Figure 5. Alcama is required for pouch bilayer formation and AJ stabilization.

(A-D) her5:mCherryCAAX fluorescence (red) and Alcama immunohistochemistry (green) show loss of Alcama protein and an aberrant multilayered pouch morphology in alcama-MO embryos (C and D) compared to un-injected controls (A and B). Scale bar = 20 μM.

(E and F) her5:mCherryCAAX labeling shows that the elongated morphology of pouch cells (arrows) resulting from CA-Cdc42 misexpression (n=49/72) is suppressed by Alcama depletion (n=0/67). Insets show schematics of pouch cell morphology.

(G and H) Immunohistochemistry shows that E-cadherin still localizes to cell-cell junctions in the absence of Alcama protein yet the apical enrichment seen in wild-type mature pouches (arrow) is missing.

(I-L) Imaging of α-catenin localization during three phases of wild-type pouch formation (I-K) and during a comparable phase of alcama-MO development (L) when wild-type pouches would have matured into bilayers. Crosshairs show target regions before FCS laser illumination (I-L) and 25 seconds after (I’-L’). Scale bar = 5 μM.

(M and N) Alcama immunohistochemistry in wild-type Gt(ctnna-citrine)^ct3a embryos shows that the appearance of Alcama (red) at apical and lateral cell-cell junctions corresponds to a transition from disorganized to strongly apical localization of α-catenin (green). Scale bar = 20 μM.

(O) FCS measurements of endogenous α-catenin mobility in wild-type embryos show a significant increase in mobility from pre-pouch to transitional endoderm and a subsequent decrease in mature bilayers. In alcama-MO embryos, α-catenin mobility increases from pre-pouch to transitional endoderm but fails to decrease at a stage comparable to the mature wild-type pouch. n=16 for each. Data represent mean ± SEM and p-values are shown for each comparison.

See also Figure S4.