MicroRNA-1 Regulates Neural Crest Cells And Affects Craniofacial Development

Objective: Establishing a zebrafish model of microRNA-1(miR-1)gene knock out to explore the regulatory mechanism of miR-1 on neural crest cell differentiation and function.Methods: Zebrafish model of miR-1 gene knockout was obtained by CRISPR/Cas9 technology,at the same time,the wild type zebrafish(WT)was used as control.PCR electrophoresis was conducted to verify the knock out of miR-1 gene.Using in situ hybridization technique to observe the original expression of miR-1 in zebrafish embryo.Morphological differences between two groups were observed by stereomicroscope.Changes of cartilage morphology at 4 days(4dpf)were displayed by Alcian blue staining.The migration and differentiation of neural crest cells were detected by in situ hybridization.Using TUNEL staining to investigate the effect of miR-1 on neural crest cell apoptosis.RT-PCR was used to reveal the changes of apoptosis signal molecules.Results: The products of PCR confirmed the complete knockout of miR-1 gene inzebrafish.In situ hybridization showed that miR-1 was the highest expression at 24 hpfin zebrafish.Knockout of miR-1 resulted in cardiac edema,mandibular retrusion and reduction of its pigment at 4dpf.Alcian blue staining revealedthe backward tongue bow and cartilage in the experiment group.In situ hybridization showed that the knockout of miR-1 mainly affected the differentiation and migration of neural crest cells.TUNEL staining exhibitedthe increased apoptotic cells in the head of zebrafish.Furthermore,RT-PCR showed that the m RNA level of caspase-3 increased significantly while Bcl-2 decreased compared with the WT.Taken together,miR-1 mainly affects the function of neural crest cells by affecting the apoptosis and differentiation,migration of neural crest cells.Conclusion: Micro RNA-1 affects craniofacial development by regulating apoptosis and differentiationof neural crest cells...