EpCAM Directly Binds to Kremen1 and Disrupts the Kremen1-Dkk2 Interaction(A) EpCAM-Myc and Kremen1-HA mutually coimmunoprecipitated from HEK293T cells, negatively controlled by Wnt2bb-HA and Lrp6-HA. IP, immunoprecipitation; IB, immunoblotting.(B) Kremen1-HA expressed in HEK293T cells is specifically pulled down by a GST-EpCAM fusion protein, but not by GST fused to an irrelevant nuclear protein Geminin (GST-Gem). Bmpr1ba-HA and Wnt2bb-HA expressed in HEK293T cells are applied as negative controls. Quantities of GST-Gem and GST-EpCAM recombinant proteins used in the pull-down assays were exhibited.(C) Controlled by preimmune serum, endogenous EpCAM in the embryonic lysate at 24 hpf coimmunoprecipitates with antibodies against Kremen1.(D–F) Expressions of dkk1a (D, 24/25), dkk1b (E, 26/29), and dkk2 (F, 12/15) at 24 hpf. Note that only dkk2 is present in the bilateral LPM domains adjacent to the hepatic endoderm (arrowheads).(G–L) Under the Tg(lfabp:GFP) background at 102 hpf, defects in liver development caused by dkk2 mRNA (H, 44/73) or kremen1 mRNA (J, 47/87) are rescued by epcam mRNA (I, 53/64) and (K, 99/135). Reduced liver size in hi2151 mutants is rescued by dkk2MO (L, 50/76).(M) In HEK293T cells, along with increases in the amount of epcam-Myc plasmid used for transfection (μg), the levels of coimmunoprecipitated Dkk2-Flag with Kremen1-HA get reduced.(N) In HEK293T cells, along with increases in the amount of dkk2-Flag plasmid used for transfection (μg), the levels of coimmunoprecipitated EpCAM-Myc with Kremen1-HA get reduced.See also Figures S1, S5, and Table S1.