Fig. S1

Tissue patterning and polarity are retained in wnt5b and fak morphants.

(A-F) in situ hybridization patterns for fgf8 and pax2a expression in control MO (A,D), wnt5b MO (B,E), and fak MO (C,F) injected embryos at 18 ss. Expression patterns are normal in both wnt5b and fak morphants. RNA probes containing digoxigenin-11-UTP were synthesized from linearized plasmid DNA for pax2.1 (Krauss et al., 1991), and fgf8 (Reifers et al., 1998) as previously described (Harland, 1991). Standard methods for hybridization and for single color labeling were used as described (Sagerstrom et al., 1996). After staining, embryos were de-yolked, flatmounted in glycerol and imaged with a Nikon compound microscope. (G-L) Immunohistochemistry staining for the apical junction marker ZO-1 (G-I) and the cell polarity maker aPKC (J-L) in control MO (G,J), wnt5b MO (H.K), and fak MO (I,L) injected embryos at prim-6. Apical localization of ZO-1 appears normal in each condition and indicates establishment of apical junctions. aPKC is also normal in each condition indicating that the cells have established cell polarity. For immunostaining experiments, embryos were fixed in 4% paraformaldehyde or Dent’s (70% methanol: 30% DMSO) for ZO-1. Embryos were blocked in 2% normal goat serum, 1% BSA, and 0.1% Triton-X100 in PBT; incubated overnight at 4C in primary antibody (anti-aPKC (C-20), SC-216, Santa Cruz Biotechnology, 1:1000; anti-ZO1, 33- 9100, Invitrogen, 1:200); then incubated in secondary antibody (goat anti-rabbit or anti- mouse IgG conjugated with Alexa Fluor 488, Invitrogen, 1:500). Arrowheads indicate MHBC. Scale bars: G-I, 20 m; J-L, 35 m.