Several signaling pathways have been shown to play important functions during regeneration in different invertebrate and vertebrate models. Many of these pathways including activin, BMP, Hh, Igf, Notch, retinoic acid (RA) and Wnt/b-catenin have been shown to be required for the successful regeneration of the zebrafish fin. However, little is known yet on the tissue-specific roles of theses pathways as well as on how they interact to orchestrate fin regeneration. Now, a recent paper from the laboratory of Gilbert Weidinberg has described the mechanisms through which the Wnt/b-catenin pathway regulates blastema growth and regeneration (http://www.ncbi.nlm.nih.gov/pubmed/24485658).
Upon fin amputation, wound healing leads to the formation of a multilayered wound epidermis. Then, a blastema is formed within 48 hours and a subsequent regenerative outgrowth phase concludes in the complete regeneration of the fin in about 3 weeks. This blastema is compartmentalized in 4 main domains: (1) a non-proliferative distal part, (2) a highly proliferative proximal medial region, (3) highly proliferative bilateral zones containing osteoblast progenitors, and (4) domains directly medial to these osteoblast progenitors. In this paper, the authors have used transgenic pathway reporters and elegant tissue-specific pathway manipulations to finely characterize the function of the Wnt/b-catenin pathway. It had been previously shown that this signaling pathway is required for blastema formation and cell proliferation; however, it was not known how does it exert this function.
First, the authors used transgenic reporters of b-catenin-dependent transcription to define the sites of endogenous b-catenin activity. One first surprise was to see that the epidermis was devoid of Wnt/b-catenin signaling although lef1, a well-known Wnt target, is expressed in the epidermis. These results suggested that this pathway must indirectly regulate lef1 expression. Also, and as Wnt/b-catenin signaling is required for blastemal cell proliferation the authors wondered whether Wnt activity correlated with sites of blastema cell proliferation. Surprisingly, they found that this pathway was strongly activated in the most distal part of the blastema (a non-proliferative domain) and weaklier in lateral domains of the proximal blastema (in actinotrichia-forming cells adjacent to osteoblast progenitors). However, this pathway was not active neither in most part of the highly proliferative proximal medial blastema or in committed osteoblasts. Therefore, and because the inhibition of the Wnt/b-catenin pathway strongly reduced proliferation in the proximal blastema, it seems that this pathway regulates cell proliferation indirectly. The specific inhibition of this pathway in the highly proliferative proximal blastema slowed down regeneration but without blocking it, in contrast to the strong regeneration blockade observed when the pathway was also inhibited in the most-distal blastema. This further supported that Wnt/b-catenin activity in the distal blastema indirectly regulates proliferation in the proximal blastema. Next, the authors investigated about the function of Wnt/b-catenin activity in the actinotrichia-forming cells and found out that the activation of this pathway here was necessary to regulate (also indirectly) the commitment and differentiation of the adjacent osteoblast progenitors.
As all this data suggested that the Wnt/b-catenin pathway regulates fin regeneration largely indirectly the authors sought to find putative downstream target signals by performing gene expression profiling after inhibition of this pathway. They found that upon inhibition of the Wnt/b-catenin pathway the expression of many genes related to other signaling pathways was also significantly reduced. These putative targets included several elements of the Hh, BMP, RA, Igf, Notch and FGF signaling. However, the silencing of those pathways appeared to have very little (if any) effect on Wnt/b-catenin activity, suggesting that Wnt signaling would act upstream of a network of pathways during fin regeneration.
Finally, the authors characterized how these different pathways could interact to regulate different aspects of fin regeneration. Thus, they found that epidermal lef1 expression would be regulated by BMP and FGF signaling downstream of b-catenin activity. Similarly, they found that Wnt/b-catenin signaling acted upstream of Hh and RA to regulate blastema cell proliferation. In addition to this indirect regulation, the authors found that the expression of fgf3 and aldh1a2 (a key enzyme for RA synthesis), in the distal blastema was directly regulated by b-catenin signaling, which suggest that these 2 genes function as signals that mediate the effect of Wnt/b-catenin signaling in the distal blastema on regenerative growth and patterning of the surrounding tissues. For example, RA formed in the distal blastema would diffuse to regulate proliferation in the proximal blastema.
In summary, the results presented here suggest that within the fin blastema, Wnt/b-catenin signaling may define organizing centers that would control regeneration by regulating the function of several downstream signaling pathways that would mediate the effects of these organizers on surrounding tissues.