Zebrafish sp7:EGFP: a transgenic for studying otic vesicle formation, skeletogenesis, and bone regeneration - PubMed (original) (raw)

Fig. 3

Expression of GFP in the Tg(sp7:EGFP)b1212 transgenic line in living zebrafish embryos, larvae, and adults. Samples in panels e–o and s are stained with vital Alizarin red to label mineralized matrix. a–d, f, h, j, l, m, and o–s are lateral views (A = anterior, P = posterior, D = dorsal, V = ventral) and e, g, i, and k are ventral views (A = anterior, L = left, R = right). All panels are projections of confocal z-stacks, except figs. b, c and d, which are single z-sections. a and b: GFP is expressed in the otic anlagen and otic placode, respectively. c and d: GFP is expressed in the otic vesicle, and in the statoacoustic ganglion anterior to the otic vesicle (indicated by arrows). e and f: No GFP expression is detected in cartilaginous elements (i.e. ch), but GFP is expressed in cells forming the op. g and h: GFP is strongly expressed in cells of the bsrp and the op, but is not associated with the ch. i–l (k and l are high-magnification images of i and j, respectively): GFP is expressed in osteoblasts of the growing op and bsrp, and also in perichondral osteoblasts of the ch (indicated by arrows in i and k). m: At larval stages, GFP expression is present in growing bones of the jaw and jaw supporting structure. n: schematic showing location of amputation of adult tail fin. o: non-amputated adult fin ray showing GFP-positive cells associated with mineralized matrix. p–s: time series of GFP-positive cell association with the site of amputation (indicated by dotted line) between 1–5 dpa (indicated by white arrows). At 5 dpa, GFP-positive cells are also detected in forming ray bone away from the site of amputation (indicated by white double arrow). Orange arrows indicate the extent of new bone growth at 5 dpa. Compared with the unamputated control (Fig. 3o), uncut bone adjacent to the amputated site does not stain with Alizarin Red. The reason for this remains unclear and requires further investigation. After 72hpf, green fluorescence is detected in the skin of fish (not shown). This does not interfere with confocal imaging of skeletal structures of fish at any stage, because bones are below the plane of focus of the skin. Abbreviations: bsr = branchiostegal rays, where bsra = anterior, bsrm = middle, bsrp = posterior, ch = ceratohyal, cl = cleithrum, d = dentary, iop = interopercle, op = opercle, pop = preopercle, sop = subopercle. Scale bars: a = 100µm, b = 50µm, c = 500µm, d = 100µm, e–j = 100µm, k and l = 50µm, m = 500µm, o–s = 50µm.