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. 2015 Feb 2;208(3):351–366. doi: 10.1083/jcb.201408058

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© 2015 Roarty et al.

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Figure 6. — Ror2 loss disrupts actin cytoskeletal dynamics during branching morphogenesis. (A) Heat map illustrating the differential alterations in genes associated with actin cytoskeletal dynamics in Ror2-depleted luminal and basal epithelial compartments. (B) Brightfield DIC images of shLUC and shRor2 organoids. Days 4 and 8 are shown to illustrate early and late stages of branching. (C) Quantification of the number of branches emanating from shLUC versus shRor2 organoids (n > 40 organoids per group). (D) Maximum-intensity projections of confocal z stacks and cross-sections depicting F-actin (green) and K5 (red) localization within shLUC and shRor2 organoids. Panels on the right (enlarged from the boxed regions) depict representative areas of F-actin patterns (arrowheads) between shLUC and shRor2 branches. (E) ZO-1 (green) IF depicting microlumens (arrowheads) associated with shRor2 branching organoids. Panels on the right represent magnified representative areas (taken from the boxed regions) of ZO-1 within a branch. (F) Western blot analysis of Rho pathway proteins in shLUC and shRor2 organoids. (G) pERM IF (green) within shLUC and shRor2 ducts. Shown are unbranched and highly branched sections for shRor2 to depict the extremes of the pERM alterations. K8 (red) denotes the luminal cells of the duct. Arrowheads indicate atypical punctate localization of pERM between luminal and basal layers in shRor2 ducts. Bars: (B) 50 µm; (D and E, left) 50 µm; (D and E, right) 8 µm; (G) 25 µm.