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First published online 28 May 2008
doi: 10.1242/dev.019802

Development 135, 2227-2238 (2008)
Published by The Company of Biologists 2008
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Control of cell flattening and junctional remodeling during squamous epithelial morphogenesis in Drosophila

Karen L. Pope and Tony J. C. Harris*

Department of Cell and Systems Biology, University of Toronto, 25 Harbord Street, Toronto, ON, M5S 3G5, Canada.


Figure 1
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  		Fig. 1. The autonomy of amnioserosa morphogenesis. (A-L) Wild-type embryos. Stage 7-8 (A) and stage 8-9 (B). AS is bracketed. (C) Stage 10-11. Fully extended AS (bracketed) is present between folded germband. (D-L) Embryo surface views at the level of AJs. (D-F) Stage 6. (D) DE-Cadherin. Two hexagonal AS cells are outlined. Inset shows a cross-section. (E) Lateral MT bundles in cross-section (arrows). (F) F-actin, AS cells are outlined. (G-I) Stage 8. (G) DE-Cadherin. Two AS cells elongating along DV axis are outlined. (H) MTs along cell elongation axis (arrows). (I) Actin, AS cells are outlined. (J-L) Stage 10. (J) DE-Cadherin. Two fully elongated AS cells are outlined. Inset shows a cross-section. (K) MT bundles along cell elongation axis (arrows). (L) Actin, AS cells are outlined. (M) Cell dimensions pre/post-elongation. (N,O) Stage 9-10 zen mutant. (N) Non-elongated dorsal cells are bracketed. (O) Projection. The germband ventral midline (arrow) extended beneath dorsal surface. (P,Q) armm/z mutant. Elongated dorsal cells are bracketed. (P) After cellularization. (Q) Stage 9-10. (R,S) bcd nos tsl mutant. Elongated dorsal cells are bracketed. (R) Stage 7-8. (S) Stage 9-10. (T) Higher magnification wild-type, zen mutant, armm/z mutant and bcd nos tsl mutant cells. Two cells are outlined in each. (U) Quantification of stage 10 wild-type, zen mutant, armm/z mutant and bcd nos tsl mutant cell lengths. (V) Bright-field timelapse imaging. Early bcd nos tsl mutant AS morphogenesis. Two cells are outlined. (W,X) dl mutant. DE-Cadherin. AS is bracketed. Ectopic furrows are indicated by arrows. (W) Stage 7-8. (X) Stage 9-10. (Y) Stage 9-11 AS cell orientations. bcd nos tsl mutants, white; dl mutants, red. White scale bar, 5 µm; gray scale bar, 50 µm.

 

Figure 2
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  		Fig. 2. Amnioserosa cell elongation involves rotation of the MT cytoskeleton. (A) Stage 7-8 AS 3D projections. DE-Cad (red). Dlg (green). Purple line and box show a cross-section. Basolateral membranes of two cells are outlined. (B) Three-dimensional projections of Tubulin-GFP as live AS cells elongate. Smooth MT rotation from apical basal axis (0:00, arrows show bundle end-on views) to cell elongation axis (4:24-26:20, arrows). Centrosomes are detected (arrowheads). Gamma adjustments are made to visualize all MTs. (C) Rotation direction quantification. (D) Single rotating cell. Side view. Rotating MT bundles (asterisks). (E,F) Pre-elongation live embryo in single plane surface view (E) or in cross section (F). Tubulin-GFP staining. MT bundles are in cross-section (E, arrow) or seen longitudinally (F, arrow). Centrosomes (F, arrowheads) above elongated nuclei (dark ovals). MTs above centrosomes are bracketed (F). (G,H) During elongation live embryo in single plane surface view (G) or in cross section (H). Tubulin-GFP staining. MT bundles are seen longitudinally (G, arrow) or in cross-section (H, arrow). Centrosomes (G, arrowhead) at one end of elongated nuclei (G, dark ovals). MT extension past centrosomes (G, brackets). (I-K) DE-Cad (red), centrosomes ({gamma}tubulin, green). (I) Pre-elongation, surface view. Centrosomes (arrowhead) above nuclei (inset, cross-section). (J,K) During elongation. Centrosomes at one end of nuclei (arrows, dark circles). (L-N) DE-Cad (red), YFP-ER targeting sequence (green). (L) Pre-elongation, surface view. ER bulk above nuclei (inset, cross-section). (M) During elongation the bulk of the ER is at one cell end (arrows) but evens out later (N). (O,P) Projections. AS cells are at early elongation. Apical Tubulin-GFP (O) and Moesin-ABD-GFP (P). (Q) Pre-elongation, cross-section. DE-Cad, red; acetylated tubulin, green. (R) Elongating, surface view. DE-Cad, red; acetylated tubulin, green; tubulin, green/red. Acetylated microtubules along cell elongation axis (arrows). (S) During elongation, surface view. EB1-GFP staining. Kymogram of box of one cell side. White lines indicate two EB-1 complexes. Scale bars: 5 µm.

 

Figure 3
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  		Fig. 3. MTs function in initiating amnioserosa cell elongation. DE-Cadherin marks cell outlines. (A) Embryo stage/region measured. Transverse furrows below surface AS. PMGI, posterior midgut invagination. (B,E,G) 30-minute treatment with carrier. (C,F,H) 30-minute treatment with colchicine. (B,C) Stage 7-8 [transverse furrow cells columnar (not shown)]. Initial AS cell elongation occurs with carrier (B) but not with colchicine (C). (D) Cell elongation quantification. (E,F) Stage 8-9. Elongated surface cells with carrier (E), but not with colchicine (F). Elongated cells in transverse furrows in both (bracketed). (G,H) Stage 9-10. The full AS cell length is surface-exposed with carrier (G). Elongated cells show less surface exposure with colchicine (H, arrows). Scale bar: 5 µm.

 

Figure 4
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  		Fig. 4. Actin inhibits apical MT protrusion. (A-F) Stage 7-8. DE-Cadherin marks cell outlines. (A) Carrier. (B) Cytochalasin D. AS apical cell protrusions (bracketed, arrows). (C,D) AS cells in A,B are magnified. (E) Colchicine alone. (F) Simultaneous colchicine and cytochalasin D. (G,H) Stage 7-8 AS cells. DE-Cad (green). Dlg (red). (G) Carrier. (H) Cytochalasin D. (I-L) Stage 10 germband. (I-K) DE-Cad. (I) Carrier. Normal DE-Cad (bracketed). Normal contractile region (arrow). (J) Cytochalasin D. Clustered DE-Cad-positive protrusions (bracketed). (K) Simultaneous colchicine and cytochalasin D. Protrusions are suppressed (bracketed). (L) Cytochalasin D. MTs (red) in DE-Cad (green)-positive protrusions (arrow). Lateral MTs embryo surface-exposed (outlined). White/black scale bars 5 µm; gray bars, 25 µm.

 

Figure 5
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  		Fig. 5. Amnioserosa extension through rapid cell elongation without intercalation. (A) Three-dimensional projections. DE-Cad-GFP. AS cell elongation. Four marked neighboring cells retain contacts. (B) Initial AS cell elongation rates. Blue lines indicate average elongation rate for five cells, DE-Cad-GFP embryos. Black lines indicate average elongation rate for five cells, Moesin ABD-GFP embryos. Red line indicates the all embryo average. (C) DE-Cadherin-GFP level quantification through confocal stacks. Background-corrected AP and DV contact values normalized to DV contact value for each cell. Five cell averages per embryo are shown (four embryos, different colors). Squares, DV contacts; circles, AP contacts (arrows). (D) Three-dimensional projections. DV contacts (white arrows), DE-Cad-GFP looping (yellow arrows). Scale bars: 5 µm.

 

Figure 6
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  		Fig. 6. Myosin II and Bazooka planar polarization during amnioserosa extension. (A-C) DE-Cad (red), GFP (green). Sqh-GFP embryos. (A) Stage 6. Sqh localizes at AJs and above (bracketed). (B) Stage 7-8. Sqh localizes along AP contacts (arrows) and at contact ends (arrowheads). (C) Stage 9-10. (D-F) DE-Cad (red). Baz (green). Wild-type embryos. (D) Stage 6. Baz colocalizes with DE-Cad. (E) Stage 7-8, DV Baz enrichment (arrows). (F) Stage 9-10. (G) Stage 7-8. Live. DV Baz-GFP enrichment and partial colocalization with Arm-CFP looping (arrows) occurs. Scale bar: 5 µm.

 

Figure 7
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  		Fig. 7. Bazooka and Myosin II synergy during amnioserosa AJ remodeling. (A) zipper heterozygosity enhances baz AS AJ phenotype. (B) Stage 8 AJ defect. DE-Cad puncta (arrowheads), large clusters (arrows). Dlg (red). (C) Enhanced DE-Cad image in B. (D) Stage 10 AJ defect. DE-Cad (green) puncta (arrowheads) and large clusters (arrows). Dlg (red). (E) Enhanced DE-Cad image in D. (F) Stage 10 wild type. (G,H) bcd nos tsl mutants. (G) Stage 7-8. DE-Cad (green). Baz (red) planar polarity is abnormal or absent (arrow). (H) Stage 10. DE-Cad (green) clustering (arrows). Baz (red). Scale bar: 5 µm.

 

Figure 8
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  		Fig. 8. Models of tissue extension by rotary cell elongation. (A) A model of amnioserosa rotary cell elongation. (B) A model of amnioserosa junctional remodeling.

 

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© The Company of Biologists Ltd 2008