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Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson H, Kumar S - J. Cell Biol. (2000)

Bottom Line: Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects.RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos.These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

View Article: PubMed Central - PubMed

Affiliation: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia.

ABSTRACT
Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

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In situ analysis of debcl expression during development. A debcl antisense RNA probe labeled with digoxygenin was used to detect debcl expression in situ. A, Uniform staining is evident in a stage 5 cellularized embryo. B, In germ band extended embryo (stage 11), staining is evident in the anterior (arrow) proctodeum and posterior midgut (arrowhead), which are regions that show higher levels of TUNEL positivity (F). C, A lateral view of a germ band retracted embryo (stage 14) showing staining in the gut, particularly in the anterior and posterior midgut (arrows) and staining in the head corresponding to tissues of the clypeolabrum (*) and of the pharynx (**). Staining is also observed in a segmental reiterated pattern (examples indicated by arrowheads), that may correspond to cells in the central and peripheral nervous system, which show positive TUNEL at this stage (G). D, A dorsal view of a stage 16 embryo showing staining in regions in the head and gut (arrow indicates a strong stripe of staining that occurs in the foregut–midgut junction). E, A lateral view of an embryo at stage 16, showing staining in regions of the gut (arrowhead indicates the foregut–midgut junction and arrow indicates the hindgut), and in tissues of the clypeolabrum (*) and pharynx (**). F, TUNEL of an embryo at stage 11, showing a higher level of TUNEL positive cells in the region of the anterior midgut (arrow) and the proctodeum, posterior midgut (arrowhead). G, TUNEL of a stage 14 embryo, showing TUNEL positive cells in a segmentally reiterated pattern in cells of the nervous system (examples indicated by arrowheads) and in the region of the clypeolabrum (*) and pharynx (**). H, TUNEL on a stage 16 embryo showing higher numbers of TUNEL positive cells in the gut (midgut indicated by the arrow, and hindgut indicated by the arrowhead), and in head. I, A stage 16 embryo hybridized with a control sense probe. J, Antisense probe on third instar larval brain lobes showing stronger staining in rows of cells in the region of the outer proliferative center of the brain hemispheres (indicated by arrows), a region that also labels with TUNEL (see Fig. 4 E). K, Antisense probe on a third instar larval eye-antennal disc showing weak staining. The arrowhead indicates the morphogenetic furrow, after which higher levels of staining are observed in some cells corresponding with the region where TUNEL positive cells are observed (see Fig. 4 G). L, Antisense probe on third instar larval salivary glands showing positivity in the duct (arrow). M, Sense control probe on third instar larval gut showing no staining. Also, sense controls on other larval tissues and adult ovaries showed no staining (data not shown). N, Antisense probe on late third instar larval gut showing high levels of staining. O, TUNEL of a late third instar larval gut showing most cells are positive at this stage. P, Antisense probe on ovaries showing high levels of debcl expression in the nurse cells (on the left) and in the oocyte (right) of stage 10a egg chambers. Lower levels of staining are observed subsequent to stage 10 (not shown).
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Figure 3: In situ analysis of debcl expression during development. A debcl antisense RNA probe labeled with digoxygenin was used to detect debcl expression in situ. A, Uniform staining is evident in a stage 5 cellularized embryo. B, In germ band extended embryo (stage 11), staining is evident in the anterior (arrow) proctodeum and posterior midgut (arrowhead), which are regions that show higher levels of TUNEL positivity (F). C, A lateral view of a germ band retracted embryo (stage 14) showing staining in the gut, particularly in the anterior and posterior midgut (arrows) and staining in the head corresponding to tissues of the clypeolabrum (*) and of the pharynx (**). Staining is also observed in a segmental reiterated pattern (examples indicated by arrowheads), that may correspond to cells in the central and peripheral nervous system, which show positive TUNEL at this stage (G). D, A dorsal view of a stage 16 embryo showing staining in regions in the head and gut (arrow indicates a strong stripe of staining that occurs in the foregut–midgut junction). E, A lateral view of an embryo at stage 16, showing staining in regions of the gut (arrowhead indicates the foregut–midgut junction and arrow indicates the hindgut), and in tissues of the clypeolabrum (*) and pharynx (**). F, TUNEL of an embryo at stage 11, showing a higher level of TUNEL positive cells in the region of the anterior midgut (arrow) and the proctodeum, posterior midgut (arrowhead). G, TUNEL of a stage 14 embryo, showing TUNEL positive cells in a segmentally reiterated pattern in cells of the nervous system (examples indicated by arrowheads) and in the region of the clypeolabrum (*) and pharynx (**). H, TUNEL on a stage 16 embryo showing higher numbers of TUNEL positive cells in the gut (midgut indicated by the arrow, and hindgut indicated by the arrowhead), and in head. I, A stage 16 embryo hybridized with a control sense probe. J, Antisense probe on third instar larval brain lobes showing stronger staining in rows of cells in the region of the outer proliferative center of the brain hemispheres (indicated by arrows), a region that also labels with TUNEL (see Fig. 4 E). K, Antisense probe on a third instar larval eye-antennal disc showing weak staining. The arrowhead indicates the morphogenetic furrow, after which higher levels of staining are observed in some cells corresponding with the region where TUNEL positive cells are observed (see Fig. 4 G). L, Antisense probe on third instar larval salivary glands showing positivity in the duct (arrow). M, Sense control probe on third instar larval gut showing no staining. Also, sense controls on other larval tissues and adult ovaries showed no staining (data not shown). N, Antisense probe on late third instar larval gut showing high levels of staining. O, TUNEL of a late third instar larval gut showing most cells are positive at this stage. P, Antisense probe on ovaries showing high levels of debcl expression in the nurse cells (on the left) and in the oocyte (right) of stage 10a egg chambers. Lower levels of staining are observed subsequent to stage 10 (not shown).

Mentions: Because of low expression of debcl, we used tyramide amplification after hybridization to detect debcl mRNA expression in situ (Fig. 3). In early embryos, debcl mRNA was present uniformly, but became more concentrated in the tissues of the gut later in embryogenesis (Fig. 3, A–D). The relatively high levels of debcl RNA in early embryos (Fig. 3 A; data not shown for precellularized embryos) are likely to be derived maternally, as zygotic transcription does not begin until stage 5. From stage 14 embryos, debcl mRNA was detected in regions in the head that correspond to the pharynx and clypeolabrum, where many TUNEL positive cells are detected (Fig. 3 G). Expression could also be detected in a segmentally reiterated pattern in stage 14 embryos (Fig. 3 C) that may correlate with the TUNEL positive cells that are detected in the nervous system at this stage (Fig. 3 G and 4 C). During 3rd instar larval development, debcl expression was detected in the brain lobes in the outer proliferative center (Fig. 3 J), in the posterior part of the eye imaginal disc (Fig. 3 K), and in the gut (Fig. 3 N) where TUNEL positive cells were clearly seen (Fig. 3 O and 4, E and G). debcl expression was also clearly evident in the salivary glands, particularly in the ducts (Fig. 3 L). Because of background staining problems in salivary glands, acridine orange staining instead of TUNEL was used to detect apoptotic cells in this tissue. Using this technique, no apoptotic cells were detected in 3rd instar salivary glands (Fig. 4 K), suggesting that debcl expression may precede cell death in this tissue. High levels of debcl expression was detected in the nurse cell compartment of stage 10a ovaries (Fig. 3 P), which undergo apoptosis at stage 10b (Foley and Cooley 1998). Thus, debcl expression late in embryogenesis, during larval development, and during oogenesis significantly correlates with tissues undergoing apoptosis.


Debcl, a proapoptotic Bcl-2 homologue, is a component of the Drosophila melanogaster cell death machinery.

Colussi PA, Quinn LM, Huang DC, Coombe M, Read SH, Richardson H, Kumar S - J. Cell Biol. (2000)

In situ analysis of debcl expression during development. A debcl antisense RNA probe labeled with digoxygenin was used to detect debcl expression in situ. A, Uniform staining is evident in a stage 5 cellularized embryo. B, In germ band extended embryo (stage 11), staining is evident in the anterior (arrow) proctodeum and posterior midgut (arrowhead), which are regions that show higher levels of TUNEL positivity (F). C, A lateral view of a germ band retracted embryo (stage 14) showing staining in the gut, particularly in the anterior and posterior midgut (arrows) and staining in the head corresponding to tissues of the clypeolabrum (*) and of the pharynx (**). Staining is also observed in a segmental reiterated pattern (examples indicated by arrowheads), that may correspond to cells in the central and peripheral nervous system, which show positive TUNEL at this stage (G). D, A dorsal view of a stage 16 embryo showing staining in regions in the head and gut (arrow indicates a strong stripe of staining that occurs in the foregut–midgut junction). E, A lateral view of an embryo at stage 16, showing staining in regions of the gut (arrowhead indicates the foregut–midgut junction and arrow indicates the hindgut), and in tissues of the clypeolabrum (*) and pharynx (**). F, TUNEL of an embryo at stage 11, showing a higher level of TUNEL positive cells in the region of the anterior midgut (arrow) and the proctodeum, posterior midgut (arrowhead). G, TUNEL of a stage 14 embryo, showing TUNEL positive cells in a segmentally reiterated pattern in cells of the nervous system (examples indicated by arrowheads) and in the region of the clypeolabrum (*) and pharynx (**). H, TUNEL on a stage 16 embryo showing higher numbers of TUNEL positive cells in the gut (midgut indicated by the arrow, and hindgut indicated by the arrowhead), and in head. I, A stage 16 embryo hybridized with a control sense probe. J, Antisense probe on third instar larval brain lobes showing stronger staining in rows of cells in the region of the outer proliferative center of the brain hemispheres (indicated by arrows), a region that also labels with TUNEL (see Fig. 4 E). K, Antisense probe on a third instar larval eye-antennal disc showing weak staining. The arrowhead indicates the morphogenetic furrow, after which higher levels of staining are observed in some cells corresponding with the region where TUNEL positive cells are observed (see Fig. 4 G). L, Antisense probe on third instar larval salivary glands showing positivity in the duct (arrow). M, Sense control probe on third instar larval gut showing no staining. Also, sense controls on other larval tissues and adult ovaries showed no staining (data not shown). N, Antisense probe on late third instar larval gut showing high levels of staining. O, TUNEL of a late third instar larval gut showing most cells are positive at this stage. P, Antisense probe on ovaries showing high levels of debcl expression in the nurse cells (on the left) and in the oocyte (right) of stage 10a egg chambers. Lower levels of staining are observed subsequent to stage 10 (not shown).
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Figure 3: In situ analysis of debcl expression during development. A debcl antisense RNA probe labeled with digoxygenin was used to detect debcl expression in situ. A, Uniform staining is evident in a stage 5 cellularized embryo. B, In germ band extended embryo (stage 11), staining is evident in the anterior (arrow) proctodeum and posterior midgut (arrowhead), which are regions that show higher levels of TUNEL positivity (F). C, A lateral view of a germ band retracted embryo (stage 14) showing staining in the gut, particularly in the anterior and posterior midgut (arrows) and staining in the head corresponding to tissues of the clypeolabrum (*) and of the pharynx (**). Staining is also observed in a segmental reiterated pattern (examples indicated by arrowheads), that may correspond to cells in the central and peripheral nervous system, which show positive TUNEL at this stage (G). D, A dorsal view of a stage 16 embryo showing staining in regions in the head and gut (arrow indicates a strong stripe of staining that occurs in the foregut–midgut junction). E, A lateral view of an embryo at stage 16, showing staining in regions of the gut (arrowhead indicates the foregut–midgut junction and arrow indicates the hindgut), and in tissues of the clypeolabrum (*) and pharynx (**). F, TUNEL of an embryo at stage 11, showing a higher level of TUNEL positive cells in the region of the anterior midgut (arrow) and the proctodeum, posterior midgut (arrowhead). G, TUNEL of a stage 14 embryo, showing TUNEL positive cells in a segmentally reiterated pattern in cells of the nervous system (examples indicated by arrowheads) and in the region of the clypeolabrum (*) and pharynx (**). H, TUNEL on a stage 16 embryo showing higher numbers of TUNEL positive cells in the gut (midgut indicated by the arrow, and hindgut indicated by the arrowhead), and in head. I, A stage 16 embryo hybridized with a control sense probe. J, Antisense probe on third instar larval brain lobes showing stronger staining in rows of cells in the region of the outer proliferative center of the brain hemispheres (indicated by arrows), a region that also labels with TUNEL (see Fig. 4 E). K, Antisense probe on a third instar larval eye-antennal disc showing weak staining. The arrowhead indicates the morphogenetic furrow, after which higher levels of staining are observed in some cells corresponding with the region where TUNEL positive cells are observed (see Fig. 4 G). L, Antisense probe on third instar larval salivary glands showing positivity in the duct (arrow). M, Sense control probe on third instar larval gut showing no staining. Also, sense controls on other larval tissues and adult ovaries showed no staining (data not shown). N, Antisense probe on late third instar larval gut showing high levels of staining. O, TUNEL of a late third instar larval gut showing most cells are positive at this stage. P, Antisense probe on ovaries showing high levels of debcl expression in the nurse cells (on the left) and in the oocyte (right) of stage 10a egg chambers. Lower levels of staining are observed subsequent to stage 10 (not shown).
Mentions: Because of low expression of debcl, we used tyramide amplification after hybridization to detect debcl mRNA expression in situ (Fig. 3). In early embryos, debcl mRNA was present uniformly, but became more concentrated in the tissues of the gut later in embryogenesis (Fig. 3, A–D). The relatively high levels of debcl RNA in early embryos (Fig. 3 A; data not shown for precellularized embryos) are likely to be derived maternally, as zygotic transcription does not begin until stage 5. From stage 14 embryos, debcl mRNA was detected in regions in the head that correspond to the pharynx and clypeolabrum, where many TUNEL positive cells are detected (Fig. 3 G). Expression could also be detected in a segmentally reiterated pattern in stage 14 embryos (Fig. 3 C) that may correlate with the TUNEL positive cells that are detected in the nervous system at this stage (Fig. 3 G and 4 C). During 3rd instar larval development, debcl expression was detected in the brain lobes in the outer proliferative center (Fig. 3 J), in the posterior part of the eye imaginal disc (Fig. 3 K), and in the gut (Fig. 3 N) where TUNEL positive cells were clearly seen (Fig. 3 O and 4, E and G). debcl expression was also clearly evident in the salivary glands, particularly in the ducts (Fig. 3 L). Because of background staining problems in salivary glands, acridine orange staining instead of TUNEL was used to detect apoptotic cells in this tissue. Using this technique, no apoptotic cells were detected in 3rd instar salivary glands (Fig. 4 K), suggesting that debcl expression may precede cell death in this tissue. High levels of debcl expression was detected in the nurse cell compartment of stage 10a ovaries (Fig. 3 P), which undergo apoptosis at stage 10b (Foley and Cooley 1998). Thus, debcl expression late in embryogenesis, during larval development, and during oogenesis significantly correlates with tissues undergoing apoptosis.

Bottom Line: Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects.RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos.These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

View Article: PubMed Central - PubMed

Affiliation: The Hanson Centre for Cancer Research, Institute of Medical and Veterinary Science, Adelaide, SA 5000, Australia.

ABSTRACT
Bcl-2 family of proteins are key regulators of apoptosis. Both proapoptotic and antiapoptotic members of this family are found in mammalian cells, but no such proteins have been described in insects. Here, we report the identification and characterization of Debcl, the first Bcl-2 homologue in Drosophila melanogaster. Structurally, Debcl is similar to Bax-like proapoptotic Bcl-2 family members. Ectopic expression of Debcl in cultured cells and in transgenic flies causes apoptosis, which is inhibited by coexpression of the baculovirus caspase inhibitor P35, indicating that Debcl is a proapoptotic protein that functions in a caspase-dependent manner. debcl expression correlates with developmental cell death in specific Drosophila tissues. We also show that debcl genetically interacts with diap1 and dark, and that debcl-mediated apoptosis is not affected by gene dosage of rpr, hid, and grim. Biochemically, Debcl can interact with several mammalian and viral prosurvival Bcl-2 family members, but not with the proapoptotic members, suggesting that it may regulate apoptosis by antagonizing prosurvival Bcl-2 proteins. RNA interference studies indicate that Debcl is required for developmental apoptosis in Drosophila embryos. These results suggest that the main components of the mammalian apoptosis machinery are conserved in insects.

Show MeSH
Related in: MedlinePlus