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First published online 5 October 2005
doi: 10.1242/dev.02056

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Rho1 regulates Drosophila adherens junctions independently of p120ctn

¹ Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599 USA
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA

View larger version (152K): [in a new window]   Fig. 1. Loss of p120 slows but does not disrupt dorsal closure. Embryos. Unless noted, in all figures anterior is left. (A,B) p120 RNAi depletes p120:GFP. GFP fluorescence, p120::GFP embryos injected with control dsRNA (A) or p120 dsRNA (B). (C-J) Anti-phosphotyrosine. (K,L) Phalloidin. Wild type (C,E,G,I,K). p120 null mutants (D,F,H,J,L). (M-N) Stills, movies of representative wild-type (M) and p120 null mutants (N), both expressing moesin-GFP. Times, lower left. (O) Mean time to complete dorsal closure ±s.d. Scale bars: 25 µm.

View larger version (95K): [in a new window]   Fig. 2. Rho1 localization during oogenesis. Egg chambers. Rho1 (red). DE-Cad (green). (A) Cross-section, stage 2-3. Rho1 accumulates laterally in follicle cells (white arrowhead) and stalk cells (red arrowhead). (B) Stage 10. Rho1 at germ cell boundaries (arrowhead). (C) DE-Cad at apical AJs of follicle cells (blue arrowhead) and in polar follicle cells (white arrow). Rho1 does not preferentially accumulate in either place. (D) Cross-section, stage 10B. DE-Cad and Rho1 elevated apically in centripetal follicle cells (red arrowhead). Rho1 accumulates basolaterally (blue arrowhead). (E) Grazing section basal to AJs, stage 10B follicle cells. DE-Cad is absent from tricellular junctions, where Rho1 accumulates (arrows). (F-I'') Cross-sections, stage 5 (F,G) and 10B (H,I). Wild type (F,H). p120 mutants (G,I). In both Rho1 is enriched along lateral membranes. DE-Cad is enriched at apical AJs (arrowheads). (J,K) Grazing sections, stage 10B. Rho1 at tricellular junctions. Wild-type (J). p120 (K). (L,M) Migrating border cells. Rho1 enriched at plasma membrane and in intracellular vesicles (M, arrowhead). Scale bars: 20 µm in A-F,I,K,L; 5 µm in M.

View larger version (127K): [in a new window]   Fig. 3. Rho1 localization during embryogenesis. Rho1 (red). DE-Cad (green). (A-C) Cross-sections, cellularization. Wild type (A,B). p120 (C). (A) Rho1 enriched at furrow canals (A, blue arrowhead). DE-Cad accumulates at basal junctions (A, yellow arrowhead) and apical AJs (A, white arrowhead). (B,C) Loss of p120 (C) does not alter Rho1 localization. (D-E) Cross-section, posterior midgut (E, close-up). Rho1 in basal puncta in invaginating midgut cells (arrowhead). DE-Cad (green, E) at sites of apical constriction. (F) Ventral furrow. DE-Cad accumulates at AJs of apically constricting cells (arrowheads) while Rho1 does not. (G-I) Cross sections, stage 8. Rho1 accumulates where ectodermal and mesodermal cells meet (G,H, arrowheads). H, close-up of G. (I) ßPS1 integrin. (J-L) Basolateral sections. Stages 5 (J), 9 (K), 15 (L). Rho1 cortical enrichment decreases. (M-N'') Cross-sections, stage 15 hindgut. Wild type (M). p120 mutant (N). Rho1 accumulates basally (arrowheads). DE-Cad accumulates in apical AJs. Scale bars: 20 µm.

View larger version (155K): [in a new window]   Fig. 4. Neither p120 nor core AJ components are essential for Rho1 localization during dorsal closure. (A,B) Wild type. DE-Cad (A,B) versus Rho1 (A',B') in apical (A,A') and basolateral (B,B') sections. (A,A') Apically, DE-Cad (A) accumulates at AJs. Leading edge (arrowhead). Rho1 (A') is at low levels in the cytoplasm and is not enriched at the leading edge (arrowhead). (B,B') Basally, DE-Cad (B) accumulates at AJs in segmental grooves (arrowhead). Rho1 (B') levels are higher and it is cortically enriched (arrowhead). (C,C') p120 mutant. Rho1 is not elevated at the leading edge (arrowheads, C' versus A'). (D,D') Apical RhoGEF2. Arrowheads, leading edge. (E-G) Basolateral Rho1 (arrowhead) is similar in wild type (E), armYD35 (F), or shgR69 zygotic mutants (G). (G') DE-Cad in a portion of G. (H,H') RhoGEF2 is enriched basolaterally and is more cortical than Rho1 (H' versus B') at both amnioserosal (arrow) and epidermal cell (arrowhead) boundaries. (I-M'') Rho1 in shgR69 mutant follicle cell clones. (I-K') GFP, green; Arm, blue; DE-Cad, red. (I) Cross-section, early egg chamber. shgR69 mutant clone (bracket) indicated by lack of GFP. Arm accumulates at AJs at this stage despite loss of DE-Cad. (J-K') Cross-section (J,J'), grazing section (K,K'), later egg chamber. shgR69 mutant clone (bracket). Arm and DE-Cad are severely reduced. (L-M'') Grazing section, similar stage egg chamber as J,K. GFP, green; Rho1, blue; DE-Cad, red. (L) Rho1 accumulates normally in shgR69 clone. (M,M'') Close-up of L. Scale bars: 20 µm in A; 50 µm in I-L,M''.

View larger version (88K): [in a new window]   Fig. 5. p120 loss of function does not substantially enhance or suppress Rho1. (A-I) Cuticle preparations, anterior up. Rho1 (A-D), wild type (E), or p120 Rho1 (F-I). Embryos were divided into phenotypic classes of increasing severity (Table 3). The percentage of embryos in each class is listed below a representative cuticle. (J-R) F-actin (Phalloidin). Wild type (J,M,P). Rho1 (K,N,Q). p120 Rho1 (L,O,R). (J-L) Lateral view. Early dorsal closure. (M-R) Dorsal views. (M-O) Late dorsal closure. (P-R) After closure. Scale bars: 25 µm in A-I; 20 µm in J-R.

View larger version (53K): [in a new window]   Fig. 6. Rho1 enhances a weak shg allele. Cuticle preparations, anterior up. (A) Wild type. (B) shgG119. Note head hole and small ventral scar. (C) Rho1 shgG119. Note head and ventral holes. (D) Schematic of shgG119 and shg2 lesions, and sequence alignment of relevant region in shgG119. Gbl-cad, Gryllus bimaculatus (cricket), DE-cad homolog. Scale bar: 25 µm.

View larger version (81K): [in a new window]   Fig. 7. Rho1 suppresses both strong and null shg alleles. Cuticle preparations, anterior up. Major phenotypic classes (>10% of cuticles), shg or Rho1 shg. (A-D) shg2. Range of defects includes ventral holes (A), fragmentary ventral cuticle (B), dorsal cuticle only (C) and dorsal cuticle with holes (D). (E,F) Rho1 shg2. More than 80% of embryos have ventral holes (E) or fragmentary ventral cuticle (F). (G) shg2 lesions in lamininG repeat and motif 5 of cadherin tail. DE-Cad versus Gbl-Cad, or mouse cadherins CAD-6 (Type II) and E-CAD (Type I). (H-J) shgR69. Range of defects includes intact dorsal cuticle only (H), dorsal cuticle with holes (I) or U-shaped dorsal cuticle (J). (J-M) Rho1 shgR69. Range of defects includes fragmentary ventral cuticle (K), dorsal cuticle only (L) or dorsal cuticle with holes (M). Scale bar: 25 µm.

View larger version (128K): [in a new window]   Fig. 8. Rho1 regulates DE-Cad but not Arm localization. (A,B) Wild-type (A) or Rho1 (B) during dorsal closure. DE-Cad (red). Arm (green). Arrow, leading edge; arrowhead, amnioserosa. (C-F) DE-Cad in wild type (C,E) and Rho11B (D,F). Stage 9 (C,D; arrows, amnioserosa). Stage 17 (E,F; arrows, dorsal midline). (G) p120 Rho1 double mutant during dorsal closure. Scale bar: 20 µm.

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