Fig. 4

hand2 overexpression requires npas4l function to inhibit pronephron formation. In situ hybridization demonstrates atp1a1a.4 (A?D) expression throughout the pronephron tubules, etv2 (E?H) expression in vessel progenitors, and flk1 (I?L) expression in the vasculature. Dorsal views, anterior to the left, of wild-type (B,F,J) and npas4ls5 mutant embryos (D,H,L) carrying hs:hand2 were compared with their corresponding wild-type control (A,E,I) and npas4ls5 mutant (C,G,K) nontransgenic siblings. Heat shock was performed at tailbud in all embryos shown. Compared to wild-type control embryos (A), atp1a1a.4 expression in the pronephron tubules was expanded in npas4ls5 mutants (C; n = 17/19). Unlike the dramatically decreased atp1a1a.4 expression in hs:hand2 embryos (B), atp1a1a.4 expression in the pronephron of npas4ls5;hs:hand2 embryos is only relatively mildly reduced (D; n = 34/38). Compared to wild-type (E), etv2 expression appears more intense and is observed in an increased number of cells, especially within the territory normally occupied by the IM, in hs:hand2 embryos (F). etv2 (G,H) and flk1 (K,L) expression are largely absent in both npas4ls5 mutants (G,K), and npas4ls5;hs:hand2 embryos (H,L). Note the faint etv2 expression in the tailbud of the npas4ls5 mutants (arrowheads) and flk1 expression in the posterior trunk of the npas4ls5 mutants (arrows) are consistent with prior reported phenotypes of npas4l mutants (Liao et al., 1997; Reischauer et al., 2016; Sumanas et al., 2005). n = 65 (A), 129 (B), 19 (C), 38 (D), 119 (E), 118 (F), 37 (G), 33 (H), 19 (I), 8 (K), 6 (L). Scale bars: 100 ?m.