The Expression,Pluripotent Activity And Transcriptional Regulation Of Oct4 From Nile Tilapia

The origin and evolution of the pluripotent regulatory mechanism are the essential issues in cell biology researches. Extensive studies show that the key transcriptional factor Oct4(octamer-binding transcription factor-4) also called Pou5f1, can maintain pluripotency of the mammalian inner cell mass(ICM) as well as embryonic stem cells(ESCs)in vitro, and play important roles in PGC survival, spermatogonial and ovary development. The embryos of Oct4-deficient mouse can only develop to blastula and form the trophectoderm cells not ICM; conditional depletion of Oct4 leads to apoptosis of PGCs rather than to differentiation into a trophectodermal lineage; the four factors Oct4, Sox2, Klf4 and c Myc can induce the differentiated cells to induced pluripotent stem cells(i PS) by cotransfection, which reveals that the significant function of Oct4 in pluripotency maintenance and induction. However, whether the pluripotent expression and acivities of Oct4 in fish is similar to that of mammal has remained to be determined.It has been shown that both zebrafish pou2 and medaka oct4 are the homolog of mammal. Nevertheless, there are some differences of Oct4 between fishes and mammals in expression pattern and bioactivities. Unlike mammalian Oct4, zebrafish Pou2 expresses in neural plate and brain during embryo development stages, and does’ t express in PGCs, while pou2 deficient zebrafish can form the ICM and develop to gastrula stage. Transfection Oct4-dificient mouse with Pou2 fails to rescue the mouse stemness, while medaka Oct4 is substantially the same as mammals except for the brain expression. Whether fish Oct4 in expression pattern and pluripotent activity is common to mammals remains unknown. Nile tilapia belongs to Perciformes, Cichlidae, Oreochromis niloticus, and is the world’s commercial fishes in aquaculture, however, the pluripotent factor Oct4 in tilapia has not been elucidated. In the present study, the expression pattern, pluripotent activity and transcriptional regulation of Oct4 from tilapia have been carried out by immunohistochemistry, morpholino depletion, and promoter activity analysis.1、Oct4 expression pattern and pluripotent activity1.1 Oct4 expression at m RNA levelOct4 expression in different adult tissues was observed in ovaries and testes, but not in other tissues by RT-PCR; while the Oct4 expression in embryos at different developmental stages appeared dynamic expression pattern. Strong signals occurred at 6, 12, 24 and 36 hpf(hours post fertilization), but not in 48, 96, 120 and 144 hpf.1.2 Oct4 expression pattern at protein levelOct4 expression pattern at protein level also appeared dynamic expression pattern in embryos at different developmental stages. The signals appeared at 0.5(1-cell), 2(4-cell) and 8 hpf(blastula), no signals were found at 18(gastrula) and 45 hpf(somitogenesis), then signals appeared again at 50(brain differentiation) then increased at 52(the hatching period), peaked at 60 hpf, while disappeared at 72 hpf. Meanwhile the Oct4 expression was observed in 5 dph(days post hatching) femal and male PGCs, different developmental oocytes and spermatogonia.1.3 The pluripotent activity of Oct4The pluripotent activity of Oct4 was investigated through microinjecting the tilapia embryos with MOoct(oct4 m RNA antisense morpholino) and MOmoct(5-base mismatch morpholino). Our results suggest that the 85% embryos injected with MOoct failed to develop to gastrula satge and died, while more than 90% embryos injected with MOmoct successfully developed to gastrula and survived. The dissociated cells derived from blastula embryos injected with MOoct cannot form the stem cell-like cells and appear apoptosis at 2-3 days. While the cells derived from blastula embryos injected with MOmoct can adhere, proliferate and form stem cell-like cells in 2-3 days. These results suggest that Oct4 is essential for embryogenesis and pluripotency maintenance of ESCs.2、The transcriptional regulation of Oct42.1 The analysis of oct4 promoterThe oct4 promoter sequences uptream of translation initiation site were cloned, and constructed into the green fluorescence reporter vector p T2AL-Oroct4-EGFP, moreover, four different lengths promoter luciferase reporter vectors, named p Or-219luc-reporter, p Or-726luc-reporter, p Or-1306luc-reporter and p Or-3056luc-reporter, respectively, were constructed as well.2.2 The oct4 promoter activity analysisAfter injected the medaka and tilapia embryos with vector p T2AL-Oroct4-EGFP, obvious green fluorescence signals were predominantly observed at blastula. The green fluorescence signals were observed in TES3(tilapia embryonic stem cells) and MES1(medaka embryonic stem cells) cells transfected with vector p T2AL-Oroct4-EGFP, but no signals were found in differentiated SCO1(southern catfish ovarian granulosa cells) cells transfected with vector p T2AL-Oroct4-EGFP. These results suggest that the 3084 bp sequences upstream the oct4 translation initiation site have promoter activity in vitro as well as in vivo, which suggest that p T2AL-Oroct4-EGFP can be a valid tools for monitoring the cell pluripotency.2.3 Luciferase activity analysis of four different length vectorsAfter transfected MES1 cells with vectors p Or-3056luc-reporter, p Or-1306luc-reporter, p Or-726luc-reporter and p Or-219luc-reporter, the fluorescence intensity was detected to test the promoter activity quntatively. The results indicate that the promoter activity of p Or-726luc-reporter was the highest, while the less was p Or-1306luc-reporter. These results indicat that positive regulatory elements might exist between 726 and 219 bp, and the negative regulatory elements might exist between 1306 and 726 bp.2.4 The positive regulation of Sox2 and Oct4After cotransfected MES1 cells with p Or-1306luc-reporter vector and eukaryotic expression vectors pc DNA3.1-sox2 or pc DNA3.1-oct4, the results indicate that the fluorescence intensity was up-regulated in dose-dependent manner.2.5 The negative regulation of RAAfter treated with 10 n M or 100 n M RA(retinoic acid, has a role in promoting cell differentiation) for 5 days, MES1 cells were transfected with p Or-1306luc-reporter vector and the fluorescence intensity was detected. The results show that the fluorescence intensity was significantly downregulated, which suggests that RA can negatively regulate the expression of Oct4.The present study illustrate that tilapia Oct4 is similar to medaka Oct4 in expression and pluripotent function. Our functional analyses suggest that tilapia Oct4 plays a significant role in embryogenesis and pluripotency maintenance of ESCs, which suggests that the pluripotent expression and activities of Oct4 might be not specific to medaka but common in fish. The 3084 bp sequences upstream of oct4 translation initiation site have promoter activity in vitro as well as in vivo. Moreover, our results suggest that Oct4 expression might be positively regulated by Sox2 and Oct4 and negatively by RA. The study not only lays the foundation of the molecular regulatory mechanisms for fish pluripotent stem cells, but also offers a valid tool for monitoring the cell pluripotency.