Firstly, by using RT-PCR, Whole-mount in situ hybridization and Immunohistochemistry, we detected FoxO3a expression in various adult tissues and in zebrafish embryos at the mRNA level. In adult zebrafish, FoxO3a expression at the mRNA level was detectable in the brain, eyes, gills, spleen, intestine, ovary, testis, heart and liver, but it was absent or very low in the muscle and swimming bladder. Furthermore, the mRNA level of FoxO3a was obviously increased at 4 hpf. Thereafter, FoxO3a expression at the mRNA level gradually decreased and was maintained at a comparatively low level until 72 hpf, as compared to embryos at the 1- to 2-cell stage. FoxO3a expression aggregated towards the head at 11 hpf. At 22 hpf, FoxO3a expression was obvious in central nervous system, including the brain and lateral spinal cord. The presence of FoxO3a transcripts at 48 hpf was remarkable in the telencephalon, diencephalon, mesencephalon, MHB, cerebellum and rhombencephalon. At 72 hpf, FoxO3a expression was also detected clearly in the brain and retina. These results indicated that FoxO3a was related to neural development in zebrafish.By using FoxO3a morpholino antisense oligonucleotides to knockdown the expression of FoxO3a in zebrafish embryos, we observed that FoxO3a loss-of-function achieved by 8ng FoxO3a-MO injection led to neural developmental defects, including increased neural apoptosis as detected by acridine orange(AO) and terminal deoxynucleotidyl transferase biotin-dUTP nick end labeling(TUNEL). These defects could be partially rescued by the injection of FoxO3a mRNA. In this study, we found that"loss-of-function"of FoxO3a resulted in the decreased expression of neuronal markers as determined by in situ hybridization and relative quantitative real-time PCR. Furthermore, the activation of FoxO3a was required for the maintenance of neuron survival but not necessary for the induction of neurogenesis.To reveal further the possible mechanism of FoxO3a in regulating neural development, we performed the construction of A3 mutant of zebrafish FoxO3a and the synthesis of the capped A3 mRNA (the putative Ser/Thr phophorylation sites by akt replaced by alanines). Our results showed that around 400pg capped mRNA of FoxO3a or A3 injection leads to an increased developmental defect. Moreover, the rate of abnormality in group of A3 mRNA overexpression was significantly higher than that in FoxO3a control group.FoxO5, also called FoxO3b, is also orthologue of vertebrate FoxO3a in zebrafish. Therefore, we also detected the expression pattern of FoxO3b in zebrafish by RT-PCR and the whole embryo in situ hybridization, which showed a large of overlapping expression distribution with FoxO3a in zebrafish embryonic development, especially in development brain. Consequently, the knockdown of FoxO3b through its anti-MO injection in zebrafish embryogenesis was also achieved in our experiments, which showed FoxO3b inhibition by 6 ng FoxO3b-MO induced an increased neural apoptosis similar to those observed in FoxO3a-MO injected embryos. Furthermore, co-injection of FoxO3a-MO and FoxO3b-MO repressed largely the expression of neural markers by relative quantitative real-time PCR detection and triggered more severe defects, which suggested that FoxO3b is synergized with FoxO3a for neural development. In addition, co-blocking of FoxO3a and FoxO3b induced increased p-Akt as indicated by Western blot using either anti p(473)-Akt or Akt antibodies at 24 hpf in zebrafish.In conclusion, these findings are firstly proven in vivo that the FoxO3 is essential for neural development during zebrafish embryonic development.