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First published online 29 April 2009
doi: 10.1242/dev.030866

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Mechanical control of global cell behaviour during dorsal closure in Drosophila

¹ Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, UK.
² Department of Physiology, Development and Neuroscience, University of Cambridge, Downing Street, Cambridge CB2 3DY, UK.

View larger version (56K): [in this window] [in a new window]   Fig. 1. Dynamics of amnioserosa contraction in pooled wild-type and mutant Drosophila embryos. (A-C,G-I,M-O) Still images from a time-lapse movie of a wild-type embryo (A-C; see Movie 1 in the supplementary material), an enGal4/UAS-spastin-EGFP embryo (G-I; see Movie 2 in the supplementary material) and an ASGal4/UAS-p35 embryo (M-O; see Movie 3 in the supplementary material), carrying the ubiECadGFP transgene at 20, 80 and 140 minutes after the start of dorsal closure (DC), defined as the onset of amnioserosa (AS) contraction. Anterior is to the left in these and all subsequent images. Cells that undergo basal extrusion (before being reached by the zippering epidermis) are labelled in red. (D-F) Data are pooled from four wild-type embryos (orange ribbons). Staging of embryos was by comparing the morphogenesis of the posterior spiracles. (J-L) Data are pooled from four different enGal4/UAS-spastin-EGFP embryos (blue ribbons). (P-R) Data are pooled from three different ASGal4/UAS-p35 embryos (magenta ribbons). For the shading code of the ribbons, see Materials and methods. (D,J,P) Mean apical cell area. (E,K,Q) Proportional rate of change in apical area of AS cells. In E, the transition from the slow phase (0-70 minutes) to the fast phase (70 minutes to end) is highlighted (dashed red line). (F,L,R) Cumulative proportional area change. Data from single embryos are shown in Fig. S1 in the supplementary material. Fluctuations in contraction rates in K and Q are considered to be noise due to experimental error and enhanced variability in the phenotype of mutant embryos (developmental timings are known to be more variable between mutants than between wild-type embryos). Although fluctuations in apical cell area do exist at early stages of development (data not shown), they occur on a smaller time scale than those shown in these graphs.

View larger version (11K): [in this window] [in a new window]   Fig. 2. Correlation between the zippering rate and the rate of contraction of AS cells. (A) Zippering rate (kz) and gradient of proportional change in area/minute (see Materials and methods) for the different embryos analysed. There are significant differences in the kz and in the gradient of proportional change in area/minute between wild-type and enGal4/UAS-spastin Drosophila embryos (kz: t=3.04, df=6, P=0.023; gradient of proportional change in area/minute: t=-3.41, df=6, P=0.014) as well as between wild-type and ASGal4/UAS-p35 embryos (kz: t=5.02, df=5, P=0.004; gradient of proportional change in area/minute: t=-5.12, df=5, P=0.004) and between wild-type and mys embryos (kz: t=7.87, df=6, P<0.001; gradient of proportional change in area/minute: t=-6.13, df=6, P<0.001). (B) The relationship between these two measures, using linear regression.

View larger version (16K): [in this window] [in a new window]   Fig. 3. Preferential apical contraction of AS cells in the mediolateral axis. (A) Proportional rates of size change of AS cells in mediolateral (ML, red) and anteroposterior (AP, blue) orientations for data pooled from four wild-type Drosophila embryos. (B) Schematic of data from A. Only towards the end of DC is there a significant AP-oriented contribution to apical cell contraction. Ribbons show mean±s.e.

View larger version (51K): [in this window] [in a new window]   Fig. 4. Differences in cell behaviour along the AP axis in wild-type Drosophila embryos. (A) Proportional change in area/minute (area strain rate) of AS cells as a function of their AP location over time. The arrow indicates the transition between the slow and the fast phases. (B,C) Mean proportional rates of change in the ML orientation for cells across the AS for the periods 30-60 minutes and 60-90 minutes after the start of dorsal closure, respectively. Colours represent the mean behaviour of cells that fall within each tile of AS tissue. (B',C') The same data as in B,C are presented as averages over the ML (red) and the AP (light blue) axis, for the same periods of DC. Data are pooled from four wild-type embryos in all graphs. (D-F) Still images from an animation of an example wild-type embryo, showing the relative magnitude and orientation of the long axis of AS cells. (G-I) Cell orientation data are pooled from four aligned wild-type embryos, and regional averages are shown for three epochs of DC: 0-30, 60-90 and 120-150 minutes after the onset of DC. The orientation of the red lines represents the mean (elongation ratio-weighted) orientation of the long axis of cells in each grid square. The lengths of red lines represent the elongation log-ratio of the long to short axes of cell shapes. A line length equal to the size of a grid square equals a log-ratio of 1.0 (a ratio of 2.718:1).

View larger version (39K): [in this window] [in a new window]   Fig. 5. A gradient of AS cell contraction from the leading edge towards the dorsal midline depends on the zippering of the epidermis. (A-C) Proportional rates of change of cell shape in the ML orientation summarized by radial location. Cells at both canthi are removed from this analysis because they are known to be directly affected by zippering. Data are pooled from four wild-type (A), four enGal4/UAS-spastin-EGFP (B) and three ASGal4/UASp35 (C) Drosophila embryos. Note the `stair-like' distribution of shape strains in A, indicating that external cells are contracting their apical surface areas faster and earlier than central cells. (A'-C') The same data as in A-C presented as time averages for 40-100 minutes after the onset of DC (arrows in A-C). In enGAL4/UAS-spastin embryos, there is only a peripheral gradient of apical contraction, whereas in ASGal4/UAS-p35 embryos there is no gradient of apical contraction along the radial axis (all cells contract at approximately the same rate).

View larger version (64K): [in this window] [in a new window]   Fig. 6. Zippering defects in mys mutant Drosophila embryos is not a consequence of defective filopodia, lack of cell extrusion or attachment to the yolk. (A-F) Still images from a time-lapse movie of a mys mutant embryo carrying the ubiECadGFP transgene (see Movie 4 in the supplementary material) at 20, 80, 140, 190, 220 and 250 minutes after the onset of DC. After the epidermis and the AS tear apart, the AS keeps on contracting (D-F). (G) Image from a time-lapse movie of a mys mutant embryo carrying the enGal4 and UASactinGFP transgenes (see Movie 5 in the supplementary material). Dorsal-most epidermal cells form filopodia at their dorsal end (inset). (H,I) Still images from a time-lapse movie of a P0180/UAS-TorsoβPScyt embryo carrying the ubiECadGFP transgene (see Movie 6 in the supplementary material) at 80 and 140 minutes after the onset of DC. When expressed in wing imaginal discs, this construct produces blistering of the wing (Dominguez-Gimenez et al., 2007) (data not shown).

View larger version (19K): [in this window] [in a new window]   Fig. 7. Dynamics of AS contraction in mys mutant Drosophila embryos. (A) Mean apical cell area. (B) Proportional change in apical area. (C) Cumulative proportional area change. Data are pooled from four aligned mys embryos (green ribbons), with wild-type (orange) and ASGal4/UASp35 (magenta) ribbons presented as in Fig. 1. Both ASGal4/UASp35 and mys embryos lack a fast phase of AS contraction (B). (D) Proportional rates of size change of AS cells in ML (red) and AP (blue) orientations for data pooled from four mys embryos. (E,F) The radial pattern of ML-oriented cell shape strain rates is shown as in Fig. 4 for pooled mys data. Central cells contract their apical surface area in the ML orientation faster than peripheral cells during the period of 40-100 minutes.

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