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Fig. 7
wt1b expressing cells are closely associated with vertebral development after injury.
(a) Images of Tg(wt1b:gfp) zebrafish following needle injury at 3 dpf and raised to 28 dpi. n > 10; experimental replicates = 4. Scale bar left panels: 100 µm; scale bar right panels: 200 µm. (b) α-GFP staining of 28 dpi larvae at the site of the healing notochord wound and in the kidney. n = 5; experimental replicates = 1. Scale bar left panels: 50 µm. (c) Image of fish from Figure 5a,c, stained with alizarin red and imaged for wt1b:gfp expressing cells. GFP positive cells are found within the ectopic vertebra (white arrow and inset). n = 4; experimental replicates = 1. Scale bar left panels: 100 μm. (d) Long-term follow up of alizarin red stained Tg(wt1b:gfp); casper larvae shows that chordacentra formation is delayed around the site of injury. GFP cells mark the site of the future vertebra. n = 6; experimental replicates = 2. Scale bar: 100 µm; scale bar zoomed images: 50 µm. (e) Confocal imaging of 15, 21 and 28 dpi larvae reveals an overlapping expression between the wt1b:gfp expressing cells and the forming chordacentra (alizarin red stained) in the injured Tg(wt1b:gfp); casper larvae. n > 10; experimental replicates = 3. Scale bar: 100 µm. Imaging views are lateral, angled and cross-section view. (f) Confocal imaging highlights the overlapping presence of bone (alizarin red stained) and wt1b:gfp cells at the wound in 18 dpi larvae (arrow). n > 10; experimental replicates = 3. Scale bar: 100 µm. (g) Confocal scans of 24 dpi Tg(wt1b:gfp) larvae stained with alizarin red and expressing GFP at the injury site following notochord injury compared with uninjured control fish. GFP positive cells are present within the vertebrae at the injury site (arrow). Scale bar left fish: 1000 µm; scale bar on vertebrae images: 100 µm.