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Fig. 2. Progression of the apoptotic phenotype in th⁵ embryos. (A-D) Wild-type or homozygous th⁵ embryos were hand-selected at the end of cellularisation and fixed after ageing for another 30 minutes (T1), 1 hour (T2) or 1.5 hours (T3). Fixed embryos are stained for either Engrailed (A), activated Drice (B,C) or TUNEL (D), and photographed using Nomarski optics. (A) T1 shows a th⁵ embryo that is still morphologically normal: it has initiated germ-band extension and exhibits regular Engrailed stripes. T2 shows a th⁵ embryo that has completed cell dispersal: the cells have lost their columnar shape and adopted a rounded shape, and as a consequence these appear larger (compare close-ups for T1 and T2). The Engrailed cells have scattered and the yolk cell has fragmented and emerged at the surface of the embryo. The embryo in T3 shows a striking change in cell and nuclear shapes (the latter revealed by the nuclear localisation of the Engrailed protein). The nuclei have condensed and the cell bodies are smaller and more numerous, indicating that the cells have fragmented (compare close-ups for T2 and T3). (B) No staining for the activated form of Drice is detected in wild-type embryos before stage 10 (T3) at which two nuclei are found positive in the amnioserosa (arrow). This is consistent with the fact that there is no apoptosis in early wild-type embryos and that the amnioserosa is the first tissue in which apoptosis is detected at stage 10-11 (Abrams et al., 1993). Similarly, no staining was detected in early embryos stained for TUNEL (not shown). (C) T1: just prior to cell dispersal, discrete cells already show detectable levels of activated Drice. Activated Drice staining patterns appear random, except in the head, where an anterior spot is always detected. T2: once cell dispersal is complete, activated Drice is detected in every cell (unstained areas are yolk fragments). T3: the nuclei have condensed (compare close-ups for T2 and T3). (D) TUNEL staining is not detected in any th⁵ homozygous embryos prior to cell dispersal (T1). However, once cell dispersal is completed (T2), all nuclei are positive for TUNEL. In T3, nuclei condensation is clearly seen (compare close-ups for T2 and T3). (E) Stills from a time-lapse movie (supplementary material Movie 3) of a th⁵ homozygous embryo in which cell membranes have been labelled with SrcGFP and imaged by confocal microscopy. Times are indicated from the end of cellularisation. The arrow in the first frame indicates the cephalic furrow. In the following frames, arrowheads label examples of the blebs observed in this movie. The embryo appears normal at first (C+10 minutes), then the cephalic furrow starts to regress, signalling the onset of cell shape changes. The embryo contracts abruptly, the cells undergo extreme cell shape changes and small membranous blebs start to form (C+25 minutes). The cells reach a more regular, rounded shape and the blebs form dynamically at the cell surface (C+40 minutes). Later, cells start fragmenting and blebbing decreases in intensity (C+55 minutes). Towards the end of the movie (C+65 minutes), most cells are fragmenting, and blebbing is detectable in only a fraction of the cell bodies. The timing of cell fragmentation in this movie matches the timing of cell fragmentation in the fixed samples (T3). (F) Summary of the sequence of apoptotic phenotypes in th⁵ embryos. Note that two hours after the end of cellularisation (T4), Engrailed expression is not detectable anymore in fixed th⁵ embryos (data not shown).

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