Requirement for croquemort in phagocytosis of apoptotic cells in Drosophila

Science

Volumn 284, Issue 5422, 1999, Pages 1991-1994

  Franc, Nathalie C ^a   Heitzler, Pascal ^a   Ezekowitz, R Alan B ^a   White, Kristin ^a  

^a NONE

Author keywords

[No Author keywords available]

Indexed keywords

CD36 ANTIGEN;

ANIMAL CELL; ANIMAL TISSUE; ANTIBODY LABELING; APOPTOSIS; ARTICLE; DROSOPHILA; EMBRYO; IMMUNE RESPONSE; MACROPHAGE; MACROPHAGE FUNCTION; NONHUMAN; PHAGOCYTOSIS; PRIORITY JOURNAL; PROTEIN EXPRESSION;

ANIMALS; ANTIGENS, CD36; APOPTOSIS; CARRIER PROTEINS; DROSOPHILA; DROSOPHILA PROTEINS; EMBRYO, NONMAMMALIAN; ESCHERICHIA COLI; GENE DELETION; GENE EXPRESSION REGULATION, DEVELOPMENTAL; GENES, INSECT; ION PUMPS; MACROPHAGES; MEMBRANE PROTEINS; PHAGOCYTOSIS; PHENOTYPE; RECEPTORS, IMMUNOLOGIC; RECEPTORS, SCAVENGER; STAPHYLOCOCCUS AUREUS;

ANIMALIA;

EID: 0033581007     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.284.5422.1991     Document Type: Article

References (45)

1. J. Savill, Br. Med. Bull. 53, 491 (1997).
2. N. Platt, R. Da Silvia, S. Gordon, Trends Cell Biol. 8, 365 (1998).
3. D. T. Fearon and R. M. Locksley, Science 272, 50 (1996).
4. N. Franc, K. White, R. Ezekowitz, Curr. Opin. Immunol. 11, 47 (1999).
5. H. Suzuki et al., Nature 386, 292 (1997).
6. R. Jack et al., ibid. 389, 742 (1997).
7. Q. Liu and M. Hengartner, Cell 93, 961 (1998).
8. Y. Wu and H. Horvitz, Nature 392, 501 (1998).
9. _, Cell 93, 951-960 (1998).
10. U. Tepass, L. I. Fessier, A. Aziz, V. Hartenstein, Development 120, 1829 (1994).
11. N. C. Franc, J. L. Dimarcq, M. Lagueux, J. Hoffmann, R. A. Ezekowitz, Immunity 4, 431 (1996).
12. A. Nicholson, S. Frieda, A. Pearce, R. Silverstein, Arterioscler. Thromb. Vasc. Biol. 15, 269 (1995).
13. C. Erdemann et al., J. Biol. Chem. 268, 11811 (1993).
14. S. Nozaki et al., J. Clin. Invest. 96, 1859 (1995).
15. J. Savill, I. Dransfield, N. Hogg, C. Haslett, Nature 343, 170 (1990).
16. J. Savill, N. Hogg, Y. Ren, C. Haslett, J. Clin. Invest. 90, 1513 (1992).
17. Y. Ren, R. Silverstein, J. Allen, J. Savill, J. Exp. Med. 181, 1857 (1995).
18. S. W. Ryeom, J. R. Sparrow, R. L. Silverstein, J. Cell Sci. 109, 387 (1996).
19. K. Schneitz, P. Spjelmann, M. Noll, Genes Dev. 7, 114 (1993).
20. P. Heitzler, unpublished observations.
21. M. Boedigheimer and A. Laughon, Development 118, 1291 (1993).
22. L. Frank and C. Rushlow, ibid. 122, 1343 (1996).
23. Berkeley Drosophila Genome Project, unpublished data.
24. Single embryos were squished in 10 μl of 10 mM tris-HCl (pH8.0), 1 mM EDTA, and 25 mM NaCl containing Proteinase K (200 μg/ml; Boehringer Mannheim) and incubated at 37°C for 30 min, followed by 2 min at 95°C. The PCR was performed with 2.5 U of Taq polymerase and 100 ng of each primer. Two pairs of primers were designed to amplify 634 base pairs (bp) of the crq genomic sequence and 200 bp of the genomic region of the doom gene as an internal control [A. J. Harvey, A. P. Bidwaldi, L. K. Miller, Mol. Cell. Biol. 17, 2835 (1997)]. Crq-specific primers were 5′-TGCCACCGATGCTTGCAGAT-3′ and 5′-AGCCGAATATGATTCCGTACTG-3′. Doom-specific primers were 5′-AGGGTAAACGGCCACAGAATGT-3′ and 5′-GATATCGTTGTAGTTGGCCCG-3′. The PCR cycles were 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min for 30 cycles. In embryos from the W88 stock, 20 of 79 were missing the crg-specific band.
25. CRQ immunostaining was used to genotype each embryo. Peroxidasin immunostaining detected all hemocytes [R. E. Nelson et al., EMBO J. 13, 3438 (1994)]. The nuclear dye 7-AAD labeled all DNA and allowed for the identification of apoptotic corpses. Unless otherwise specified, stage 11 to 16 embryos were fixed with standard procedures (44). Fixed devitellinized embryos were incubated in phosphate-buffered saline (PBS), 0.0125% saponin, 1% bovine serum albumin, and 4% normal goat serum (PSN) for 1 hour at room temperature and then incubated with the primary antibodies at a 1:1000 dilution in PSN overnight at 4°C. After several washes in PBS, the embryos were incubated for 1 hour at room temperature with the following secondary antibodies: fluorescein isothiocyanate-conjugated goat antibody to mouse and Cy5-conjugated goat antibody to rabbit (Jackson Immunoresearch) used at a 1:1000 dilution in PSN. Finally, embryos were washed three times in PBS for 20 min and subsequently incubated with 7-AAD (5 μg/ml) in PBS for 30 min. Embryos were quickly washed twice in PBS, mounted in Vectashield (Vector), and viewed by confocal microscopy (Leica TCS NT 4D).
26. The efficiency of engulfment was quantified by counting the number of engulfed corpses per macrophage in at least five fields of four embryos of each genotype. A P.I., that is, the mean number of engulfed corpses per macrophage, was calculated for each embryo, and the mean P.I. was derived for each genotype.
27. C. Phelps and A. Brand, Methods 14, 367 (1998).
28. N. Franc and K. White, data not shown.
29. Stage 12 to 14 w, UAS-crq;hs-Gal4/+ and control w; hs-Gal4 embryos were heat-shocked for 1 hour at 39°C, aged for 2 hours at 25°C, and fixed and embedded in Spurr's resin (34). Serial 1-μm sections of two embryos of each genotype were stained with a solution of methylene, toluidine blue, and borax (44) and viewed by standard microscopy.
30. J. Pugin et al., Immunity 1, 509 (1994).
31. A. Devitt et al., Nature 392, 505 (1998).
32. 8 tetramethyl rhodamine isothiocyanate (TRITC)-labeled fluorescent E. coli (K-12 strain) or S. aureus (Wood strain) bioparticles (heat-killed bacteria; Molecular Probes) with standard microinjection procedures (44). After injection, embryos were kept in the dark at 18°C for 14 to 16 hours, incubated for 1 hour at 4°C, mounted, and viewed under Nomarski and fluorescence with a confocal microscope.
33. J. Abrams, A. Lux, H. Steller, M. Krieger, Proc. Natl. Acad. Sci. U.S.A. 89, 10375 (1992).
34. K. White et al., Science 264, 677 (1994).
35. M. E. Grether, J. M. Abrams, J. Agapite, K. White, H. Steller, Genes Dev. 9, 1694 (1995).
36. P. Chen, W. Nordstrom, B. Gish, J. M. Abrams, ibid. 10, 1773 (1996).
37. After CRQ immunostaining, the amount of fluorescence seen in five isolated macrophages of each genotype was quantified with a confocal microscope. For each macrophage, the fluorescence of serial sections of 0.5 μm was quantified from top to bottom of the cell. After subtracting the background fluorescence, the total amount of fluorescence within each macrophage was calculated.
38. M. Hortsch et al., Int. J. Dev. Biol. 42, 33 (1998).
39. J.-L Dimarcq et al., Insect Biochem. Mol. Biol. 27, 877 (1997).
40. E. Gateff et al., in Invertebrate Systems In Vitro, E. Kurstak, K. Maramorosch, A. Dubendorfer, Eds. (North-Holland, Elsevier, Amsterdam, 1980), pp. 517-533.
41. L. Nagy, P. Tontonoz, J. Alverez, H. Chen, R. Evans, Cell 93, 329 (1998).
42. A. Pearson, A. Lux, M. Krieger, Proc. Natl. Acad. Sci. U.S.A. 92, 4056 (1995).
43. V. Rodrigues, P. Cheah, K. Ray, W. Chia, EMBO J. 14, 3007 (1995).
44. M. Ashburner, Drosophila: A Laboratory Manual (Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, 1989).
45. We thank J. Fessier and M. Hortsch for providing antibodies; E. Noll, N. Perrimon, the Bloomington Stock Center, and L. Dobens for fly stocks; M. Krieger for the Dil AcLDLs; Y. Ge and W. Fowle for assistance with confocal microscopy and histology; the lab of T. Orr-Weaver for advice on nuclear dyes; I. Ando for suggesting the bacteria assay experiment; and J. Settleman and the members of the Ezekowitz and White laboratories for helpful comments on this work. This work was supported by grants from the Shiseido Company of Japan to Massachusetts General Hospital/Harvard Medical School (N.C.F. and K.W.), from NIH (K.W. and A.E.), and from the Human Frontiers in Science Program (A.E. and N.C.F.).