Rescue Of Somatostatin-3 Gene Knockdown Morphant And Functional Verification In Zebrafish Embryos

Somatostatins (SS), which mainly express in the endocrine pancreas, the gut, the peripheral and central nervous system, also in the submandibular glands, thyroid, adrenals, kidney, prostate, blood vessel walls, placenta, and immune cells with a low level, are of a family of cyclic peptide hormones with a variety of biologically activities.The in vitro research indicates that the somatostatins bind with G protein coupled receptors plays its physiological action, which include the inhibition of the secretion and release of growth hormone, the secretion of endocrine hormones and the exocrine ability of the gastrointestinal tract, and the function as a neurotransmitter or neural regulation factor to regulate the proliferation and differentiation of normal and tumor cells. In the present study, six SS genes have been identified in the zebrafish. The unction of the SSs are poorly understood in the embryo development. In the previous study, we cloned SS3 cDNA, and found that the SS3 was expressed in the zebrafish pancreatic tissue; the glucagon and insulin mRNA expression was significantly reduced in SS3 and SS4 mRNA double knock down embryos. To reveal the physiological roles of SS3 in the early embryonic development, we used the zebrafish embryos as a model for studying the regulation of SS3 to insulin expression during embryonic development and employed in vitro synthesized the SS3 mRNA to rescue SS3+SS4 knockdown embryo, Our data are useful to elucidate the function of somatostatin-3 in the embryonic development.First, to verify the validity of the SS3 MO, the pCS2+SS3 MO EGFP recombinant plasmid containing SS3 5’end of the MOs target sequence and a EGFP reporter gene was constructed.The results of this study obtained from the embryos co-injected with SS3 MOs and the pCS2+SS3 MO EGFP recombinant plasmid are based on SS3 mRNA targeting morpholino antisense oligonucleotides having a highly sequence-specific targeting in the embryonic cells can efficiently and specificially knock down gene expression of SS3. Secondly, basing on codon degeneracy, anti-SS3 MO fusion plasmids were constructed by introducing the mutant nucleotides (no change encoding amino acids). Co-injected with SS3 MO and the pCS2+SS3 EGFP recombinant plasmid, the results indicated the plasmid containing a reporter gene successfully expressed in embryonic cells, proved the combination construct with MO resistance in vivo.Co-injected with the use of pCS2+SS3 kzm recombinant plasmid of anti-SS3 in vitro synthesized SS3 mRNA and SS3 MO, The results showed that compared with wild type control, the co-injection SS3 MO+SS4 MO+anti-SS3 MO mRNA embryos had no significant difference. It indicated the SS3 mRNA synthesized in vitro could rescue the defected phenotypes of SS3 MO and SS4 MO knockdown embryos. The protein from translated anti-SS3 MO mRNA synthesized by in vitro was able to replace most of the functions of the natural SS3 mRNA.In order to verify the regulation specificity of zebrafish embryonic SS3 gene on insulin gene expression, we use DIG-labeling RNA probe to detected insulin mRNA expression by in situ hybridization. The results indicated insulin mRNA expression was significantly reduced in SS3 and SS4 double knockdown embryos. In pancreatic tissue, insulin mRNA expression restored or slightly increased by SS3 mRNA rescue. The result indicated that SS3 possibly involved in insulin gene transcriptional regulation via paracrine action of in some ways. SS3 could replace SS4 to regulated insulin expression. The regulation style of insulin transcription in embryonic stage of SS3 is combination result from the maintenance steady-state of extracellular insulin level, as well as the regulation of insulin synthesis and secretion of SSs in embryonic development. In addition, we also observed that SS3 and SS4 double knock down embryos had several significant morphological features which were different from wild type embryos including yolk extension defects, ventricular dilatation, slow blood flow et al. The abnormal phenotypic defects were recovered after rescue, indicating that SS3 is likely involved in the regulation of early embryonic development and growth through as the systemic endocrine signals.