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Importância da região 3´UTR do mRNA do deltaC no desenvolvimento de Peixe-Zebra
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A somitogénese é um processo morfogenético altamente regulado e conservado entre os vertebrados. Durante este processo ocorre a formação de sómitos de forma rítmica e sequencial ao longo do eixo ântero-posterior do embrião a partir de um tecido progenitor não segmentado denominado de mesoderme pré-somítica (PSM). Os sómitos são aglomerados de células da mesoderme, precursores do esqueleto e da musculatura axial. A periodicidade da formação de sómitos a partir da PSM é imposta por um oscilador molecular denominado de relógio molecular que leva à expressão cíclica de um conjunto de genes, que se propaga ao longo da PSM no sentido caudo-rostral. A comunicação celular e a sincronização das oscilações de expressão na PSM através da sinalização intercelular Notch-Delta são fundamentais no controlo da somitogénese e da diferenciação celular. Esta oscilação da expressão dos genes do relógio é controlada por um mecanismo de regulação de feedback negativo, onde a instabilidade do mRNA desempenha um papel fundamental. A região 3´UTR geralmente contém vários elementos reguladores que governam a expressão espacial e temporal de diferentes mRNAs.
Estudos anteriores do laboratório demostraram uma estabilização do mRNA do gene deltaC de peixe-zebra na ausência de porções da região 3´UTR. Para melhor compreensão do fenótipo, neste trabalho foi avaliado o papel da região 3’UTR do gene deltaC na somitogénese de embriões de peixe-zebra. Para isso foram utilizadas diferentes linhas mutantes em que a região 3'UTR do gene deltaC foi editada usando a tecnologia CRISPR/Cas9. Procedeu-se à caracterização do padrão de expressão dos genes her7 e deltaC e caracterização morfológica dos sómitos formados para perceber o impacto destas alterações. Os resultados do presente estudo confirmaram que a região 3´UTR de deltaC é importante para a correta expressão de deltaC e her7, bem como para a segmentação corporal do embrião de peixe-zebra. Embora nos embriões RR2 e RR3 a segmentação não é interrompida, o número total de sómitos é menor e os sómitos anteriores são ligeiramente mais curtos. Usando uma linha transgénica em que o Notch Intracellular Domain (NICD) é expresso constitutivamente após indução térmica observou-se diminuição do tamanho dos sómitos como nos embriões RR3 o que sugere que o fenótipo observado nos embriões mutantes poderá ser devido a um aumento da sinalização Notch durante a somitogénese.
Embryonic somitogenesis is a highly regulated and conserved morphogenetic process among vertebrates. During this process, somites are formed rhythmically and sequentially from a non-segmented progenitor tissue called presomitic mesoderm (PSM) along the anterior-posterior axis of the embryo. Somites are clusters of mesoderm cells that are precursors of the axial skeleton and musculature. The periodicity of somite formation from the PSM is due to an oscillator mechanism called the molecular clock that leads to the cyclic expression of a set of genes that propagate along the PSM in a caudal-rostral direction. Cellular communication and synchronization of expression oscillations in the PSM through Notch-Delta intercellular signaling are fundamental in the control of somitogenesis and cell differentiation. The oscillation of clock gene expression is controlled by negative feedback, where mRNA instability plays a key role. The mRNA 3'UTR region generally includes several regulatory elements that govern the spatial and temporal expression of mRNAs. Previous studies in the lab suggest that mRNA stabilization in the absence of portions of the 3'UTR region of zebrafish deltaC gene might be involved in the regulation of the molecular clock. For a better understanding of the phenotype, this work was designed to evaluate the role of the 3’UTR region of the deltaC gene in the somitogenesis of zebrafish embryos. For this study, different mutant lines generated by CRISPR / Cas9 were used, where the 3'UTR region of the deltaC gene was edited. The characterization of expression patterns of her7 and deltaC genes was carried out, as well as the morphological characterization of the somites formed in these mutants. The results of this study confirmed that the deltaC 3'UTR region is importante for proper expression of deltaC and her7, as well as for zebrafish embryo body segmentation. Although in RR2 and RR3 embryos segmentation is not interrupted, the total number of somites is smaller and the anterior somites are slightly shorter. Using a transgenic line where the Notch Intracellular Domain (NICD) is constitutively expressed after heat-shock was observed a decrease in somite size as observed in the RR3 embryos suggesting that the phenotype observed in the mutant embryos could be due to enhanced Notch signaling during somitogenesis.
Embryonic somitogenesis is a highly regulated and conserved morphogenetic process among vertebrates. During this process, somites are formed rhythmically and sequentially from a non-segmented progenitor tissue called presomitic mesoderm (PSM) along the anterior-posterior axis of the embryo. Somites are clusters of mesoderm cells that are precursors of the axial skeleton and musculature. The periodicity of somite formation from the PSM is due to an oscillator mechanism called the molecular clock that leads to the cyclic expression of a set of genes that propagate along the PSM in a caudal-rostral direction. Cellular communication and synchronization of expression oscillations in the PSM through Notch-Delta intercellular signaling are fundamental in the control of somitogenesis and cell differentiation. The oscillation of clock gene expression is controlled by negative feedback, where mRNA instability plays a key role. The mRNA 3'UTR region generally includes several regulatory elements that govern the spatial and temporal expression of mRNAs. Previous studies in the lab suggest that mRNA stabilization in the absence of portions of the 3'UTR region of zebrafish deltaC gene might be involved in the regulation of the molecular clock. For a better understanding of the phenotype, this work was designed to evaluate the role of the 3’UTR region of the deltaC gene in the somitogenesis of zebrafish embryos. For this study, different mutant lines generated by CRISPR / Cas9 were used, where the 3'UTR region of the deltaC gene was edited. The characterization of expression patterns of her7 and deltaC genes was carried out, as well as the morphological characterization of the somites formed in these mutants. The results of this study confirmed that the deltaC 3'UTR region is importante for proper expression of deltaC and her7, as well as for zebrafish embryo body segmentation. Although in RR2 and RR3 embryos segmentation is not interrupted, the total number of somites is smaller and the anterior somites are slightly shorter. Using a transgenic line where the Notch Intracellular Domain (NICD) is constitutively expressed after heat-shock was observed a decrease in somite size as observed in the RR3 embryos suggesting that the phenotype observed in the mutant embryos could be due to enhanced Notch signaling during somitogenesis.
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Keywords
somitogénese 3´UTR Peixe-zebra Notch-Delta mRNA