Fig. 5

npas4l overexpression inhibits pronephron formation in the absence of hand2 function. Dorsal views, anterior to the left, of wild-type control embryos (A,E), hand2s6 mutant embryos (B,F), and wild-type control (C,G) and hand2s6 mutant embryos (D,H) injected with npas4l mRNA, all at 26 hpf. In situ hybridization demonstrates atp1a1a.4 (A?D) expression throughout the pronephron tubules, and mrc1a (E?H) expression in the posterior cardinal vein (arrow) and posterior blood island (arrowhead) (Stachura and Traver, 2016). Compared to wild-type (A), pronephron gene expression is expanded in hand2s6 mutants (B) and greatly reduced in npas4l-overexpressing embryos (C,D). The extent of the defects seen in npas4l-overexpressing embryos was comparable between injected hand2s6 mutants (D; n = 10/28 with reduced pronephron as shown) and injected wild-type control embryos (C; n = 26/79 with reduced pronephron as shown). Compared to wild-type (E) and npas4l-overexpressing (G) embryos, mrc1a gene expression in the posterior cardinal vein (arrow) is greatly reduced in both uninjected (F) and npas4l-overexpressing (H) hand2s6 mutants. npas4l mRNA overexpression also resulted in ectopic mrc1a expression along the yolk extension in some injected animals (asterisk in G; ectopic expression in injected wild-type 41/75, in injected hand2s6 10/19). Although higher doses of npas4l mRNA led to stronger phenotypes than shown here, they also increased embryonic lethality or severe deformation. Therefore, the dose of npas4l mRNA injected was limited to 5?14.5 ng so that overall development was not severely impacted which likely limited the penetrance and expressivity of phenotypes displayed. n = 28 (A), 11 (B), 79 (C), 28 (D), 38 (E), 13 (F), 75 (G), 19 (H). Scale bars: 100 ?m.