Disruption of BMP and FGF signaling prior to blastema formation causes permanent bending and skeletal malformations in Poecilia latipinna tail fin

Abstract

Teleost fish, such as Poecilia latipinna, exhibit remarkable regenerative capabilities, making them excellent models for studying tissue regrowth. They regenerate body parts like the tail fin through epimorphic regeneration, involving wound healing, blastema formation (a pool of proliferative cells), and tissue differentiation. Bone Morphogenetic Protein (BMP) and Fibroblast Growth Factor (FGF) signaling pathways play crucial roles in this process, but their specific functions during blastema formation remain unclear. To explore this, BMP and FGF signaling were inhibited using targeted drug treatments prior to blastema formation in amputated tail fins. The treatment group of P. latipinna received drugs at set intervals, and analyses were conducted using skeletal staining, gene expression via quantitative real-time PCR, and protein analysis with Western blotting to assess blastema formation, extracellular matrix (ECM) remodeling, and skeletal patterning. Dual inhibition of BMP and FGF pathways led to significant regenerative defects, including bent blastema and disrupted bone structure, along with downregulation of essential patterning genes like sonic hedgehog (Shh) and bmp2b. Additionally, ECM remodeling and epithelial-to-mesenchymal transition (EMT) were impaired, as shown by reduced matrix metalloproteinases (MMP2 and MMP9), hindering cell migration and blastema stability. Cell proliferation was markedly decreased, as evidenced by reduced proliferating cell nuclear antigen (PCNA) expression and bromodeoxyuridine (BrdU) incorporation, while apoptosis increased, with elevated markers like caspase 3 (casp3) and higher DNA fragmentation. These findings indicate that BMP and FGF signaling are essential for blastema formation and skeletal patterning, with their inhibition causing major regenerative abnormalities.