fam20b-dependent proteoglycans do not affect dermal bone formation and fin regeneration, but Bmp signalling promotes fin regenerate outgrowth

The role of proteoglycans (PGs) in regulating growth factor signalling during endochondral ossification, or formation of bone around a cartilage template, is established. However, whether PGs regulate other skeletal processes, like intramembranous ossification or fin regeneration, has not been studied extensively. Given that endochondral ossification in PG-deficient fam20b mutant zebrafish is altered due to increased Bmp signalling, we hypothesized that PGs also normally inhibit Bmp signalling during intramembranous ossification and fin regeneration. To test this hypothesis, the functional relevance of Bmp signalling during craniofacial dermal bone formation and caudal fin regeneration was examined in wild type and fam20b mutant zebrafish. Bmp responsiveness of cells around wild-type dermal bones was verified by phospho-Smad1/5/9 immunoreactivity. Dermal bones in fam20b mutants were generally unaffected, apart from early formation of the dentary and quadrate, which actually initiate in the perichondrium of Meckel's cartilage and the palatoquadrate, respectively. Treatment of wild-type or fam20b^-/- embryos with the Bmp inhibitor DMH1 did not yield clear differences in the size or morphology of most dermal bones, but did rescue early dentary and quadrate formation in fam20b mutants. During adult fin regeneration, Bmp signalling was confirmed by careful temporal analyses of GFP expression of Tg(5xBmpRE-Xla.Id3:GFP)ir1189 and Tg(BmpRE:EGFP)pt510 zebrafish, each expressed in different tissue compartments of the regenerating fin. fam20b^-/- adults did not show fin regeneration defects. Treatment with DMH1, however, significantly reduced outgrowth of regenerating fins in distinct anatomical regions (the most dorsal and ventral principal rays) in adult zebrafish. Bony ray differentiation also was inhibited by DMH1 treatment. In total, these data do not support a major role of Bmp signalling or fam20b-dependent PGs in craniofacial intramembranous ossification, but do show that Bmp signalling is required for outgrowth of regenerating fins.