||Fig. 4. Pitx2c controls epithelial morphogenesis in left stomach wall. Frog embryos were injected with control
morpholino (control-MO; A,E,I,M,Q) or Pitx2c-MO (B,F,J,N,R) targeted to the left side of the stomach, or injected with Pitx2c- GR mRNA (C,G,K,O,S and D,H,L,P,T) targeted to the right side. (See Fig. S6A,B for morpholino validation.) In injected
embryos (A-D), the greater curvature of the stomach at stage 42 is indicated by an arrowhead (A,C); absence of curvature is
specified by an asterisk (B,D). Sections through stage 39 stomachs (E-T) were stained for β-catenin (βcat; red; E-L),
α-tubulin (αtub; green; M-P) or atypical PKC (aPKC; red; Q-T). GFP mRNA was coinjected as a lineage tracer to confirm proper targeting (green; E-H). MO depletion on the left (F) or ectopic activity of Pitx2 on the right (H) results in a loss of asymmetry
within the stomach compared with controls (E,G, respectively). In addition, compared with control-MO injected embryos, in which αtub and aPKC are concentrated at the apical surface of the left stomach wall (arrowheads in M,Q, respectively), MO
depletion of Pitx2c disrupts epithelial architecture (brackets in J,N,R). Meanwhile, dexamethasone induction of Pitx2c activity in the right wall polarizes stomach endoderm, as indicated by ectopic regions of polarized epithelial architecture (arrowheads in L,P,T), correlating with ectopic αtub (P) and aPKC (T), which are not observed in right wall of uninduced controls (K,O,S). Scale bars: 500 μm in A-D; 75 μm in E-H; 50 μm in I-T. L, left; R, right.
||Figure S4. Markers of apicobasal polarity become left-right asymmetric in the developing stomach. The
intensity of several markers of apical polarity, including aPKC, Par3, E-cadherin (E-cad), alpha-tubulin (a-tub) and
gamma-tubulin (g-tub) were measured at the left and right surfaces of the frog stomach lumen at the indicated stages using image J (A). The level of apical enrichment is represented as a ratio of left and right intensities measured
in at least 3 sections of 3-5 different embryos. The left/right (L/R) ratios of all apical markers become
significantly different by stage 39, while the L/R ratios of Beta-catenin (B-cat) and integrin are not significantly >1.
B-J) High magnification images of sections through the Stage 39 stomach stained forB-catenin B-cat; red; BD),
alpha-tubulin (atub; green; E-G), or atypical PKC (aPKC; red; H-J). Compared to control embryos (B, E, H),
in which a-tub and aPKC are concentrated at the apical surface of the left stomach wall (arrowheads in E, H,
respectively), MO depletion of Pitx2c (C, F, I) disrupts epithelial architecture (arrows in I). Meanwhile,
dexamethasone-induction of Pitx2c activity (D, G, J) in the right wall ectopically polarizes the stomach
endoderm, as indicated by ectopic regions of polarized epithelial architecture, correlating with enriched tub
and ectopic aPKC at the apical surface (arrows in G, J). Scalebars = 25uM. Left (L), Right (R).