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Figure 4
(A) Constructs of −6.35drl:Cre-ERT2 (drl:CreERT2) and −3.5ubb:LOXP-EGFP-LOXP-mCherry (ubi:Switch). Upon 4-OHT induction between 10 and 12 hpf, Cre recombinase expressed by the drl promoter cleave the loxP sites to allow ubb:mCherry expression in the cells that once expressed drl. (B) Scheme for tracing the mesodermal lineage of ectopic β-cells in control siblings and npas4l−/− Tg(ins:Flag-NTR);Tg(ubi:Switch);Tg(drl:CreERT2) zebrafish larvae. (C, D, I, and J) Quantification of the pancreatic or ectopic β-cells with or without mesodermal lineage in npas4l mutants (C, D) or etsrp morpholino (MO)-injected larvae (I, J) at 3 dpf. *p=0.0227 and ****p<0.0001 (Šidák’s multiple comparisons test); (C, D) n = 21 (control) and 14 (npas4l−/−) or (I, J) n = 21 (control) and 23 (etsrp MO). Data are represented as the mean ± SEM. Standard control MO (4 ng) and etsrp MO2 (4 ng) were injected to the control group and etsrp MO group respectively at the one-cell stage (I, J, K–L’’’). (E–H’’’ and K–L’’’) Representative confocal images of pancreatic (E, F) or ectopic β-cells (G, H) of control siblings and npas4l−/−, or ectopic β-cells in control or etsrp MO-injected (K, L) Tg(ins:Flag-NTR);Tg(ubi:Switch);Tg(drl:CreERT2) zebrafish larvae at 3 dpf after β-cell ablation by MTZ at 1–2 dpf, displaying β-cells in cyan with immunostaining for insulin and lineage-traced cells derived from drl-expressing mesodermal cells in red from the Cre-recombined ubi:Switch. Pancreata are outlined by solid white lines (E, F). Arrowheads point to ectopic β-cells derived from the mesoderm (H–H’’’ and L–L’’’). Selected areas in dashed squares in (H) and (L) are magnified in split (H’, H’’, L’ and L’’) and merged (H’’’ and L’’’) channels, respectively. Scale bars = 20 μm (E–H, K, and L) or 10 μm (H’–H’’’ and L’–L’’). Anatomical axes: D (dorsal), V (ventral), A (anterior), and P (posterior).
The ectopic β-cells are of mesodermal origin in npas4l mutants and etsrp morphants.