RESUMEN
Bone morphogenetic proteins play a pivotal role in the epimorphic regeneration in vertebrates. Blastema formation is central to the epimorphic regeneration and crucially determines its fate. Despite an elaborate understanding of importance of Bone morphogenetic protein signaling in regeneration, its specific role during the blastema formation remains to be addressed. Regulatory role of BMP signaling during blastema formation was investigated using LDN193189, a potent inhibitor of BMP receptors. The study involved morphological observation, in vivo proliferation assay by incorporation of BrdU, comet assay, qRT-PCR and western blot. Blastemal outgrowth was seen reduced due to LDN193189 treatment, typified by dimensional differences, reduced number of proliferating cells and decreased levels of PCNA. Additionally, proapoptotic markers were found to be upregulated signifying a skewed cellular turnover. Further, the cell migration was seen obstructed and ECM remodeling was disturbed as well. These findings were marked by differential transcript as well as protein expressions of the key signaling and regulatory components, their altered enzymatic activities and other microscopic as well as molecular characterizations. Our results signify, for the first time, that BMP signaling manifests its effect on blastema formation by controlling the pivotal cellular processes possibly via PI3K/AKT. Our results indicate the pleiotropic role of BMPs specifically during blastema formation in regulating cell migration, cell proliferation and apoptosis, and lead to the generation of a molecular regulatory map of determinative molecules.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Poecilia/fisiología , Aletas de Animales/efectos de los fármacos , Animales , Proteínas Morfogenéticas Óseas/genética , Ciclo Celular , Fragmentación del ADN , Matriz Extracelular , Femenino , Masculino , Pirazoles/farmacología , Pirimidinas/farmacología , RegeneraciónRESUMEN
Caudal fin regeneration in sailfin molly, Poecilia latipinna (Lesueur 1821) involves an initial wound healing stage, followed by blastema that is formed of fast proliferating cells. In order to replicate the lost fin, correct differentiation of the blastemal cells into various tissues is the prime essence. Among the molecular signals governing proper differentiation of blastemal cells, members of the bone morphogenetic protein (BMP) family are crucial. Herein, we investigated the specific effects of inhibition of BMP signaling using LDN193189 on skeletal and connective tissue formation in the regenerating tail fin of P. latipinna during early differentiation phase. It was observed that BMP inhibition leads to reduction in the length of regeneration, which can be correlated with compromised proliferation of blastemal cells. Decreased expression of cell proliferation marker like pcna together with reduced BrdU positive cells consolidate the above observation. Further, histological analysis revealed stunted progression of skeletal tissues and this correlated with the reduced expression of sox9, runx2 and dlx5, Osc and Osn genes in response to BMP inhibition. Also, defective bone patterning was observed due to BMP inhibition, which was associated with diminished levels of shh, ptc-1, gli2 and other BMP ligands. Moreover, histochemical analysis revealed that collagen, one of the most prominent components of connective tissue, was formed below par in treated fin tissues which was subsequently confirmed by biochemical and transcript level analyses. Overall our results highlight the importance of the BMP pathway in proper differentiation of skeletal and connective tissues during the differentiation stage of regenerating caudal fin.
Asunto(s)
Aletas de Animales/fisiología , Proteínas Morfogenéticas Óseas/metabolismo , Diferenciación Celular , Proteínas de Peces/metabolismo , Regeneración/fisiología , Transducción de Señal , Animales , PoeciliaRESUMEN
The tail fin of teleost fish responds to amputation by expressing few putative factors that promote scar-free wound healing, which paves the way for restoration of the lost part. Among the factors playing a role in this initial response, bone morphogenetic proteins (BMPs) are crucial. In the current study, we have analyzed the effect of BMP inhibition on wound healing in sailfin molly Poecilia latipinna. The study involved histological assessment of wound epithelium formation, an expression profile of proteins, and gelatinase activity as well as expression in response to BMP signal inhibition. LDN193189, a pharmacological inhibitor of BMP receptor, was administered to experimental fish. Our observations include incomplete wound healing and a significant reduction in the expression of a number of proteins as a result of LDN treatment at 24 h post-amputation. A pronounced effect was also seen on the gelatinases MMP-9 and MMP-2, which showed significantly reduced activities on a zymogram. Reduced expression of these MMPs after inhibitor treatment was also confirmed by western blot and real-time PCR analyses. In view of these results, we confirm that BMP signaling has a definitive role in the early stages of fin regeneration in P. latipinna. The effect of BMP inhibition is especially seen on the expression of MMP-9 and MMP-2, which are very important effectors of tissue remodeling immediately following amputation.