| MicroRNAs (miRNAs) are a kind of endogenous noncoding small RNAs that have 19-25 nucleotides in length and are highly conservative in sequence. It is well known that miRNAs can induce the breakdown of downstream messenger RNAs (mRNAs) and/or the inhibition of translation by binding to their target sites, mainly within the 3’untranslated region (3’-UTR), of downstream target mRNA by base pairing, and have been recognized as a major post-transcriptional regulators of gene expression. A great number of studies have proved that miRNAs play important roles in diverse developmental processes, such as the body pattern formation, organogenesis, immune response, cell proliferation, differentiation, apotosis and division cycle control. Disturbances of miRNAs expression can lead to various diseases in animals and humans, including tumorigenesis, tumor progression and metastasis. However, only a small part of miRNAs have been confirmed their target genes and functions. Identification of more miRNAs that participate in some vital developmental processes and their downstream target genes are important in systematically revealing the regulatory mechanisms of animal development and tumor pathogenesis, as well as figuring out the functions and application value of different miRNAs.In vertebrates, multiple functional regulatory genes are expressed in different embryonic cells at different developmental stages and regulate diverse developmental processes. These genes are initially transcribed in a wide embryonic region but restricted within distinct cell types at subsequent stages during development. Therefore, post-transcriptional regulation is required for the transition from one developmental stage to the next and establishment of different cell identities. However, how the multiple functional regulatory genes are regulated at post-transcription level remains largely unknown.visual system homeobox-1 (ysxl) is such a multiple functional regulatory gene. At blastula and gastrula stages, vsxl is ubiquitously expressed at a low level and plays crucial roles in axial-paraxial mesoderm patterning and prechordal mesendoderm, the head organizer, formation in zebrafish. During neurogenesis, vsx1 is expressed in presumptive neurons of hindbrain and spinal cord, suggesting that it might be involved in regulation of the central neural system formation and the V2 interneurons development. During retinogenesis of examined vertebrates, Vsxl is limited to presumptive bipolar cells, and plays roles in regulating the proliferation and differentiation of retinal progenitors, as well as the functional maintenance of bipolar cells. It has been demonstrated that miRNAs play a role in regulating Xenopus vsx1 expression at post-transcription level during retinogenesis. Nevertheless, it is still unclear whether the miRNA is involved in the post-transcriptional regulation of vsx1 and has effects on the functional transformations of vsx1 during embryogenesis. Therefore, we identified a miRNA that can regulate vsxl expression at post-transcriptional level during embryogenesis and further investigated its role in establishing the spatiotemporal expression pattern of Vsxl and regulating normal embryogenesis. The main results and conclusions are as follows:1. By comparing the diverged sequences and potential miRNA binding sites on the 3’-UTRs of homologous vsxl genes differentiated after genome duplication, we found that vsx1 might be the target gene of miR-20a in goldfish and zebrafish. Then we further analyzed the effect of miR-20a on the expression of vsx1 3’-UTR mediated report gene and endogenous Vsxl, as well as on direct downstream target gene of Vsxl, and validated that goldfish and zebrafish vsxl is a direct target gene of miR-20a indeed. We also revealed that miR-20a represses Vsxl expression mainly via inducing the breakdown of vsx1 mRNA by binding to its 3’-UTR.2. We examined the spatiotemporal expression patterns of miR-20a during embryogenesis in goldfish and zebrafish with real-time quantitative RT-PCR and whole mount in situ hybridization methods, and compared it with the spatiotemporal expression patterns of GFP report gene mediated by vsx1 3’-UTR in both the wild type and miR-20a decoyed embryos. These results exposed that the spatiotemporal expression patterns of endogenous miR-20a and Vsxl protein correlate negatively, vsx1A1 3’-UTR mediated protein expression is inhibited when and where miR-20a is3.expressed. These observations suggested that miR-20a plays an essential role in establishment of spatiotemporal expression pattern of Vsxl.4. Decoying miR-20a resulted in severely impaired head, eye and trunk formation in association with excessive generation of vsxl marked V2 interneurons in the central tier of spinal cord and defect of somites, illustrating that miR-20a is involved in the regulation of fish embryonic development.Taken together, our results indicated that miR-20a is required for normal embryogenesis by restricting Vsxl expression in goldfish and zebrafish, in addition, post-transcriptional regulation is a significant mechanism for Vsxl acting different roles on diverse developmental processes. |
PDF Full Text Request