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Drac1 and Crumbs participate in amnioserosa morphogenesis during dorsal closure in Drosophila

Nicholas Harden^1,, Michael Ricos², Kelly Yee¹, Justina Sanny¹, Caillin Langmann¹, Hong Yu¹, William Chia^2, and Louis Lim^3,4

¹ Department of Molecular Biology and Biochemistry, Simon Fraser University, 8888 University Drive, Burnaby, BC, V5A 1S6, Canada
² Drosophila Neurobiology, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
³ Glaxo-IMCB Laboratories, Institute of Molecular and Cell Biology, 30 Medical Drive, Singapore 117609, Republic of Singapore
⁴ Department of Neurochemistry, Institute of Neurology, 1 Wakefield St., London WC1 1PJ, UK
Present address: MRC Centre for Developmental Neurobiology, King's College London SE1 1UL, UK

View larger version (142K): [in a new window]   Fig. 1. Dorsal views of the amnioserosa showing morphogenesis of this tissue during dorsal closure. Anterior is to the left in this and subsequent figures. Dashed yellow lines demarcate clusters of apically constricted cells. (A,C,E,G) Embryos at progressively later stages of dorsal closure stained with anti-phosphotyrosine antibodies. (B,D) Embyros stained with anti-nonmuscle myosin heavy chain antibodies (Kiehart and Feghali, 1986). (F,H) Embryos stained with anti-ß-galactosidase antibodies. (A) A stage 13 embryo prior to commencement of dorsal closure showing amnioserosa as a flat sheet with cells elongated perpendicular to the A-P axis of the embryo. (B) Embryo at similar developmental stage to that in A, showing no enrichment of myosin in the amnioserosa. (C,D) Early stage 14 embryo double-stained to show elevated levels of phosphotyrosine (C) and myosin (D) in cells at the anterior and posterior ends of the amnioserosa. These cells are apically constricted relative to cells in the middle of the amnioserosa (the apical ends of the amnioserosa cells are at the surface of the embryo). The hindgut can be seen as a stained structure under the posterior end of the amnioserosa. (E) Late stage 14 embryo midway through dorsal closure showing apically constricted cells at the ends of the amnioserosa and loss of original elongation of middle amnioserosa cells. (F) UAS-lacZ1-71;GAL4332.3 embryo early in dorsal closure with anti-ß-galactosidase staining revealing amnioserosa as an elliptical sheet of cells. Note the lack of expression of the lacZ reporter gene in the epidermis. That no epidermal cells were expressing the lacZ reporter gene was confirmed by double staining UAS-lacZ1-71;GAL4332.3 embryos with anti-phosphotyrosine antibodies to show cell boundaries (data not shown). (G) A stage 15 embryo late in dorsal closure showing that the middle amnioserosa cells are both elongated along the A-P axis and apically constricted. (H) A UAS-lacZ1-71;GAL4332.3 embryo after the completion of dorsal closure with ß-galactosidase staining revealing amnioserosa as a tubular structure. Note lacZ expression appearing in epidermal cells.

View larger version (127K): [in a new window]   Fig. 2. Constitutively active Drac1 transgene expression causes contraction of the amnioserosa. Embryos were stained with phalloidin to detect F-actin (A) or with the anti-phosphotyrosine antibody (B-D). (A) An early stage 14 GAL4332.3;UAS-Drac1V12 embryo showing dramatic contraction of amnioserosa into the rear half of dorsal hole. (B) A stage 15 GAL4332.3;UAS-Drac1V12 embryo late in dorsal closure showing bunched closure of the epidermis around contracted amnioserosa. Phosphotyrosine staining in the amnioserosa is very intense. (C) An early stage 14 Hs-GAL4M-4/UAS-Drac1V12 embryo showing an amnioserosa phenotype similar to that in A. Attachments of the amnioserosa to the epidermis can be seen (arrowhead). (D) Late stage 14 Hs-GAL4M-4/UAS-Drac1V12 embryo showing bunched closure of the epidermis around contracted amnioserosa.

View larger version (137K): [in a new window]   Fig. 3. Expression of constitutively active Drac1 causes an increase in F-actin and myosin staining in the amnioserosa. Embryos were stained with phalloidin to detect F-actin (A,B) or anti-nonmuscle myosin heavy chain antibody (C,D). (A) Dorsal view of the amnioserosa of a stage 15 wild-type embryo late in dorsal closure. There is heavy accumulation of F-actin along the leading edge (arrowhead). (B) Dorsal view of the amnioserosa of a stage 15 Hs-GAL4M-4/UAS-Drac1V12 embryo showing elevated F-actin staining as compared to the embryo in A. (C) Lateral view of a stage 14 wild-type embryo showing the boundary between the amnioserosa (top of micrograph) and the epidermis. There is accumulation of myosin along the leading edge (arrowhead). (D) Lateral view of a stage 14 Hs-GAL4M-4/UAS-Drac1V12 embryo showing the boundary between the amnioserosa (top of micrograph) and the epidermis. Compared with the embryo in C, there is increased myosin staining in the amnioserosa and parts of the epidermis. In this embryo the amnioserosa has begun to contract and pull away from the epidermis, although points of adhesion between the two tissues remain (arrowhead).

View larger version (124K): [in a new window]   Fig. 4. Dorsal closure defects are seen following expression of dominant-negative Drac1 in the amnioserosa and in embryos homozygous for a hypomorphic allele of crb. Dorsal views of embryos stained with anti-ß-galactosidase antibodies to label amnioserosa cells (A,B) or anti-phosphotyrosine antibodies to show morphology (C-H). (A,C) A double-stained stage 15 UAS-lacZ1-71;GAL4332.3 control embryo late in dorsal closure showing amnioserosa in transition from an elliptical to tubular morphology. (B,E) A double-stained UAS-lacZ1-71;GAL4332.3;UAS-Drac1N17 embryo similar in age to that in A and C but in which the amnioserosa is still elliptical in shape. The hindgut has ruptured the amnioserosa in this embryo. (D) A wild-type embryo at the end of dorsal closure. (F) UAS-lacZ1-71;GAL4332.3;UAS-Drac1N17 embryo similar in age to that in D showing persistence of a small dorsal hole. (G) A stage 15 embryo homozygous for crbS010409 showing germband retraction defect and impaired dorsal closure. There is robust phosphotyrosine staining along the leading edge. (H) An embryo homozygous for crbS010409, similar in age to that in D, showing persistence of dorsal hole.

View larger version (57K): [in a new window]   Fig. 5. (A) The dorsal hole of stage 15 GAL4332.3;UAS-Drac1N17 embryo late in dorsal closure showing strong leading edge phosphotyrosine staining. The amnioserosa cells near the top of the figure have changed shape properly (arrowhead), but the cells toward the bottom of the figure have not (arrow). The leading edge has progressed further toward the dorsal midline on the side of the embryo at the top of the figure. The amnioserosa cells and dorsal hole of this embryo can be compared with those of the wild-type embryo late in dorsal closure shown in Fig. 1G. (B) Dorsal surface of stage 16 GAL4332.3;UAS-Drac1N17 embryo showing successful but distorted dorsal closure.

View larger version (86K): [in a new window]   Fig. 6. Gains or losses of Crb function cause dorsal closure defects. (A) A dorsolateral view of wild-type embryonic cuticle. (B) A dorsolateral view of cuticle of GAL4332.3/UAS-crbwt embryo showing a dorsal hole resulting from overexpression of wild-type Crb in the amnioserosa. (C) Dorsal view of wild-type embryonic cuticle. (D) Dorsal view of cuticle of embryo homozygous for crbS010409, showing dorsal hole and germband retraction defect.

View larger version (177K): [in a new window]   Fig. 7. Premature apical constriction of amnioserosa end cells is induced by overexpression of Crb or by expression of Drac1V12 in a crbS010409 mutant background. Dashed yellow lines demarcate clusters of apically constricted amnioserosa cells. Embryos were stained with anti-nonmuscle myosin heavy chain antibodies (A), antiphosphotyrosine antibodies (B-E) or phalloidin (F). (A,C) A doublestained stage 13 GAL4332.3/UAS-crbwt embryo showing clusters of apically constricted cells with strong myosin staining at the anterior and posterior ends of the amnioserosa. (B) A stage 14 GAL4332.3/UAS-crbwt embryo showing dumbbell-shaped amnioserosa. (D) A stage 13 GAL4332.3/UAS-crbwt;UAS-Drac1N17 embryo showing that impairment of Drac1 signaling does not block induction of premature cell constriction by overexpressed Crb. (E) A stage 13 crbS010409, UAS-Drac1V12/crbS010409, Hs-GAL4M-4 embryo showing premature apical cell constriction at the anterior end of the amnioserosa. The posterior end of the amnioserosa in this embryo is obscured by the unretracted germband. (F) A stage 13 Hs-GAL4M-4/UAS-Drac1V12 embryo is included for comparison, showing contraction of amnioserosa into the posterior end of dorsal hole.

View larger version (64K): [in a new window]   Fig. 8. Overexpression of Crb in the amnioserosa disrupts the leading edge cytoskeleton. Lateral view of leading edge of stage 14 GAL4332.3/UAS-crbwt embryo double-stained with anti-nonmuscle myosin heavy chain antibodies (A) and anti-phosphotyrosine antibodies (B). Phosphotyrosine and myosin are depleted in patches along the leading edge (arrowheads), and there is little elongation of epidermal cells. Compare the leading edge myosin staining in A with the wild-type leading edge myosin staining in Fig. 3C.

View larger version (104K): [in a new window]   Fig. 9. Dorsal closure defects are seen following expression of Drac1 and crb transgenes with the GAL4c381 driver. (A-C) Dorsal views of UAS-lacZ1-71;GAL4c381 embryos stained with anti-ß-galactosidase antibodies to show expression of the driver in the amnioserosa but not the epidermis during (A,B) and after dorsal closure (C). (D) Lateral view of cuticle of UAS-Drac1V12;GAL4c381 embryo showing germband retraction failure and puckers extending out from the dorsal surface. (E) Dorsolateral view of the cuticle of the UAS-Drac1N17;GAL4c381 embryo showing the large dorsal hole. (F) Dorsolateral view of cuticle of UAS-crbwt;GAL4c381 embryo showing large dorsal hole. (G) Dorsal view of stage 14 UAS-Drac1V12;GAL4c381 embryo stained with anti-phosphotyrosine antibodies to show contraction of the amnioserosa, germband retraction failure and bunching of the epidermis. (H,I) Lateral views of the leading edge in embryos stained with anti-phosphotyrosine antibodies. (H) Stage 14 UAS-Drac1V12;GAL4c381 embryo showing robust accumulation of phosphotyrosine at the leading edge and extensive epidermal cell elongation. (I) A stage 14 UAS-crbwt;GAL4c381 embryo showing loss of leading edge phosphotyrosine nodes and lack of epidermal cell elongation. The arrowhead marks a portion of the leading edge where phosphotyrosine nodes are intact.

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