Fig. 2

Fig. 2 mmp9+ Cells Are the Osteoblast Progenitor Cells during Fin Regeneration (A) Confocal image of a longitudinal section of Cre-labeled Tg fin at 2 days post amputation (dpa) that were stained with Sp7, Zns5, and EGFP antibodies. Sp7 proteins and Zns5 antigens are localized in the nucleus and on the cell surface, respectively. Respective positive cells were also confirmed by nuclear staining with TO-PRO-3 (not shown). Many of the migrated mmp9+ joint cells became Sp7+ or Zns5+ cells in the regenerates. Dashed lines, amputation sites. Scale bar, 20 μm. (B) Magnification of boxed area in (A). Progenies of mmp9+ joint cells produced Sp7+ osteoblasts (white arrowheads), Sp7− BLCs (red arrowheads), or regenerated OPCs (yellow arrows). Scale bar, 20 μm. (C) Quantification of joint-derived (EGFP+) cells among Sp7+ or Zns5+ cells. Cells were counted on confocal optical sections in area distal to the amputation plate. n = 20 optical sections (different fin rays, total 5 fish). Error bars indicate mean ± SEM. (D) Ratios of contribution of joint-derived cells to osteoblasts (OBs), BLCs, or OPCs in the regenerated tissue. n = 15 fin rays (total 5 fish). Error bars indicate mean ± SEM. Cell count was performed on confocal z-stack images to confirm respective antibody staining in individual cells. OPCs and BLCs, which are Sp7−, were distinguished by their cell morphologies and tissue localization: OPCs, elongated shape along proximal-distal axis with localization in fin ray joints; BLCs, a flattened irregular shape with non-joint distribution. (E) Confocal image of a regenerating Cre-labeled Tg fin at 4 dpa, which also carries the sp7:mcherry transgene. Dashed lines indicate planes of optical sections shown on the right. Arrow, growing bone matrix; red arrowheads, BLCs; white arrowheads, OBs. Scale bar, 10 μm.