| As the original gene of the somatostatin (SS) gene family, SSI is synthesized through propersomatostatin 1 (PPSS1) and is two bioactivity polypeptides which is produced with the catalysis of tissue-specific protease. The SS-14 and SS-28 type, which are common among all the vertebrates, are the same between the 14 position and 28 position of the C-terminal. With hybridization in situ, the pervious studies had already confirmed that SSI was expressed in both CNS and pancreas in the development period of zebrafish embryo, but its role during early embryonic development is unknown. On the basis of our past experiment, SS1 gene expression was targeted knockdown the sequence near start codon of SS1 gene by microinjection in the morpholino oligo nuclear acid. The results showed that the locomotion activity and the development had significantly decreased after knockdown morphants. However, the SSI mRNA synthesized in vitro can partly rescue the morphant defect phenotype. In order to verify whether other genes’expression was also influenced by the down-regulation expression of SS1 gene and to further explore the function of SS1 gens, the present study using developmental genetic techniques, such as targeted knockdown mRNA expression, in vitro transcription synthesis mRNA and expression in vivo and the microinjection zygote etc, to acquire the zebrafish embryos of SSI knockdown and rescue, subsequently using the next generation depth sequencing technology to analysis the gene expression profile of 30hpf embryos of SSI knockdown and rescue compared with wild type and screening variation expressed genes. The results will be of importance to elucidate the function of SSI gene during embryonic zebrafish.SS1 gene was knockdown and then rescue. Total RNA were extracted from the 30 hpf embryos including knockdown, rescue, and wild type embryos. Digital Gene Expression Profiling (DGE) analysis was performed. Each group replicated two times. Illumina high throughput sequencing was performed for each replicate of each group. Each library output about 11.77 M raw reads. After filter of the low quality data, a total 77763126 clean reads were produced. The average clean reads of each sample was 11.66 M-15.02 M; and with a similar GC content (～47%). About 11 M～14.11 M of each sample reads match the zebrafish’s genome map, taking up about 93.52%-94.30%, indicating that zebrafish’s genome is an ideal reference. The Pearson’s correlation coefficient within zfSS1 MR group and zfWT group was similar (R2=0.98 and R2=0.973). This results suggested a highly repetitiveness of this technology. Besides, zfSS1MR group and zfWT group had the similar variation tendency of gene expression level, while the gene expression level variation of zfSSIM group was much higher than these two group, up-regulation or down-regulation. With respect to the biological repetitive experiment, DESeq had already eliminated the biological variation, and then the standard of differentially expressed genes was padj< 0.05. Compared to zfWT group, zfSSIM group had 935 genes appeared significantly variation expression, which 334 genes were up-regulation and 601 genes were down-regulation. However, compared to zfWT group, zfSS1MR group had only 1 gene appeared to be variation expressed, which suggested the embryo gene expression of rescue group was similar to the control group. Meanwhile, compared to zfSS1M group, zfSS1MR group had 1086 variation expressed genes, among which 691 genes were up-regulation and 395 genes were down-regulation, indicating a significant variation expression between rescue group and knockout group. Therefore, at the same time of rescue, the variation expressions of other genes were also influenced not only for the gene variation expression of knockdown. The variation genes between zfSSIM and zfWT, zfSS1MR and zfSSIM were selected. The same variation genes among the two group selected genes were then the successful rescued genes. The former selected group had 705 genes, and the latter group had 935 genes, which showed 705 genes were successfully rescued.The results of DGE analysis are as follows:(1) the Insulin induced protein family (insigl), Insulin-like growth factor Ⅱb(igf2b) and Insulin-like growth factor binding protein(igfbps) were significantly up-regulated after SS1 gene knockdown; but after SS1 rescue, the gene expression level recovered to the no significant differences level of the control group and negatively correlated with SS1 expression. As we known, GH/IGF and its signal member is the key to promote growth and development, the present results shows that SS1 regulated the growth-promotion function of GH and IGF by negative regulation. The knockdown of SS1 caused the significant up-regulation of thyrotropin releasing hormone (trh) gene expression; TRH was mainly to promote the release of thyroxin and thyroxin would promote the growth and development of brain and reproductive organs. Moreover, thyroxin is also major regulator of the release of GH by pituitary gland. Our results also suggested that SS1 could suppress the expression of trh and indirectly regulate the release of GH.(2) After SS1 knockdown, the embryos phenotype shows fuzzy brain room and under development. The DGE analysis shows significant down-regulation of Pou3fl, but after SS1 mRNA rescued, it recover to the no significant differences level of the control group. Pou3f1 transcription factor induce the stem cell in the dermal layer into neural precursor cells and then into Ectoderm. These neural precursor cells continue to differentiate into neuron and glial cells, and finally develop into brain and neural systems. All of that suggested that SS1 possibly influence zebra fish brain and neural systems by positive regulating the expression of POU III gene family. soxl9a, soxl9b, sox21b and soxl2 were significantly down regulated after SS1 gene knockdown, but these gene expression level recovered to the no significant differences level of the control group after SS1 mRNA rescue. The two members of SoxB1, sox19a and sox19b, can promote neurons differentiation. Induction of Sox21 was another requirement of neurogenesis, and Sox21 may inhibit SoxB1 protein by competitive binding target gene. The significant down regulation of soxl9a, soxl9b, sox21b and sox12 after SS1 knockdown suggested SSI was positive correlation with SOX transcription factor expression and may have function in neural differentiation.(3)foxo3b was significantly up regulated after SS1 gene knockdown, and the gene expression level recovered to the same level of the control group after SS1 mRNA rescue. However, FoxO, as the sub-family of the Fox transcription factor family, could induce G1 block, G2 delay, DNA repair and apoptosis inducing. Embryo defect after SS1 knockout was possibly related to the Fox transcription factor. Thus, we can infer that SSI correlated with Fox transcription factor maintains the embryo development, cell recycle procedures, apoptosis inducing and metabolism.In conclusion, SS1 through the direct way acting on GH/IGF axis to negative regulating development in zebrafish embryonic, and also by up-regulating trh gene expression to effect on GH action. SS1 may involve in zebrafish brain, nervous system development and neuronal differentiation via positive regulating pou and sox gene families expressions. SS1 may by negative regulating the expression of several fox transcription factors to maintain embryonic development, cell cycle progression, apoptosis induction and metabolism. |
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