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First published online 6 February 2008
doi: 10.1242/dev.016337

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Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis

¹ Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.
² Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599-3280, USA.

View larger version (130K): [in this window] [in a new window]   Fig. 1. Dia localization during morphogenesis. Wild-type Drosophila embryos: anterior leftwards, antigens indicated. (A-C) Cellularization. (D,E) Gastrulation. (A-B',D-D''') Cross-sections. (C,E) Surface views. (E') Diagram of A-E. (F-F''') Epidermis at stage 8. Sqh-GFP and AJs enriched at A/P (yellow arrows) or D/V (blue arrows) cell borders. Dia expression is symmetrical. (G) Mitotic domains. Cytokinetic furrows (arrows); MidBody (arrowhead). (H) Stage 10. (I) Stage 12. (J-K''',M,N) Dorsal closure. (J,J') Leading-edge AJ (arrow) and cortex (arrowhead). (K-K''') Amnioserosa showing drop-out cells (arrows). (L) Immunoblot: wild-type or e22c-GAL4xEGFP::Dia embryonic extract, probed for Dia or tubulin control. (M-O2) e22c-GAL4xEGFP::Dia live. (M) Inset shows donut-shaped structures in amnioserosa cytoplasm. Similar puncta are seen with Dia antibody (J,L). (N) Leading-edge AJs (arrows). Cell cortex (arrowheads). (O,O2) Segmental groove cells. Dia D/V planar polarization (O1, arrows). (P) Quantitation of EGFP::Dia planar-polarity in Groove cells. Error bars indicate s.d. Scale bars: 20 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 2. DiaCA triggers precocious amnioserosal apical constriction. Drosophila embryos: anterior leftwards, genotypes and antigens indicated. (A) Dia and DiaCA. (B-H,J,K,M-Q) Dorsal closure. (B) DiaCA expression in entire amnioserosa (c381-GAL4) (inset, DE-cad). (C-D'') DiaCA expression in individual cells. Constricted cells expressing DiaCA (arrowheads) and stretched wild-type cells (arrows). (E,E') Wild-type amnioserosal cells have irregular shapes. (F-G'') DiaCA expression in entire amnioserosa (F) versus wild type (G). (H,H',J,J') DiaCA expression in large group of amnioserosal cells showing Arm (H) and Myosin (J) accumulation. (I) Apically constricting amnioserosal cells (surface view left, cross-section right). (K) Myosin in wild-type drop-out cell (arrow). (L) Immunoblot: 12-16 hour DiaCAxe22c-GAL4 and wild-type embryos probed for MHC and -catenin. Peanut is used as a loading control. (M-Q) Movie frames showing Sqh::GFP under its own promoter. (M1-M3) Wild type. (N1-O5,Q,R) DiaCA expression in amnioserosa (AS; c381-GAL4). (N1-N3) Drop-out cells (arrowheads). (P,Q) Higher magnification views. (R) Cuticle: DiaCA expression in amnioserosa. Scale bars: 20 µm.

View larger version (128K): [in this window] [in a new window]   Fig. 3. DiaCA induces planar-polarized cell shape changes in the epidermis. Drosophila embryos: anterior leftwards, genotypes and antigens indicated. (A-C) Stage 16. Deep segmental grooves (arrows). (D-M) Dorsal closure. (D) Wild-type groove cells (arrows). (E-F''') DiaCAxen-GAL4. DE-cad D/V enrichment and F-actin accumulation (arrows). (G) Myosin in elongating wild-type epidermis. (H-I'') Wild-type groove cells. Myosin, DE-cad and EGFP::Dia (e22c-GAL4) D/V enrichment (arrows). (J,J') DiaCA D/V enrichment. (K-M) DiaCAxprd-GAL4. (K'',K''') Region indicated by yellow bracket in K'. DE-Cad and Myosin D/V enrichment (arrows). (L) Movie frame showing Arm::GFP. (L,M) DiaCA-expressing cells delimited by double-headed arrows. (N,O) Quantitation of planar polarization in indicated cell types. Error bars indicate s.d. (P) Wild-type elongating and groove cells. Scale bars: 20 µm.

View larger version (155K): [in this window] [in a new window]   Fig. 4. Activating Myosin partially mimics DiaCA. Drosophila embryos: anterior leftwards, genotypes, GAL4 drivers and antigens indicated. (A-D') Laterial view of stage 13 embryos of indicated genotypes. (E-H'') Dorsal view of stage 13 embryos of indicated genotypes. (A-H'',K,K') Dorsal closure. (I-J') Stage 16. (A,A',E-E'') Wild-type. (B,B',D,D') DiaCA or MLCKCA expression induces deep grooves (arrowheads). (F-F'',H-H'') DiaCA- or MLCKCA-expressing amnioserosal cells. (C,C') SRF-VP16 expression. Elevated Myosin without deeper grooves (arrowheads). (G-G'') SRF-VP16-expressing amnioserosal cells. (I) Wild-type; grooves are no longer present (arrowheads). (K,K') DiaCA expression in zip zygotic mutants still leads to amnioserosal cell rounding. (J-K') DiaCA expression in zip zygotic mutant. Persistent grooves (J,J', arrowheads). Cortical actin alterations are slightly reduced (see Fig. 3E). Scale bars: 20 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 5. Activating Myosin induces grooves without altering planar polarity. Drosophila embryos: dorsal closure, anterior leftwards, genotypes, GAL4 drivers and antigens indicated. (A-B'') Individual MLCKCA-expressing amnioserosal cells (arrows). (C-F'') MLCKCA expression delimited by double-headed arrows (C,D-D'') or yellow lines (E-F''). Scale bars: 20 µm.

View larger version (56K): [in this window] [in a new window]   Fig. 6. Reducing Dia function in Drosophila disrupts ventral furrow. Movie frames. (A) Wild type showing Moe::GFP. Red arrows indicate coordinated apical constriction. Blue arrows indicate adjacent cells stretching. (B) dia5M, Moe::GFP. Red arrows indicate non-constricting cells. Blue arrows indicate cells rupturing. Scale bars: 20 µm.

View larger version (95K): [in this window] [in a new window]   Fig. 7. Reducing Dia function destabilizes AJs. Drosophila embryos: anterior leftwards, genotypes and antigens indicated. (A,B) Wild type. (C,D,I) Zygotically-rescued dia5M. (E-H) dia5M/Z; note less continuous AJs. (I-K) DE-cad. (J-K) dia5M/Z. (I2,J2 insets) Cross-sections. (L) Immunoblot showing 6-8 hour wild-type and dia5M/Z embryo extracts. -tubulin is used as a loading control. (M-N',P,P',R-S') dia5M/Z. (O,Q,Q') Wild-type. (P,P') Increased apical Rab5-positive vesicles; some colocalize with DE-cad (arrows, lines). (R,S) Abnormal cortical blebbing (arrows). Scale bars: 20 µm.

View larger version (126K): [in this window] [in a new window]   Fig. 8. Reducing Dia function disrupts tissue borders and increases protrusiveness. Drosophila embryos: germband retraction, anterior leftwards, genotypes and antigens indicated. (A,C) Wild-type. (A) Entire germband. (C,C') Higher magnification of the tail. Note smooth amnioserosa/epidermal tissue boundary (arrows). (B,D-G) Abnormal amnioserosal protrusions (arrows). (B,D,E-E'') dia/Rho1. (F,F') dia5M/Z or dia5M. (G) Progeny of dia2 +/+ RhoGEF204291 mothers. (H1-H3) Movie frames showing Moesin::GFP-expressing dia/Rho1 embryos. Abnormal protrusions over other amnioserosal cells (blue arrows), lateral and head epidermis (red arrows), or caudal epidermis (yellow arrows). (I) Movie frame showing Moesin::GFP-expressing wild type. (J-K'') MLCKCA expression rescues protrusiveness of dia/Rho1 (J,J', arrow). (L) Epidermal cell protrusions. Scale bars: 20 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 9. Reducing Dia function alters protrusive activity during dorsal closure. Drosophila embryos: dorsal closure, anterior leftwards, genotypes and antigens indicated. (A-C) Movie frames. Abnormal amnioserosal protrusions, dia/Rho1 (B) and dia5M/Z (C). Arrows indicate filopodia (A) or lamella (B,C). (D,E) Abnormal amnioserosal protrusions (arrows). (F,G) Drop-out cells appear prematurely in dia/Rho1 (G, arrows). (H) dia5M/Z with zippering defects (arrow). Scale bars: 20 µm.

View larger version (22K): [in this window] [in a new window]   Fig. 10. Models of the roles of Dia in actin and Myosin regulation. (A) Cell biological model of actin-myosin regulation at AJs by Dia. Dia induces formation of actin-myosin cables at AJs, leading to AJ stabilization either directly or/and by blocking access of endocytic machinery to AJ proteins. In the absence of Dia, AJs are destabilized and Myosin activity outside AJs stimulates cell protrusiveness. (B) Mechanistic model of coordinated actin and Myosin regulation by the Rho pathway.