Cells Isolated from Regenerating Caudal Fin of Sparus aurata Can Differentiate into Distinct Bone Cell Lineages.

Teleosts have the ability to regenerate their caudal fin upon amputation. A highly proliferative mass of undifferentiated cells called blastema forms beneath wound epidermis and differentiates to regenerate all missing parts of the fin. To date, the origin and fate of the blastema is not completely understood. However, current hypotheses suggest that the blastema is comprised of lineage-restricted dedifferentiated cells. To investigate the differentiation capacity of regenerating fin-derived cells, primary cultures were initiated from the explants of 2-days post-amputation (dpa) regenerates of juvenile gilthead seabream (Sparus aurata). These cells were subcultured for over 30 passages and were named as BSa2. After 10 passages they were characterized for their ability to differentiate towards different bone cell lineages and mineralize their extracellular matrix, through immunocytochemistry, histology, and RT-PCR. Exogenous DNA was efficiently delivered into these cells by nucleofection. Assessment of lineage-specific markers revealed that BSa2 cells were capable of osteo/chondroblastic differentiation. BSa2 cells were also found to be capable of osteoclastic differentiation, as demonstrated through TRAP-specific staining and pit resorption assay. Here, we describe the development of the first successful cell line viz., BSa2, from S. aurata 2-dpa regenerating caudal fins, which has the ability of multilineage differentiation and is capable of in vitro mineralization. The availability of such in vitro cell systems has the potential to stimulate research on the mechanisms of cell differentiation during fin regeneration and provide new insights into the mechanisms of bone formation.

Lineage tracing of col10a1 cells identifies distinct progenitor populations for osteoblasts and joint cells in the regenerating fin of medaka (Oryzias latipes).

The caudal fin of teleost fish regenerates fully within two weeks of amputation. While various cell lineages have been identified and characterized in the regenerating fin, the origin of bone cells remains debated. Here, we analysed collagen10a1 (col10a1) expressing cells in the regenerating fin of the medaka (Oryzias latipes) and tested whether they represent an alternative progenitor source for regenerating osteoblasts. Under normal conditions, col10a1 cells are positioned along fin ray segments and in intersegmental regions. Lineage tracing in the amputated fin revealed that col10a1 cells from the stump contribute to the regenerating bony fin rays. However, ablation of col10a1 cells did not abolish fin regeneration suggesting that col10a1 expressing osteoblast progenitors are dispensable for regeneration. Intriguingly, however, after ablation of osterix (osx)/sp7-col10a1 double-positive osteoblasts, col10a1 cells exclusively gave rise to joint cells in the intersegmental region thus identifying a pool of lineage-restricted joint progenitor cells. To identify additional sources for regenerating osteoblasts, we performed clonal lineage analysis. Our data provide the first evidence that after ablation of mature osteoblasts in medaka, transdifferentiation does not account for de novo osteoblast generation. Instead, our findings suggest the presence of lineage restricted progenitor pools in medaka, similar to the situation in zebrafish. After osteoblast ablation, these pools become activated and give rise to fin ray osteoblasts and intersegmental joint cells during regeneration. In summary, we conclude that col10a1-positive cells do not represent an exclusive source for osteoblasts but are progenitors of joint cells in the regenerating fin.

Bone morphogenetic protein signaling promotes morphogenesis of blood vessels, wound epidermis, and actinotrichia during fin regeneration in zebrafish.

Zebrafish fin regeneration involves initial formation of the wound epidermis and the blastema, followed by tissue morphogenesis. The mechanisms coordinating differentiation of distinct tissues of the regenerate are poorly understood. Here, we applied pharmacologic and transgenic approaches to address the role of bone morphogenetic protein (BMP) signaling during fin restoration. To map the BMP transcriptional activity, we analyzed the expression of the evolutionarily conserved direct phospho-Smad1 target gene, id1, and its homologs id2a and id3. This analysis revealed the BMP activity in the distal blastema, wound epidermis, osteoblasts, and blood vessels of the regenerate. Blocking the BMP function with a selective chemical inhibitor of BMP type I receptors, DMH1, suppressed id1 and id3 expression and arrested regeneration after blastema formation. We identified several previously uncharacterized functions of BMP during fin regeneration. Specifically, BMP signaling is required for remodeling of plexus into structured blood vessels in the rapidly growing regenerate. It organizes the wound epithelium by triggering wnt5b expression and promoting Collagen XIV-A deposition into the basement membrane. BMP represents the first known signaling that induces actinotrichia formation in the regenerate. Our data reveal a multifaceted role of BMP for coordinated morphogenesis of distinct tissues during regeneration of a complex vertebrate appendage.