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Fig. 2 Notum 1a is an inhibitor of Wnt signaling. (A) Overexpression of notum1a produces phenotypes resembling and synergistic with dkk1 overexpression. (Left) Representative embryos following injection of increasing amounts of notum1a. Phenotypic classes are marked by colored squares. (Right) Injection of a quantity of notum1a mRNA that does not produce overt phenotypes enhances the severity of phenotypes seen in embryos injected with dkk1. (B,C) tbx6, which is expressed around the margin at 60% in wild-type embryos (B) is eliminated following notum1a overexpression (C). (D,E) gsc expressed on the presumptive dorsal side of the embryo in wild-type shield stage embryos (D), is expanded following notum1a injection (E). (F) Notum 1a interacts with the Wnt/β-catenin pathway. Injection of wnt8 or wnt1 mRNA or dkk1 MO produces embryos lacking eyes (lightest blue) or with smaller eyes (light blue) than wild-type embryos (dark blue) at 24 hpf. Co-injection of notum1a suppressed wnt8 and wnt1 mRNA, and dkk1 MO-induced eye reduction. MO-mediated depletion of notum1a with a splice-site-directed MO enhanced eye reduction induced by wnt1 mRNA; this enhancement was rescued by injection of notum1a mRNA. (G-J) Stimulation of Wnt signaling by LiCl partially restored wild-type forebrain rx3 expression following notum1a overexpression. (G) Flat-mounted control, non-heat shocked Tg(hs:notum1a) embryo at eight somites with in situ hybridization against rx3 (labeling forebrain), pax2a (midbrain-hindbrain boundary and pronephric mesodern), egr2b (krox20) (rhombomeres three and five) and myod1 (adaxial cells and somites) (H) Tg(hs:notum1a) embryos heat shocked at 50% epiboly display expanded rx3 domain. (I) Eight minutes of LiCl treatment at 75% epiboly eliminated rx3 expression in non-heat-shocked embryos. (J) Heat-shock at 50% epiboly before the same LiCl treatment at 75% epiboly partially restores rx3 expression.