Conservation of zebrafish miRNA-145 and its role during neural crest development.

STEEMAN, TOMÁS J.; CALCATERRA, NORA B.; WEINER, ANDREA M. J.

Congreso; Congreso conjunto SAIB-SAMIGE 2021; 2021

The neural crest (NC) is a transient and multipotent cell population that gives rise to a diverse cell lineage. Its formation starts early in development at the border of the neural tube. After closure of the neural tube, NC cells (NCCs) experience an epithelial-to-mesenchymal transition in order to delaminate and migrate away to some of the most distant positions of any embryonic cell type. NCCs differentiate into a variety of derivatives, including neurons, pigment cells, chromaffin cells, bone and cartilage of the face, endocrine cells, cardiac structures, smooth muscle cells, and tendons. A complex gene regulatory network controlling the specification, delamination, migration, and differentiation of this cell type has been thoroughly described. However, the role of post-transcriptional factors, such as miRNAs, has not been deeply characterized yet. miRNAs bind target mRNAs’ 3’UTR and regulate their expression by inhibiting translation or promoting degradation of the target mARN. miRNAs comprise 1-2% of all genes in animals, and since each miRNA is predicted to regulate hundreds of targets, it is thought that half of protein-coding genes are under their control. One such miRNA is miR-145, associated with inhibiting chondrogenic differentiation of murine embryonic mesenchymal cells, as well as critically affecting human articular chondrocyte function. miR-145 was reported to act as a tumor suppressor and has been shown to be downregulated in several types of cancer. It was also predicted to target key genes during NC development such as sox9a/b, col2a1a and ltk. Our in silico analysis showed and strikingly high conservation of miR-145 sequence across 25 animal species. Using zebrafish as a model organism, we modified miR-145 expression levels to assess its role during NC development. Overexpression was achieved by microinjecting in vitro synthetized dsRED-pre-miR145 which is later processed into mature miRNA by the endogenous Dicer1. We also generated a mutant miR-145-/- line with CRISPR/Cas9 genome editing. In both cases we observed and change in the development pattern of iridophores, pigmented cells in charge of light reflection. Overexpression also resulted in aberrant melanocyte (dark pigment cells) development. Another type of NCC derivates, craniofacial cartilage, was also affected. As we showed with Alcian Blue cartilage stain, multiple lower jaw cartilages were shorter when miR-145 levels were altered. By in situ hybridization, NC markers sox9a, sox9b, and collagen type II col2a1a showed reduced or altered spatiotemporal expression patterns. In addition, our in vivo assay shows that the presence of miR-145 affects the amount of dGFP for sox9b-3′UTR and suggests that sox9b expression is regulated by miR-145. All these results reinforce the notion that miR-145 is involved in chondrocyte differentiation and craniofacial development, as well as supporting the use of zebrafish as a valuable tool for disease modeling.