While mammals have a limited capacity to repair bone defects, zebrafish can completely regenerate amputated bony structures of their fins. Fin regeneration is dependent on formation of a blastema, a progenitor cell pool accumulating at the amputation plane. It is unclear which cells the blastema is derived from, whether it forms by dedifferentiation of mature cells, and whether blastema cells are multipotent. We show that mature osteoblasts dedifferentiate and form part of the blastema. Osteoblasts downregulate expression of intermediate and late bone differentiation markers and induce genes expressed by bone progenitors. Dedifferentiated osteoblasts proliferate in a FGF-dependent manner and migrate to form part of the blastema. Genetic fate mapping shows that osteoblasts only give rise to osteoblasts in the regenerate, indicating that dedifferentiation is not associated with the attainment of multipotency. Thus, bone can regenerate from mature osteoblasts via dedifferentiation, a finding with potential implications for human bone repair.

Pax3/7 paired homeodomain transcription factors are important markers of muscle stem cells. Pax3 is required upstream of myod for lateral dermomyotomal cells in the amniote somite to form particular muscle cells. Later Pax3/7-dependent cells generate satellite cells and most body muscle. Here we analyse early myogenesis from, and regulation of, a population of Pax3-expressing dermomyotome-like cells in the zebrafish. Zebrafish pax3 is widely expressed in the lateral somite and, along with pax7, becomes restricted anteriorly and then to the external cells on the lateral somite surface. Midline-derived Hedgehog signals appear to act directly on lateral somite cells to repress Pax3/7. Both Hedgehog and Fgf8, signals that induce muscle formation within the somite, suppress Pax3/7 and promote expression of myogenic regulatory factors (MRFs) myf5 and myod in specific muscle precursor cell populations. Loss of MRF function leads to loss of myogenesis by specific populations of muscle fibres, with parallel up-regulation of Pax3/7. Myod is required for lateral fast muscle differentiation from pax3-expressing cells. In contrast, either Myf5 or Myod is sufficient to promote slow muscle formation from adaxial cells. Thus, myogenic signals act to drive somite cells to a myogenic fate through up-regulation of distinct combinations of MRFs. Our data show that the relationship between Pax3/7 genes and myogenesis is evolutionarily ancient, but that changes in the MRF targets for particular signals contribute to myogenic differences between species.

SummaryThe somitic compartment that gives rise to trunk muscle and dermis in amniotes is an epithelial sheet on the external surface of the somite, and is known as the dermomyotome. However, despite its central role in the development of the trunk and limbs, the evolutionary history of the dermomyotome and its role in non-amniotes is poorly understood. We have tested whether a tissue with the morphological and molecular characteristics of a dermomyotome exists in non-amniotes. We show that representatives of the agnathans and of all major clades of gnathostomes each have a layer of cells on the surface of the somite, external to the embryonic myotome. These external cells do not show any signs of terminal myogenic or dermogenic differentiation. Moreover, in the embryos of bony fishes as diverse as sturgeons (Chondrostei) and zebrafish (Teleostei) this layer of cells expresses the pax 3 and 7 genes that mark myogenic precursors. Some of the pax7-expressing cells also express the differentiation-promoting myogenic regulatory factor Myogenin and appear to enter into the myotome. We therefore suggest that the dermomyotome is an ancient and conserved structure that evolved prior to the last common ancestor of all vertebrates. The identification of a dermomyotome in fish makes it possible to apply the powerful cellular and genetic approaches available in zebrafish to the understanding of this key developmental structure.

Fibroblast growth factors (Fgfs) have long been implicated in regulating vertebrate skeletal muscle differentiation, but their precise role(s) in vivo remain unclear. Here, we show that Fgf8 signalling in the somite is required for myod expression and terminal differentiation of a subset of fast muscle cells in the zebrafish lateral somite. In the absence of Fgf8, lateral somite cells transiently express myf5 but fail to make muscle and remain in a dermomyotome-like state characterised by pax3 and meox expression. Slow muscle fibres form and commence normal migration in the absence of Fgf8, but fail to traverse the expanded undifferentiated lateral somite. The Fgf8-independent residual population of medial fast muscle fibres is not Hedgehog dependent. However, Fgf8-independent medial fast muscle precursors are lacking in floatinghead mutants,suggesting that they require another ventral midline-derived signal. We conclude that Fgf8 drives terminal differentiation of a specific population of lateral muscle precursor cells within the early somite.

Hedgehog (Hh) signalling has been implicated in the development of osteoblasts and osteoclasts whose balanced activities are critical for proper bone formation. As many mouse mutants in the Hh pathway are embryonic lethal, questions on the exact effects of Hh signalling on osteogenesis remain. Using zebrafish, we show that there are two populations of endochondral osteoblasts with differential sensitivity to Hh signalling. One, formed outside the cartilage structure, requires low levels of Hh signalling and fails to differentiate in Indian hedgehog mutants. The other derives from chondrocytes and requires higher levels of Hh signalling to form. This latter population develops significantly earlier in mutants with increased Hh signalling, leading to premature endochondral ossification, and also fails to differentiate in Indian hedgehog mutants, resulting in severely delayed endochondral ossification. Additionally, we demonstrate that the timing of first osteoclast activity positively correlates to Hh levels in both endochondral and dermal bone.