|
Figure 5
(A) Schematics of npas4lPt(+36-npas4l-p2a-Gal4-VP16)bns423 (npas4l:Gal4), UAS:Cre and −3.5ubb:LOXP-EGFP-LOXP-mCherry (ubi:Switch). (B, C) Quantification of the pancreatic or ectopic β-cells with or without npas4l-positive mesodermal origin in control or transheterozygous npas4ls5/bns423 mutants at 3 dpf. ***p=0.0001 and ****P<0.0001 (Šidák’s multiple comparisons test); n = 20 (control) and 21 (npas4ls5/bns423). Data are represented as the mean ± SEM. (D–E’’’) Representative confocal images of ectopic β-cells and npas4l-positive lineage-traced cells in control siblings and npas4ls5/bns423 Tg(ins:Flag-NTR);Tg(ubi:Switch);Tg(npas4l:Gal4);Tg(UAS:Cre) zebrafish larvae at 3 dpf after β-cell ablation by MTZ from 1 to 2 dpf, displaying β-cells in cyan with immunostaining for insulin and lineage-traced cells derived from npas4l-expressing mesodermal cells in red from the Cre-recombined ubi:Switch. The selected area in a dashed square in (E) is magnified in split (E’ and E’’) and merged (E’’’) channels, respectively. Arrowheads point to ectopic β-cells derived from the mesoderm (E–E’’’). (F) Schematics of npas4lPt(+36-npas4l-p2a-Gal4-VP16)bns423 (npas4l:Gal4) and UAS:EGFP. (G–G’’’) Representative confocal images of ectopic β-cells losing npas4l expression in npas4ls5/bns423 Tg(ins:Flag-NTR); Tg(npas4l:Gal4);Tg(UAS:EGFP) zebrafish larvae at 3 dpf after β-cell ablation by MTZ from 1 to 2 dpf, displaying β-cells in magenta with immunostaining for insulin and cells expressing npas4l in green. The selected area in a dashed square in (G) is magnified in split (G’ and G’’) and merged (G’’’) channels, respectively. Arrowheads point to ectopic β-cells without npas4l expression (G–G’’’). Scale bars = 20 μm (D, E, and G) or 10 μm (E’–E’’’ and G’–G’’). Anatomical axes: D (dorsal), V (ventral), A (anterior), and P (posterior).
The mesodermal cells lose npas4l expression after differentiating into ectopic β-cells.