The role of bone morphogenetic protein signaling in zebrafish caudal fin regeneration

Abstract

Zebrafish possess the remarkable ability to regenerate many organs and tissues following damage, including their fins. Understanding the mechanisms behind regeneration in these organisms may be the key to promoting regeneration of tissues in humans. Using the zebrafish caudal fin as a model we examined the role of the Sonic hedgehog (Shh) and Bone morphogenetic protein (Bmp) signaling pathways in fin regeneration. The expression patterns of shh, its receptor ptc1, and downstream target bmp2b within the fin regenerate revealed their potential roles in bone regeneration. To test their involvement we performed a functional analysis by ectopically expressing and inhibiting Shh and Bmp2b signaling within the regenerate. Ectopic expression of shh and bmp2b within the blastema, using an in vivo transfection method, resulted in ectopic bone formation. This effect was blocked when we co-injected shh and chordin, a Bmp antagonist, indicating that Shh signaling requires a Bmp factor to induce ectopic bone formation. These findings link Shh and Bmp signaling to scleroblast proliferation, differentiation and/or function during fin regeneration. Two Bmp family members are expressed within the regenerate in a pattern suggesting their role in bone regeneration: bmp2b, and the newly characterized bmp6. On the other hand, bmp4 expression, in cells of the distal blastema suggests its possible function in growth of the regenerate. To establish a direct role for Bmp signaling, we inhibited signaling by injecting chordin into the blastema of individual fin rays, resulting in reduced matrix secretion attributed to a defect in scleroblast function. We identified Bmp targets with known function in bone formation, runx2a/b and col10a1, which are both expressed in scleroblast cells and downregulated following Bmp inhibition and are likely mediating the function of Bmp signaling in scleroblast differentiation/function. Interestingly, within the regenerate we detected the expression of 'cartilage-specific' genes involved in endochondral bone formation although fin rays form through dermal ossification, and many of these factors are regulated by Bmp signaling. These findings suggest that fin rays may be made of an intermediate type of bone or that intramembranous and endochondral bones are more closely related than previously thought. Also, inhibition of Shh and Bmp signaling pathways, using cyclopamine and chordin misexpression respectively, led to a decrease in blastema proliferation, and a downregulation of the msxb and msxc transcription factors. Overall our studies indicate a critical role for the Shh and Bmp signaling pathways in fin regeneration, mediating both scleroblast differentiation/function and blastemal proliferation and growth.