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First published online May 16, 2007
doi: 10.1242/10.1242/dev.02849

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Enabled plays key roles in embryonic epithelial morphogenesis in Drosophila

¹ Lineberger Comprehensive Cancer Center and Department of Biology, University of North Carolina at Chapel Hill, Chapel Hill, NC 27599, USA.
² Department of Biology, Bucknell University, Lewisburg, PA 17837, USA.
³ Department of Cell Biology and Program in Neuroscience, Harvard Medical School, Boston, MA 02115, USA.
⁴ Massachusetts Institute of Technology, Department of Biology, Cambridge, MA 02139, USA.

View larger version (74K): [in this window] [in a new window]   Fig. 1. FP4mito alters Ena localization and phenocopies ena loss-of-function. (A) FP4mito. (B-C'') S2 cells, antigens indicated. (B) In wild-type cells (asterisk), Ena is diffusely cytoplasmic and enriched at leading edge (arrowhead) and cell contacts (white arrow). In FP4mito-expressing cells, GFP-FP4mito colocalizes with MitoTracker and Ena (yellow arrow). (C) FP4mito does not recruit WASP. (D-H) Embryonic CNS, BP102 antibody. (D) Control. (E,G) FP4mito x e22c-Gal4. (F) ena23/enaGC1. (H) ena210/enaGC1. Longitudinal (arrowhead) and commissural axons (arrow). (I) ISNb motor axon projections; wild type (left), ena mutants (right). (J-N) Projections of ISNb (arrows, anti-Fasciclin II) in embryos expressing indicated constructs (elav-Gal4). Scale bars: 10 µm.

View larger version (120K): [in this window] [in a new window]   Fig. 2. Ena localization. Wild-type embryos, anterior left, antigens indicated. (A, inset) Cellularization, surface view. (A) Gastrulation, ventral view. (B-D) Stages 10 and 12. Arrows, amnioserosa; blue arrowheads, tricellular junctions. White arrowhead in D', leading edge. (E,H) Onset of dorsal closure, (F,G,I) mid-late dorsal closure: arrowheads, Ena at leading-edge AJs; blue arrowheads, tricellular junctions. Arrows in F,G, segmental groove cells; arrows in H,I, dorsal-fold. (J,K) Live embryos. GFP-Ena at filopodial tips (J, arrowhead) and leading-edge AJs (K, arrowhead). (L,L') FP4mito (paired-Gal4). FP4mito recruits Ena from cytoplasm and AJs to punctate structures (blue arrows). Adjacent wild-type cells retain Ena at amnioserosal cell cortex (white arrow) and leading edge AJs (arrowhead). (M) AP4mito (engrailed-Gal4) does not recruit Ena. (N,O) FP4mito (green, paired-Gal4). (N',O') Expression boundaries (arrowheads). Unaltered cortical actin and actin/myosin cable (N), DE-cad (O). (P) UAS-Ena (engrailed-Gal4) elevates Ena levels (P'') without altering cable or cortical actin (P'). Scale bars: 20 µm.

View larger version (122K): [in this window] [in a new window]   Fig. 3. Both FP4mito and ena23 disrupt Ena localization. (A) Wild-type Ena, stage 13. (B-C'') mat-FP4mito, stages 10 and 13. Mislocalized Ena. (D-E'') Stage 13 wild type and M/Zena23. Arrows, AJs of leading-edge cells. Scale bars: 10 µm.

View larger version (69K): [in this window] [in a new window]   Fig. 4. Ena is essential for morphogenesis. Cuticles. Wild-type (A,J) and representative ena mutant (C,H,I) or FP4mito-expressing (B,D-G,K) embryos illustrating phenotypic classes. (J,K) Close-ups of A,B. Arrowheads, wild type (A,J) or defective dorsal midline (B,D,G,K). Arrows in A,C,D, wild type (A) or failed head involution (C,D). Arrow in E, germband retraction failure. Arrows in F,H, small or large ventral holes. Bottom, frequency of defects for each genotype. Scale bars: 100 µm.

View larger version (168K): [in this window] [in a new window]   Fig. 5. Inactivating M/Z Ena does not disrupt cell adhesion. F-actin (except I,J, DE-cad). (A-J) Extended germband. mat-FP4mito and M/Zena23. Morphology (A-D), cortical actin (E-H) and DE-cad (I,J) normal. (K,L) Early germband retraction. mat-FP4mito (L) amnioserosal cells (brackets) do not overlap posterior epidermis (arrows) to the same extent as wild type (K). Smaller cell size in mat-FP4mito is probably because of overall smaller size of embryos, because of defects in nurse cell dumping. (M-P) Germband retraction. (M,N) Wild-type. Amnioserosa and epidermis maintain close contact throughout (arrows). (O,P) mat-FP4mito. Arrows, amnioserosa detached from epidermis. Arrowheads in P, deep segmental grooves. Scale bars: 20 µm.

View larger version (148K): [in this window] [in a new window]   Fig. 6. Inactivating M/Z Ena disrupts morphogenesis. F-actin. (A-D) M/Zena23. Late germband retraction. Arrowheads, deep segmental grooves; arrow, uneven leading edge. (E-N) Onset of dorsal closure. (E-H) Deep segmental grooves (E-H, arrowheads). Arrows, failure of epidermal-zipping relative to wild type (E) or paternally rescued mutant (G). (I-L) Close-ups. Arrows, actin cable; arrowhead, cells elongating; brackets, occasional cells with disrupted actin cable and splayed-open leading edges. (M,N) More severely affected mat-FP4mito embryo. Note deep segmental grooves (M, arrowheads) and defects in cell-shape changes (N, arrowheads). Actin cable is largely normal (N, arrow). (O-U) Mid-late dorsal closure. Wild-type (Q), paternally rescued mutants (O,R), mat-FP4mito (T,U, close-up) and M/Zena23 mutant (P,S). Note head involution failure (S,T, brackets), persistent deep grooves (T,U, arrowheads) and slow epidermal-zipping (Q-S, arrows). (V,W) End of dorsal closure. Arrows, dorsal midline. (X) Terminal-stage mat-FP4mito. Bracket, severe head defects. Scale bars: 20 µm.

View larger version (112K): [in this window] [in a new window]   Fig. 7. GFP-Ena in filopodia. Movie stills showing embryos expressing GFP-Ena alone (A,B) or with GFP-actin (C) using engrailed-Gal4. Time, minutes:seconds. (A) GFP-Ena at filopodial tip as it extends (arrows). (B) See Movie 1 in the supplementary material. Arrows, GFP-Ena remains at filopodial tip during retraction; arrowheads, GFP-Ena spots move rearward. (C) See Movie 2 in the supplementary material. Arrowheads and arrows, large lamellipodial fan with actin microspikes that fuse at distal tips to form filopodia. Scale bars: 5 µm.

View larger version (171K): [in this window] [in a new window]   Fig. 8. Ena inactivation slows epithelial zippering. Movie stills showing (A) embryos expressing GFP-actin (see Movie 3 in the supplementary material) or (B) FP4mito+GFP-actin using e22c-Gal4 (see Movie 4 in the supplementary material). Time, minutes:seconds.

View larger version (112K): [in this window] [in a new window]   Fig. 9. Altering Ena levels or localization alters membrane protrusions. (A-E,G) Movie stills showing embryos expressing GFP-actin alone (A) or with FP4mito (B,C), UAS-Ena (Ena overexpression; D,G) or FP4CAAX (E) using engrailed-Gal4. Asterisk, engrailed stripe in high-magnification series. Time, minutes:seconds. (A) See Movie 5 in the supplementary material. Wild-type. Arrow, lifecycle of lamellipodial-based protrusion. (B) See Movie 5 in the supplementary material. FP4mito. Lamellipodia and rare, short filopodia (arrow, 4:00). (C) FP4mito. Arrow, lamellipodia extended for several minutes; arrowhead, second lamellipodium emerging as first retracts. (D) See Movie 7 in the supplementary material. Ena overexpression. Arrows, fusion of microspikes to form filopodia. (E) FP4CAAX. Longer filopodia. (F) Box and whisker plot, maximum filopodial length in leading-edge cells, genotypes indicated. Box, 25th-75th quartile; line across middle, median; diamond, mean±s.d.; broken lines, 10th and 90th percentiles; dots, outliers. P values, Student's t-test. (G) Ena overexpression induces filopodia on lateral cells. Scale bars: 5 µm.

View larger version (98K): [in this window] [in a new window]   Fig. 10. Filopodial regulation. (A-D) Upper panels: F-actin in D16-C3 cells treated for 7 days with indicated dsRNAs; lower panels: histograms showing total F-actin (fluorescent phalloidin) in arbitrary units versus total cell number. Red line, mean actin levels in control cells. (E-L) Actin (green); Ena (red). (E,F) Control and abl dsRNA. (G-L) M/Zabl4 and wild type stained in parallel: frames in G and H show areas enlarged in I-L. Scale bars: 5 µm.