Browsing by Author "Santos, Mariana Cabral Monteiro Matos"
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- Characterization of microRNAs expression during zebrafish skeletal developmentPublication . Santos, Mariana Cabral Monteiro Matos; Gavaia, PauloSkeletogenesis is a highly conserved developmental process that involves osteogenic and chondrogenic mechanisms, and vast number molecular determinants. High conservation of skeletogenesis and the numerous technical advantages of zebrafish, make this organism a suitable scientific model for the study of this process. In this context, miRNAs recently emerged as important and highly conserved regulators of skeleton formation in vertebrates. MiRNAs are a sub-class of ncRNAs with ~22 nucleotides that function as negative or positive regulators of gene expression and are involved in crucial biological mechanisms. Not surprisingly, miRNAs have been implicated in several physiological and pathological processes, including in skeleton formation. Nevertheless, their in vivo effects in zebrafish have now started to be demonstrated. In our lab, three miRNAs, miR-214, miR-20a, and miR-29a, were recently implicated in both fish and mammalian skeletogenesis. However, there is lack of information regarding the effects of these miRNAs in vivo. Therefore, the purpose of this study was to investigate the skeletogenic effects of these miRNAs using zebrafish as model. We started by investigating levels and sites of expression of miR-20a and miR-29a (already performed for miR-214) throughout zebrafish development, using qPCR and in situ hybridization techniques. Simultaneously, we proceeded to 1) the creation of transgenic zebrafish lines overexpressing miR-214, miR-20a, and miR-29a, in a constitutive manner (using constructs containing cmv promoters); and 2) the creation of transgenic zebrafish lines overexpressing miR-20a, in skeleton tissues (using cartilage-specific collagenXa1 promoter and bone-specific osteocalcin promoter). So far, only one founder specimen, for cmv-miR-29a construct, was obtained. As an alternative approach, we forced the overexpression of miR-29a in zebrafish larvae by microinjecting embryos with up to 9 μM of miRNA mimic of dre-miR-29a in eggs, and investigated skeleton phenotypes at 6 dpf.