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Drosophila RhoA regulates the cytoskeleton and cell-cell adhesion in the developing epidermis

Developmental, Cell and Molecular Biology Group, Department of Biology, B330 LSRC Building, Duke University, Durham, NC 27708-1000, USA

View larger version (68K): [in a new window]   Fig. 1. Striped epidermal expression of GMA reveals cell shape during morphogenesis. UAS-GMA expression driven by either enGAL4 (A,D-G) or prdGAL4 (B,C) demonstrates cell shape in epidermal stripes, as well as in some amnioserosal cells (arrows in B,C) that continue these stripes dorsally. (D-G) Frames from a time-lapse video (Movie 1 at http://dev.biologists.org/supplemental/) showing that epidermal stripes generally meet in register at the dorsal midline. However, mismatches are observed (small arrowheads in D-F) and in some cases suturing along the dorsal midline continues for a substantial distance prior to mismatch resolution (E,F, arrows indicate progression of dorsal closure).

View larger version (177K): [in a new window]   Fig. 2. RhoAN19 expression disrupts dorsal closure and ventral integrity. UAS-RhoAN19 expression using either enGAL4 (A-D) or prdGAL4 (E,F), causes defects in the dorsal and ventral cuticle. All embryos exhibit anterior holes (arrowheads) and some sort of lateral scarring. Expression of RhoAN19 (A,E), and co-expression with GMA (B) can result in the formation of a posteriorly located dorsal hole (arrows indicate extent of the hole). Alternatively embryos exhibit puckering at the dorsal midline (C,F; white arrows indicates dorsal midline) and ventral cuticular holes (D,F).

View larger version (152K): [in a new window]   Fig. 3. RhoAN19 expression disrupts actin organization and epidermal cell morphology. Embryos expressing UAS-RhoAN19 under the control of prdGAL4 (A,B,F-H) or enGAL4 (C-E) stained with rhodamine-phalloidin to reveal F-actin. In each panel, white bars indicate representative stripes of RhoAN19 expression. (A,B) Two focal planes of the same high magnification lateral field from a stage 12 embryo undergoing germband retraction; RhoAN19-expressing cells within the prdGAL4 stripe have flattened and spread (indicated by asterisks in A) over each other and wild-type epidermis (indicated by arrows in B). (C) Stage 13 embryo prior to dorsal closure, extensive filopodia emanate from RhoAN19-expressing leading edge cells (arrow). Leading edge cells expressing RhoAN19 (D,F) or co-expressing RhoAN19 and GMA (H) appear to extend across the amnioserosa and continue to put out extensive filopodia (arrows) as dorsal closure proceeds. These filopodia (arrows) are easily seen at higher magnification (E,G,H).

View larger version (175K): [in a new window]   Fig. 4. RhoAN19 disrupts nonmuscle myosin II localization. Embryos expressing UAS-RhoAN19 under the control of enGAL4 (A,B,D,F) or prdGAL4 (C,E) were stained for nonmuscle myosin II. In all panels, bars indicate the extent of representative RhoAN19-expressing stripes. (A,B) During germband retraction, cells expressing RhoAN19 fail to ingress at the ventral midline and rather bulge, anteriorly and posteriorly, along the groove formed by neighboring wild-type cells. At higher magnification (B), nonmuscle myosin II appears less cortical and more cytoplasmic when compared with wild type. (C,E) During dorsal closure, nonmuscle myosin II is no longer highly concentrated at the leading edge of RhoAN19 stripes and these cells seem to spread forward over the amnioserosa. Arrowheads in the high-magnification view (E) indicate wild-type nonmuscle myosin II localization. (D,F) Leading edge cells expressing RhoAN19 also extend laterally, forming cell bridges. At this stage, cortical nonmuscle myosin II is clearly reduced in RhoAN19-expressing cells.

View larger version (116K): [in a new window]   Fig. 5. Loss of RhoA function affects puc expression. (A) Lateral view of an embryo carrying the pucE69 enhancer trap, stained with rhodamine-phalloidin to detect F-actin and anti-ß-gal antibody to detect puc expression. In wild-type embryos, puc expression is strictly limited to the single row of leading edge cells. (B-D) Embryos carrying the pucE69 enhancer trap and expressing RhoAN19 in enGAL4 stripes, stained as above. (B) Dorsal view showing that, although the leading edge is ragged, puc expression occurs along its entire length. In some places, however, puc expression is seen away from the leading edge. (C) High-magnification view showing stripes of RhoAN19 expression (indicated by white bars and detected by abnormal actin organization, arrow) in which either cell rearrangement (stripes marked by an asterisk) or ectopic puc expression results in cells lateral to the leading edge being positive for ß-gal staining. (D) Ectopic expression of puc is also detected within cells of the ventral epidermis.

View larger version (74K): [in a new window]   Fig. 6. RhoAN19 expression alters leading edge cell behavior. Lateral (A,B) and dorsal (C-F) views of embryos co-expressing UAS-RhoAN19 and UAS-GMA via prdGAL4 (A) or enGAL4 (B-F). In stage 12 embryos undergoing germband retraction, stripes of RhoAN19-expressing cells do not narrow dorsally (A, compare with Fig. 1B; compare Movie 3 with Movie 1 at http://dev.biologists.org/supplemental/), bringing RhoAN19-expressing stripes into close proximity and allowing leading edge filopodia to initiate cell bridge formation. Regardless of whether cell bridges are formed, extensive leading edge filopodia are clearly observed by the completion of germband retraction (B, compare with Fig. 3C). (C-F) Subsequently, RhoAN19-expressing leading edge cells spread dorsally and laterally (C) and when filopodia come into contact (arrows), they form ectopic lateral cell-cell adhesions and cell bridges (D-F).

View larger version (188K): [in a new window]   Fig. 7. Loss of RhoA function causes defects in DE-cadherin localization and epidermal cell polarity. Embryos expressing RhoAN19 driven by enGAL4 (indicated by bars) that were stained either with an antibody against DE-cadherin (A-D) or anti-ßHeavy-spectrin (E-G). (A) RhoAN19 expression causes loss of cell surface DE-cadherin at dorsal closure stages. Higher magnification images show complete loss of DE-cadherin surface staining is first apparent in ventral epidermal cells (C), when compared with the dorsal epidermis (B). (D) At earlier stages, punctate spots of DE-cadherin staining fail to coalesce into bands of apical staining in cells expressing RhoAN19. (E) Lateral view of a stage 12 embryo; RhoAN19-expressing cells lose apicolateral staining of ßHeavy-spectrin. (F) Dorsal view of a stage 11 embryo showing loss of ßHeavy-spectrin in RhoAN19-expressing stripes at the ventral midline (midline indicated by an arrow). (G) Later, loss of apicolateral staining is clearly seen in RhoAN19-expressing leading edge cells (arrowheads) that push beyond wild type neighbors. (H) DE-cadherin staining of a stage-14 RhoA mutant embryo depicting leading edge disorganization, but no loss of DE-cadherin staining. (J) At stage 15, when dorsal closure is complete, some RhoA mutants exhibit loss of cell surface DE-cadherin at sites of dorsal puckering (arrows; compare with wild-type embryo, I).

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