Fig. 4

EC specification is induced by Wnt3a and mediated via Lef1. (A,B) Morpholino (MO)-mediated knockdown of wnt3a reduced the EC number (A, n = 14) in Tg(kdrl:GFP)^s843 embryos compared to control (ctr.) MO (A, n = 19). Knockdown of other Wnt ligands, expressed in the zebrafish posterior LPM, did not significantly change EC numbers compared to ctr. MO injected embryos (A): wnt9a (n = 13), wnt9b (n = 14), wnt8 (n = 19) or wnt3 (n = 7). (C) MO-mediated knockdown or transient CRISPR-Cas9-mediated knockdown of wnt3a significantly reduced the EC volume in Tg(kdrl:GFP)^s843 embryos (ctr. MO: n = 5; wnt3a MO: n = 8; ctr. CRISPR: n = 5; wnt3a CRISPR: n = 8). Agarose gel electrophoresis> of wnt3a CRISPR genotyping showed an undigested putatively mutated PCR product (wnt3a CRISPR fragment). Non-mutated wild type fragments were cut by PstI digest. (D,E) MO-mediated knockdown of lef1 reduced the EC number (D, n = 10) compared to ctr. MO (D, n = 38) in Tg(kdrl:GFP)^s843 embryos. Knockdown of any of the other Tcf-transcription factors did not significantly change EC numbers compared to ctr. MO injected embryos (D): tcf7 (n = 30), tcf7l1a (n = 23), tcf7l1b (n = 28) or tcf7l2 (n = 32). (F) Heatshock induced Wnt ligand overexpression using Tg(hsp70l:wnt8-GFP)^w34 (indicated as hs:wnt8) increased the EC volume in Tg(kdrl:mCherry)^s896 embryos (F, n = 7) compared to control siblings (F, n = 12). In contrast, heatshock induced overexpression of Wnt signaling inhibitor Dkk1 using Tg(hsp70l:dkk1-GFP)^w32 (indicated as hs:dkk1) decreased the EC volume (F, n = 5) compared to control siblings. (G) EC and erythrocyte cell volume analysis in lef1^u767 mutant embryos. Embryos carrying heterozygous and homozygous lef1^u767 mutations showed reduced EC volumes (G, upper panel, wt sibling: n = 14; lef1^u767/+: n = 18; lef1^u767/u767: n = 10), but increased erythrocyte volumes (G, lower panel, wt sibling: n = 14; lef1^u767/+: n = 19; lef1^u767/u767: n = 9) compared to wild type siblings. (H) MO-mediated knockdown of lef1 decreased the EC volume (H, upper panel, n = 6) compared to ctr. MO (H, upper panel, n = 7) in Tg(kdrl:GFP)^s843 embryos, but increased the erythrocyte volume (H, lower panel, n = 7) compared to ctr. MO (H, lower panel, n = 7) in Tg(gata1:GFP)^la781 embryos. Overactivation of Wnt signaling in lef1-deficient embryos using BIO restored EC volume and erythrocyte volume nearly to control levels (H, upper and lower panel, n = 7). All values represent mean±SD. *p<0.05, **p<0.01, ***p<0.001; Student's t-test.