abl^MZ mutants have defects in epithelial morphogenesis. Cuticle preparations, anterior up. In A–D, dorsal is to the right. (A) Wild-type. (B–D) The range of phenotypes in abl⁴ maternal/zygotic mutants, a similar range is observed in abl¹ . (B) Approximately 7% of abl^MZ mutants have head involution defects and completely fail to germband retract. (C) ∼14% of abl^MZ mutants partially fail to germband retract and have variable dorsal closure defects. Note the dorsal hole (arrow). (D) Approximately 67% of abl^MZ mutants have dorsal closure defects. (E) Wild-type dorsal hair pattern. (F) Misaligned dorsal hairs in abl^MZ mutant.

Complete loss of Abl alters Ena and actin localization during dorsal closure. Lateral view. Embryos double-labeled with anti-Ena (red) and phalloidin (green), labeling F-actin. (A) Stage 13 wild-type embryo with leading edge cells initiating elongation. Ena (left, red) is enriched at vertices of cell–cell contact. Actin (middle, green) outlines all cell membranes and is beginning to accumulate at the leading edge. Actin and Ena colocalize at cell junctions. (B) Stage 13 abl^MZ mutant. Ena (left, red) and Actin (middle, green) enrichment is not uniform at the leading edge. Both are enriched in some cells (arrows) and depleted in others (brackets). (C) Stage 14 wild-type embryo. More lateral cells have undergone uniform elongation. Ena is uniformly enriched at adherens junctions of leading edge cells (left, red). Actin forms a tight cable along the leading edge (middle, green). Ena and actin colocalize at adherens junctions as actin expands along the entire leading edge. (D) Stage 14 abl^MZ mutant. Nonuniform localization of Ena and Actin persists. Cells with excess Ena (left, arrow) often accumulate excess Actin (middle, arrow), whereas cells with diminished Ena levels (left, bracket) have diminished levels of Actin (middle, bracket) Bars, 10 μm.

Ena and Arm colocalize at adherens junctions throughout development. All images (except I) are anterior to the right. Ena is green and Arm is red in merged images. (A) Stage 3 egg chamber. Ena and Arm are enriched in apical adherens junctions of follicle cells. (B and C) Stage 10 egg chamber. Levels of Ena and Arm drop, but both remain at adherens junctions. Anterior (border cells) and posterior polar follicle cells are enriched in both Ena and Arm (B, arrows). Ena and Arm also colocalize to nurse cell membranes (B, arrowhead). (D–G) During embryogenesis Ena and Arm colocalize to adherens junctions of epithelial tissues. (D) Cross-section, stage 9 embryo. Ectodermal adherens junctions (arrow). (E) Adherens junctions of polarized cells of the invaginating hindgut (arrow). (F) Apical view, stage 9 embryo. Ena is enriched at vertices of cell–cell contact (arrow), whereas Arm is more uniform. (G) During dorsal closure Ena is enriched at adherens junctions of leading edge cells, but it is also found in the cytoplasm of cells at the segment boundary (arrowheads). (H) Ena and Arm both localize to axons. (I–K) Imaginal discs. (I) Apical surfaces of two epithelial sheets opposed to one another in the wing imaginal disc (arrow). Ena and Arm colocalize to apical adherens junctions, and are also found at the apical surface. (J and K) In eye imaginal discs cell differentiation occurs after the morphogenetic furrow passes. In undifferentiated cells, Ena and Arm colocalize to cell boundaries (J, arrowhead). As groups of cells begin differentiating as photoreceptors (J, arrow), Ena localizes uniformly to all cells of the precluster. Arm, in contrast, accumulates at high levels in a subset of these cells. Later, Ena and Arm colocalize in photoreceptor rhabdomeres (K, arrow). Bars: (A–J) 10 μm; (K) 50 μm.

DE-cadherin (shg) genetically interacts with Abl. Cuticle preparations, anterior up. (A) Wild-type. (B) abl⁴/abl⁴ × shg²/CyO. abl⁴ maternal mutants are zygotically rescued, with all hatching as larvae and most appearing wild-type. shg heterozygosity prevents zygotic rescue of abl maternal mutants and leads to morphogenesis defects. Note defects in dorsal closure and head involution (arrow). (C) abl^MZ mutants die during embryogenesis with defects in epithelial morphogenesis. (D and E) shg heterozygosity enhances the abl^MZ phenotype. (D) 70% of lethal progeny of abl⁴/abl⁴ × shg²/+; abl⁴/+ have cuticles that are reduced in size with a large dorsal-anterior hole. (E) 30% of the lethal progeny of abl⁴/abl⁴ × shg^R69/+; abl⁴/+ have a prominent dorsal-anterior hole.

Total levels of Arm and α-catenin are reduced in abl mutants. abl germline mutant females were mated to abl heterozygous males and progeny were picked at the cellular blastoderm stage and aged for the indicated time postblastoderm (PBD). Wild-type embryos (Canton S [CS]) served as a control. Cell extracts were fractionated by SDS-PAGE and immunoblotted with the indicated antibodies. Molecular weight markers are to the right. Bicaudal D (BicD) or Peanut (Pnut) are loading controls. Each vertical set of samples represents sequential reprobing of the same blot. (A and B) Stage 11/12 embryos (6 h postblastoderm). (C) Stage 13/14 embryos (9 h postblastoderm).

Complete loss of Abl disrupts CNS development. (A) Cell extracts from 3-h-old wild-type embryos or embryonic progeny of females with abl mutant germlines, immunoblotted with anti-Abl antibody. Wild-type Abl is ∼180 kD (top arrow). abl⁴ does not produce a protein recognized by this antibody. abl¹ produces a truncated protein product of ∼80 kD (bottom arrow). (B–F) Embryos (anterior up) labeled with mAb BP102, which labels all axons. (B) Wild-type CNS, with a scaffold of longitudinal (arrowhead) and commissural (arrow) axons. (C and D) Maternally abl mutant but zygotically-rescued embryos have relatively wild-type CNS development, with occasional collapsed longitudinal axons (D, arrowhead) and gaps in axon bundles (D, arrow). (E and F) abl^MZ mutants exhibit severe disruptions in CNS development. Most exhibit loss of commissural axons (arrow, E) and some have defects in both longitudinal and commissural axons (F). Bar, 25 μm.

abl^MZ mutants fail to undergo coordinated changes in cell shape during dorsal closure. Embryos labeled with antiphosphotyrosine. Anterior is to the left. (A–E) Wild-type at progressively later stages of dorsal closure. (A–C) Lateral views. (D and E) Dorsal views. (A) Leading edge cells have begun to uniformly elongate (arrow). (B and C) Successive lateral cell rows uniformly elongate (arrow). (D) Lateral epithelial sheets zip together. (E) Closure is complete, with lateral epithelial cells evenly matched at the midline (arrow). (F–J) abl^MZ mutants at progressively later stages of dorsal closure. (F–H) Lateral and (I–J) dorsal views. (F) Leading edge cells do not elongate uniformly (arrow). Some cells have broadened or constricted leading edges (arrowheads). (G and H) Lateral cells have begun to elongate, but do so nonuniformly (arrow). Some cells have broadened or narrowed leading edges (arrowheads). Other groups of cells completely fail to elongate (asterisks). (I) abl^MZ mutants that proceeded through dorsal closure. Small groups of cells have still completely failed to change shape (asterisks). (J) abl^MZ mutant that completed closure. Epithelial sheets often fail to align properly at the midline (arrow). (K and L) Some abl^MZ mutants initiate dorsal closure even though they have not completed germband retraction. Cell shape defects are also seen in these embryos (arrows). (M and N) arm^XP33 mutants have cell shape defects similar to abl^MZ mutants. Cells fail to elongate uniformly (arrow) and have broadened or narrowed leading edges (arrowheads). (O) Cell shape defects in abl^MZ mutants are not caused by multinucleate cells. abl^MZ, double-labeled with antiphosphotyrosine and with propidium iodide, labeling nuclei. Mononucleate cells have defects in shape (arrow). Bars: (A–J and L–O) 10 μm; (K) 50 μm.

Dorsal closure is substantially slowed in abl^MZ mutants. Dorsal view of embryos expressing moesin-GFP, anterior to the right. Time is in minutes. Insets in (B, H, and N) display actin-rich filopodia extending from amnioserosa or leading edge cells. (A–D) Wild-type embryo at 30 min intervals during dorsal closure. (A) The leading edge of the dorsal closure front is uniformly enriched in actin. Lateral epithelial sheets elongate uniformly. (B) 30 min. Amnioserosa cells are undergoing apical constriction and the embryo is zipping together at the anterior and posterior ends (arrows). (C) 60 min. Amnioserosa cells have constricted apically and remain in the same plane of focus as the lateral epithelial sheets. (D) 90 min. Dorsal closure is complete. (E–K) abl^MZ mutant. The amnioserosa cells in E are comparable in surface area to the wild-type in A. (F–H) As closure progresses, amnioserosa cells constrict and lateral epithelial cells elongate, but dorsal closure is delayed relative to wild-type (compare D and H). If one matches embryos based on the length of the leading edge (compare A and G), closure is still delayed. This embryo took >4 h to complete closure (K). (L–R) A different abl^MZ mutant at higher magnification, illustrating the folding-under of the leading edge and failure to complete closure. (S) Cross-section diagram depicting one interpretation of the defects of abl^MZ mutants. In wild-type embryos, the rate at which lateral cells elongate, the sheets migrate, and amnioserosa cells constrict are tightly coordinated. In abl^MZ mutants, the leading edge folds under the more lateral epidermis, perhaps because leading edge cells migrate too slowly or amnioserosa cell constriction is slowed (these events are likely coupled), forcing the sheet to fold under. Time-lapse videos supplementing this figure are available at http://www.jcb.org/cgi/content/full/jcb.200105102/DC1. Bars, 25 μm.

Mutations in ena enhance arm's dorsal closure defects. Cuticle preparations, anterior up. (A) Wild-type. (B) arm^H8.6 mutants have segment polarity defects due to defects in Wingless signaling (arrowhead), but head involution and dorsal closure are normal. (C) ena²¹⁰/ena^GC1. (D) arm^H8.6/Y; ena^GC1/ena^GC1. Note strong defects in dorsal closure and head involution (arrow), with no change in the segment polarity phenotype (arrowhead). (E) arm^YD35 mutants have a dorsal hole (arrows), as well strong segment polarity defects. (F) The dorsal side of arm^YD35; ena²¹⁰/ena²¹⁰ embryos is completely open (arrows).

Levels of junctional Arm and α-catenin are reduced in abl^MZ mutants. Embryos labeled with anti-Arm (A–D), anti–α-catenin (E and F), anti–DE-cadherin (G and H), antiphosphotyrosine (I and J), anti-Neurotactin (K and L), anti-Crumbs (M and N), and anti-Coracle (O-P). (A and B) Stage 8. (C and D) Stage 14. (A and C) Wild-type. Arm localizes to adherens junctions. (B and D) abl^MZ mutants. Arm accumulates at reduced levels in adherens junctions. (E and F) Stage 13. (E) Wild-type. α-catenin localizes to adherens junctions. (F) abl^MZ mutant. α-catenin accumulates at reduced levels in adherens junctions. (G–J) Stage 13. DE-cadherin localizes to adherens junctions, without striking differences in localization or levels between wild-type (G) and abl^MZ mutants (H). Phosphotyrosine localizes to adherens junctions, without noticeable differences between wild-type (I) and abl^MZ mutants (J). (K and L) Stage 11. Neurotactin localizes to the baso-lateral membrane without noticeable differences between wild-type (K) and abl^MZ mutants (L). (M and N) Cross-sectional views, Stage 11. Crumbs localizes to the apical membrane of epithelial cells without striking differences between wild-type (M) and abl^MZ mutants (N). (O and P) Stage 13. Coracle localizes to septate junctions with no striking difference in levels between wild-type (O) and abl^MZ mutants (P). Bar, 10 μm.