Spatiotemporal Expression Of Somatostatatin And Developmental Role In Zebrafish Embryo

Somatostatin. which is a short polypeptide with heterology. exists almost in all vertebrates. SS is usually translated to the form of preprosoamostatin. which will undergo tissue-specific tissue cleavage generating the bioactive peptides (SS-14. SS28 and other N-terminal prolonged form). SS is expressed in nervous system and various peripheral organs, such as central nervous system and peripheral nervous system neurons, gastrointestinal tract, pancreas, thyroid et al. SS exhibites numerous functions in vertebrates, such as modulate growth, energy distribution, digestion and metabolism. SS is also inbolved in development progress, which is evidenced by onset SS transcription and transient expression in mouse embryos and its capacity affect neuronal migration in the developing rodent nervous system.However, not too much is known about its role in embyos until now. Different SS genes have been characterized in vertebrates, especially in teleost, which may implicate variable and complex functions. In this article, we investigate expression pattern of SS in early developmental stages of zebrafish embryo using methods of RT-PCR. in situ hybridization, microinjection and rescue experiment. Besides, we also investigate the function of SSI in zebrafish embryos development by blocking its transcription and rescue experiment.Four complete or partial SS cDNA sequences (SS2.3.5.6) have been cloned. Comparing the deduced amino acid sequcences by software, it shows that C-terminal amino acid sequences are relatively conserved which present characterized sequences of SS and SS-like peptide:however N-terminal sequences have very low homology. Two cysteine residues could form disulfide bond which could contribute to form stable second conformation of SS polypeptide.Variable and changing expression pattern of SS could be observed during zebrafish embryo development.SS2 mRNA expressed in inner primodium. and could maintain its expression in neurosecretop. preoptic nucleus and posterior tuberculum until 3 days post fertilization, which possibly implicates certain function in nervous system development and neuroendocrine modulation. SS3 temporarily expressed in neural head region, and could maintain its expression until 3dpf in the pancreas which recapitulate expression pattern of SS4. It indicates that SS3 secreting cells are important component of pancreas and may be involved in endocrine modulation. Besides.SS3 also expressed in some neurons of diencephalon which may supplied evidence that endocrine was controlled by brain. No significant signal have been observed, however it seems that it’s not for the reason of the degradation of RNA probe. Further study will promote functional research of SS6.SS1 morpholino is designed directly to region near the initiation code, and injected into 1-2 cell zebrafish embryos. Significant morphant phenotype was observed, including decreased locomotivity and arrested ontogenesis. Comparing to wild type embryos,SS1 morphants are characterized with shorter body length, smaller eyes, yolk absorption disability and yolk extension defects. Several expression vectors have been constructed in this experiment. pCS2+EGFP is used for construction of EGFP fusion expressing vectors. Specificity and efficiency of SS1 MOs is verified by coinjection of SS1 MOs and pCS2+SSl MO chk EGFP vector into zebrafish embryos. SS1-EGFP fusion protein expressing vector has also been constructed. Besides. pCS2+SS1 vector is used for transcription of site-directed mutated SS1 mRNA in phenotype rescue experiment. After coinjection of SS1 MOs and SS1 mRNA into zebrafish embryos, it shows that the mutated SS1 mRNA could reverse phenotypes of SS1 morphant.