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First published online 23 May 2007
doi: 10.1242/dev.02860

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The Drosophila homolog of the putative phosphatidylserine receptor functions to inhibit apoptosis

¹ Cutaneous Biology Research Center, Massachusetts General Hospital, 149 13th street, Charlestown, MA 02129, USA.
² Developmental and Cell Biology Department, University of California, Irvine, 5205 McGaugh Hall, Irvine, CA 92697, USA.

View larger version (27K): [in this window] [in a new window]   Fig. 1. Cell death in Drosophila embryos that lack dPSR. Embryos from flies containing previously identified genomic deletions were screened by Acridine Orange at 8-10 hours of development. (A) In yw control embryos, apoptotic cells were observed to cluster together because of their engulfment by hemocytes. The inset shows a higher magnification image to illustrate clustering. (B) A lack of apoptotic cell clustering was observed in embryos homozygous for Df(3R)5C1, ry506. (C) dPSR null embryos have clustered AO-stained cells. (D) The genomic region of dPSR is shown. The gray arrowhead indicates the location of the P-element insertion. The dashed lines indicate the region deleted in dPSRFM1. The translation start and stop sites are indicated. The Jmjc domain and the putative transmembrane domain are indicated in green and orange, respectively. Primers PSR1 and PSR12 were used for inverse PCR and sequencing of the breakpoint. The remaining dPSR sequence does contain an ATG; however, it is located at the 3' end of the JmjC homology and putative transmembrane regions.

View larger version (28K): [in this window] [in a new window]   Fig. 2. Macrophages of Drosophila PSRFM1 embryos are competent to engulf apoptotic cells. Macrophages are marked with GFP in embryos that are srp-hemo-GAL-4, UAS-src-EGFP or srp-hemo-GAL-4, UAS-src-EGFP; dPSRFM1 (lacking both maternal and zygotic dPSR). Embryos (8-10 hours) were fixed, and the red apoptotic nuclei were observed after staining with 10 µg/ml 7-aminoactinomycin D or TUNEL and visualized by confocal microscopy. (A) Wild-type macrophages (green) can be observed to contain engulfed TUNEL-labeled apoptotic cell nuclei (red). (B) Macrophages in homozygous dPSRFM1 embryos also contain many apoptotic nuclei. (C) Engulfment was quantified as the mean number of phagocytosed apoptotic nuclei per macrophage (phagocytic index). The mean and standard error are shown. At least five macrophages from eight age-matched embryos per genotype were analyzed (total macrophages analyzed were 65 for wild type and 68 for dPSRFM1). Homozygous dPSRFM1 embryos show a slight increase in apoptotic cell engulfment by macrophages, possibly owing to increased cell death.

View larger version (38K): [in this window] [in a new window]   Fig. 3. dPSR is localized in the nucleus of the cell in Drosophila. (A) Expression of ECFP in S2 cells. ECFP fills the cytoplasm of the cell. (B) dPSR was fused to the N-terminal of ECFP and expressed in S2 cells. (C) PSR:ECFP cells stained with Hoechst 33342 and (D) the overlay of PSR-ECFP and Hoechst 33342 visualized by fluorescence microscopy. The yellow arrows indicate identical cells in each of panels B,C,D. PSR-ECFP co-localizes with the DNA dye Hoechst 33342.

View larger version (88K): [in this window] [in a new window]   Fig. 4. Overexpression of dPSR results in phenotypes similar to hid and dronc null flies. Male flies of the genotype w; act5C-GAL4/+ (A), w; act5C-GAL4/UAS-dPSR (B), w; hidP05414/H99 (C), and dronc124/dronc129(D). The black lines above the fly indicate the alignment of the genitals. Rotated genitalia can be observed in flies that lack hid or dronc as well as those that overexpress dPSR ubiquitously. (E) The penetrance of the rotation defect was observed to be similar in w; hid05014/H99 compared to w; hid05014, dPSRFM1/H99, dPSRFM1 males (n=74 and 61 males, respectively). (The penetrance of rotation in act5C-GAL4/UAS-dPSR flies was 54%, n=642, and 100% for dronc124/dronc129 escapers, n=8.) (F-H) Expression of dPSR under control of the apterous driver resulted in wing phenotypes similar to those of hid and dronc mutants. (F) yw; apterous-GAL4/UAS-dPSR, (G) w; hidP05414/H99, and (H) dronc124/dronc129. The ballooned wing phenotype (G,H) was observed in 39% and a wavy wing phenotype (F) was observed in 54% of 59 apterous-GAL4/UAS-dPSR flies analyzed. The ballooned wing phenotype was observed in 100% of eight dronc124/dronc129 flies examined, and 100% of 20 hid flies.

View larger version (92K): [in this window] [in a new window]   Fig. 5. Expression of PSR suppresses hid- or grim-induced death in the eye. Drosophila eyes are shown of (A) w; GMR-GAL4/+: GMR-hid/+, (B) w; GMR-GAL4/UAS-dPSR; GMR-hid/+,(C) w; GMR-GAL4/+; UAS-grim/+ and (D) w: GMR-GAL4/UAS-dPSR; UAS-grim/+. GMR-Hid suppression by PSR was assessed in 20 randomly paired samples; average size increase by dPSR co-expression was 19% (P=0.0002 in paired t-test analysis).

View larger version (113K): [in this window] [in a new window]   Fig. 6. Loss of dPSR enhances Rpr, Hid and Grim killing in the Drosophila eye. Male eyes are shown for (A) CyO, 2 copies GMR-rpr/+, (B) CyO, 2 copies GMR-rpr/+; PSRFM1/+, (C) CyO, 2 copies GMR-rpr/+; PSRFM1/PSRFM1, (D) SM1, GMR-hid/+, (E) SM1, GMR-hid/+; PSRFM1/+, (F) SM1, GMR-hid/+; PSRFM1/PSRFM1, (G) w; GMR-grim/+, (H) w; GMR-grim/+; PSRFM1/+ and (I) w; GMR-grim/+; PSRFM1/PSRFM1. Removing one copy of dPSR (B,E,H) enhances the small or rough phenotype caused by expressing cell death inducers in the eye. Removing both copies of dPSR (C,F,I) further enhances the rough eye phenotype.

View larger version (119K): [in this window] [in a new window]   Fig. 7. Changes in dPSR levels can alter the number of interommatidial cells late in pupal eye development. Drosophila eye discs were dissected and stained with an antibody to Discs large to visualize the membranes of the cone cells and primary pigment cells and the hexagonal lattice of secondary and tertiary pigment cells and bristle cells. Fifty hour APF eye discs from (A) yw control or (B,C) dPSRFM1. dPSRFM1 mutant discs show ommatidial fusions and disorganization, indicated by arrows. (D) dPSRFM1 at 40 hour APF appear normal, indicating that the defects seen at 50 hours are likely to be due to cell loss. Apoptosis of interommatidial cells was visualized by TUNEL. (E,F) TUNEL-labeled nuclei (red) are seen at 49 hours APF in dPSRFM1 eye discs (F), but not in yw discs (E). Lack of hid suppresses the dPSRFM1 phenotype as observed in w; hid05014, dPSRFM1/H99, dPSRFM1 (G), and results in extra interommitidial cells, indicated by arrows, similar to w; hid05014/H99 eyes that lack hid alone (H). Overexpression of dPSR results in extra interommatidial cells. These are indicated with arrows in w; GMR-GAL4/UAS-dPSR (I).

View larger version (12K): [in this window] [in a new window]   Fig. 8. The dPSR-induced genital rotation defect is suppressed through the JNK pathway in Drosophila. (A) The penetrance of the rotation defect caused by expression of dPSR was suppressed by removing one copy of puc observed in w; UAS-dPSR/act5c-GAL4 or w; UAS-dPSR/act5c-GAL4; pucE69lacZ/+ flies (observed percentage of 642 and 496 males analyzed, respectively). (B) The pathway through which dPSR may act to inhibit cell death.