Files in this Data Supplement:
Fig. S1. Developmental defects induced by the microinjection of DnFz7 mRNA at a final concentration of 1.1 µg/µl. Images taken at 72 hours post-fertilization.
Fig. S2. Relative nuclear β-catenin activity measured at different developmental stages using a TOP-Flash reporter assay. The relative level of luciferase activity was normalized to the level detected in control embryos at 10 hours post-fertilization.
Fig. S3. Expression profile of Frizzled9/10 (JN712908) (A) and Frizzled4 (JN712907) (B) analyzed by Northern blot. Stages are as follows: E, fertilized egg; 16, 16-cell stage; 60, 60-cell stage; B1, early blastula (7 hpf); B3, mid-blastula (9 hpf); B5, late blastula (11 hpf); SB, swimming blastula (12 hpf); MB, mesenchyme blastula; EG, early gastrula; G, gastrula; Pr, prism; Pl, pluteus.
Fig. S4. Developmental defects associated with downregulation (DnWnt6) or overexpression (Wnt6) of the Wnt6 ligand at indicated time points and indicated mRNA concentrations. Two first columns are DIC images of the embryos. Last column contains immunostained injected embryos labeled with an endoderm marker (blue), a skeletogenic mesoderm marker (red) and a green counterstain. DnWnt6-injected embryos developed skeletal rods but no archenteron. Wnt6-overexpressing embryos displayed enlarged archenterons and disorganized skeletons. These phenotypes are consistent with those previously described for these two constructs by Croce et al. (Croce et al., 2011).
Fig. S5. Phylogenetic analysis of Frizzled receptor proteins using maximum parsimony and the Mega5.05 program. Smoothened proteins, which are related to Frizzled receptors, were used as an outgroup. Hs, Homo sapiens; Dm, Drosophila melanogaster; Xl, Xenopus laevis; Pl, Paracentrotus lividus; Nv, Nematostella vectensis; Che, Clytia hemisphaerica; Ce, Caenorhabditis elegans. Accession numbers of all Frizzled sequences used to generate this tree are as follows: CheFz1, ABI98898; CeMom5, NP_492635; XlFz7, NP_001079354 PlFz1-2-7, HQ322502; PlFz9-10, JN712908; PlFz4, JN712907; PlFz5-8, CAJ29888; PlSmo, JN835465; HsFz1, Q9UP38; HsFz2, Q14332; HsFz3, Q9NPG1; HsFz4, Q9ULV1; HsFz5, Q13467; HsFz6, O60353; HsFz7, O75084; HsFz8, Q9H461; HsFz9, O00144; HsFz10, Q9ULW2; HsSmo, NP_005622; NvFrizzled1-2-7, c409502365; NvFrizzled9-10, c405101650; NvFrizzled4, c406601695; NvSmo, c412402615; DmFz3, AAF63250; DmFz, CAA38458; DmFz2, NP_001137971; DmFz4, AAF81195; DmSmo, AAF51518.
