Análise da função biológica da molécula orientadora por repulsão a1 (RGMA1) durante o estabelecimento de precursores musculares em somitos de embriões de galinha in vitro

Abstract

The Repulsive Guidance Molecule a (RGMa) is a protein originaly described playing role as a repulsive cue during axonogenesis, via the Neogenin and/or as co-receptors of the BMP signaling pathways. RGMa expression pattern during the chicken embryo development suggested additional roles for this protein, including during somitogenesis and myogenesis. Our group has previosly identified a RGMa isoform exclusively expressed in the somites, which are transitory structures that give rise to the skeletal muscle cells of the trunk and also to the skeletal muscle stem cells. The objective of this study was to characterize the sequence of this isoform, RGMa1, and to investigate its biological function in chicken embryo somite cells in vitro. Nucleotide sequence analysis revelead that RGMa1 is produced by a post-transcriptional mechanism that occors in the RGMa sequence, complete removing the domain that codify the Nterminal portion of the protein. These results suggested that RGMa1 plays its role as membrane-anchored protein. Functional analysis was performed in somite cells from 33-35h chicken embryos (HH10) which form the followiong groups: (i) caudal epithelial somites; (ii) caudal epitelial somites associated with the neural tube and notochord; (iii) cranial mature somites; and (iv) cranial mature somites associated with the axial structures. In addition RGMa1 overexpression could upregulate the expression of pre-myogenic (Pax3/Pax7) and myogenic (MyoD/Miogenina/Desmina) markers of cells from the groups (i), (ii) and (iii). RGMa1 could additionaly induce Myf5 and αactinin expression in the cells from group (iii). RGMa1 overexpression in the cells from group (iv), however, induced the opposite effect: all tested markers were found to be downregulated. These results suggest an important biological function for RGMa1 during the establishment of the myogenic cell lineages in the epithelial somites; and that RGMa1 expression is regulated by factors expressed by the axial structures after somite maturation. It is also important to note that RGMa1 effects on the expression of premyogenic and myogenic factors are not performed via Neogenin or BMP receptors, as it was not possible to note changes in their respective secundary messengers expression that could correlated with the observed effects. Our results contribute to include RGMa1 as a new factor involved with myogenesis, a molecular program that shows medical and biotechnological applications.