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First published online 8 October 2003
doi: 10.1242/dev.00824

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Caspase-independent cell engulfment mirrors cell death pattern in Drosophila embryos

Department of Biological Sciences and Science and Technology Center for Light Microscope Imaging and Biotechnology, Carnegie Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213, USA

View larger version (88K): [in a new window]   Fig. 1. Engulfment assay using VGAL. (A-C) A syncytial-blastoderm wild-type embryo was injected with AO and VGAL and a three-dimensional, time-lapse recording was made. Shown here is a single optical section of one time point at stage 14/15 of the AO, green fluorescence (A), VGAL, red fluorescence (B) and a composite image of both fluorescent channels (C). (A-I) The engulfing macrophages are outlined with a white line. (D-F) A syncytial-blastoderm wild-type embryo was injected with AO and DQ Red BSA and a three-dimensional, time-lapse recording was made. Shown here is a projection of two 4 µm optical sections of a single time point at stage 14/15 of the AO, green fluorescence (D), DQ Red BSA, red fluorescence (E) and a composite image of both fluorescent channels (F). (G-I) A UAS-nGFP embryo was injected with caged GAL4VP16 and VGAL and a five- to eight-cell patch of cells in the ventral furrow just anterior to the cephalic furrow was photoactivated. Shown here is a projection of two 5 µm optical sections of a single time point of a stage 14/15 embryo of the GFP fluorescence (G), VGAL fluorescence (H) and a composite of both fluorescent channels (I). (J-O) A UAS-rpr; UAS-hid embryo was injected with caged GAL4VP16, AO and VGAL and a five- to eight-cell patch of cells was photoactivated in the lateral epidermis. Shown here is a series of images that are projections of three 4 µm optical sections from a time-lapse recording at 20 minute intervals from the first appearance of the AO-positive ectopic cell death. The AO signal is shown as green and the VGAL signal as red. The photoactivated region is indicated by the white circle. Notice that the position of the circle changed over time as the embryo developed. Some of the VGAL-positive spots within 10 µm of the circle originated from the photoactivated region, further VGAL-spots are from naturally dying cells outside the photoactivated zone.

View larger version (69K): [in a new window]   Fig. 2. Coincidence of AO and VGAL signals in the epidermis. A wild-type embryo was injected with AO and VGAL. (A-I) A series of images that are projections of five 2 µm optical sections from a time-lapse recording of the lateral epidermis. The embryo is shown at (A-C) 1/2 germband retraction (GBR), 3/4 GBR (D-F) and full GBR (G-I). (A,D,G) AO fluorescence; (B,E,H) VGAL fluorescence; (C,F,I) both fluorescence channels. A gray mask was applied to cover the amnioserosa.

View larger version (96K): [in a new window]   Fig. 3. Epidermal engulfment in the absence of cell death in H99 and p35-expressing embryos. (A-F) A series of time-lapse images of (A-C) a wild-type embryo and (D-F) a homozygous H99 embryo injected with VGAL: (A,D) 1/2 GBR, (B,E) 3/4 GBR and (C,F) full GBR. (G-I) A series of time-lapse images of a UAS-p35; UAS-cGFP embryo injected with caged GAL4VP16 and VGAL and exposed to whole-embryo photoactivation: (G) 1/2 GBR, (H) 3/4 GBR and (I) full GBR. The images in A-F are projections of four 3 µm optical sections; G-I are projections of three 3 µm optical sections. (J) Correlation between AO and VGAL signals. A histogram plotting the closest distance between VGAL and AO spots was measured within 30 minutes of the appearance of the VGAL signal, n=40 VGAL spots from 11 segments in two embryos. (K) Segmental distribution of VGAL spots within the lateral abdominal epidermis. A histogram showing the distribution of VGAL spots in the anterior, middle and posterior third of the abdominal segments in wild-type (solid bar), homozygous H99 (hatched bar) and whole-embryo photoactivated UAS-p35;UAS-cGFP (checkered bar) embryos. The number of embryos samples was as follows. wild-type, n=23 segments from six embryos; homozygous H99, n=29 segments from five embryos; UAS-P35;UAS-cGFP, n=37 segments from 13 embryos.

View larger version (107K): [in a new window]   Fig. 4. Tissue-specific engulfment in wild-type, H99 and p35-expressing cells. Brain neuronal precursors were marked by photoactivation of a UAS-GFP transgene in different genetic backgrounds. The embryos were injected with caged GAL4VP16 and VGAL, photoactivated in a five- to eight-cell patch of the head neuroectoderm and followed by time-lapse microscopy. The engulfing macrophages are outlined with a thin white line. (A-C) A projection of four 6 µm optical sections of a UAS-nGFP embryo with the GFP fluorescence (A), VGAL fluorescence (B) and a composite of both fluorescent channels (C). (D-F) A projection of three 5 µm optical sections of a UAS-p35; UAS-cGFP embryo with the GFP fluorescence (D), VGAL fluorescence (E) and a composite of both fluorescent channels (F). (G-I) A projection of eight 5 µm optical sections of a UAS-p35/UAS-nGFP embryo with the GFP fluorescence (G), VGAL fluorescence (H) and a composite of both fluorescent channels (I). In the GFP channel, the contrast of several regions was adjusted to equalize the fluorescence of marked epidermal and the brain cells to show their location. In G-L, epidermal cells are outlined with a dashed line and labeled `E'; brain neurons are outlined with a dotted line and labeled `B'. (J-L) A projection of four 5 µm optical sections of a UAS-nGFP; H99 embryo with the GFP fluorescence (J), VGAL fluorescence (K) and a composite of both fluorescent channels (L).

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