Fig. S2

Waif1a acts as inhibitor of Wnt/β-catenin signaling in zebrafish embryos and HEK293T cells, related to figure 2.

(A-B) Rescue of wnt8 induced anteroposterior patterning defects by waif1a is evident at early somitogenesis.

(A) Classes of phenotypes produced by wnt8 RNA overexpression assayed at the 1 somite stage by whole mount in situ hybridization using the forebrain marker foxg1a (BF1, arrowhead), the midbrainhindbrain boundary marker her5 and the posterior mesoderm marker tbx6. Class 1, foxg1a reduction; class 2, reduction in length between anterior end of the embryo and the her5 domain; class 3, loss of foxg1a; class 4, radial expansion of tbx6.

(B) waif1a RNA (60 pg) coinjection reduces the severity of phenotypes induced by 1.6 pg wnt8 RNA as assayed by the criteria defined in A and compared to embryos coinjected with equimolar amounts of Renilla Luciferase (Rluc) negative control RNA. *, p<0.05, Chi-Square test.

(C) Overexpression of waif1a in zebrafish embryos interferes with wnt8-induced activation of a coinjected firefly luciferase reporter for β-catenin signaling.

pBAR DNA (80 pg) plus 4 pg pGL4.73 hRLuc/SV40 was injected with 40 pg wnt8 or equimolar amounts of tomato control RNA. Firefly and renilla luciferase levels were determined during gastrulation. Reporter activity was normalized to renilla levels and is shown relative to tomato RNA control injected embryos. *, p<0.05, Student´s t-test.

(D) Inhibitory ability of Waif1a towards different Wnt ligands in zebrafish embryos and HEK293T cells. n.d. = not done

(E) Waif1a-GFP interferes with Wnt8-induced pBAR activation in HEK293T cells in a dosedependent manner. Firefly reporter activity normalized to levels of ubiquitously expressed Renilla luciferase are shown relative to vector transfected controls. +, 10 ng Waif1a-GFP, ++, 80 ng, +++, 160 ng.

(F) Waif1a inhibits Wnt8-induced activation of another transcriptional reporter of β-catenin signaling, SuperTopflash. Firefly reporter activity normalized to levels of ubiquitously expressed Renilla luciferase are shown relative to vector transfected control cells. ***, p<0.001, Student′s t-test.

(G-H) Waif1a reduces Wnt3a-induced stabilization of cytosolic β-catenin.

(G) Total β-catenin was detected on Western blots of the cytosolic fraction of HEK293T cells 12h after stimulation with Wnt3a conditioned medium.

(H) Quantification of data shown in (G).

(I) Zebrafish waif1a MO1 is functional. Injection of waif1a morpholino 1 which is antisense to the waif1a 5′UTR suppresses translation of a reporter construct RNA containing the waif1a 5′UTR and GFP open reading frame.

(J) waif1a knockdown sensitizes embryos to exogenous Wnt/β-catenin signaling. Embryos injected with the indicated reagents were scored for Wnt gain-of-function phenotypes as shown in Figure 2A. Note that embryos injected with 4 ng waif1a MO1 display significantly more severe phenotypes than control embryos in response to two doses (0.4 pg, ***, p<0.001, Chi-Square-test or 1.6 pg **, p<0.01) of wnt8 RNA.

(K) Waif1/5T4 is expressed in HEK293T cells. Semiquantitative PCR for human waif1 and gapdh loading control on HEK293T cDNA and a positive plasmid control is shown.

(L-M) Western blot (L) and quantitative real-time PCR (M) showing knockdown of endogenous human Waif1/TPBG/5T4 protein/RNA in HEK293T cells transfected with hwaif1 siRNA1 or hwaif1 siRNA2 96 h after siRNA transfection.

(N) Direct Wnt/β-catenin target genes sp5l, cdx4 and vent are upregulated in embryos injected with 10 ng waif1a MO2. (sp5l 14/14, cdx4 13/13, vent 21/22 embryos). Note that the staining reaction was stopped before the wildtype expression pattern in control embryos became fully visible.