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Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

Obata F, Tanaka S, Kashio S, Tsujimura H, Sato R, Miura M - BMC Biol. (2015)

Bottom Line: Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body.With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis.Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. obataf@mol.f.u-tokyo.ac.jp.

ABSTRACT

Background: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.

Results: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.

Conclusions: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

No MeSH data available.


Related in: MedlinePlus

In vivo cell ablation of peripheral or central nervous system by Cry1Aa injection. a Adult flies injected with Cry1Aa during third instar larval stage lost their bristles. Arrows in the left panel (negative control, without Neur-Gal4) indicate bristle positions (pDC, posterior Dorsocentral; aSC, anterior Scuteller; pSC, posteior Scuteller) and arrows in the right panel (Neur > CryR) indicate the presence (aSC) or absence (pDC, pSC) of bristles. b Survival curve of male flies with CryR expression throughout the whole body (da) or only in neurons (Elav) with and without Cry1Aa; n = 50 for each condition. c Confocal images of Propidium iodide (PI) staining of larval brain from Elav > CryR, with and without Cry1Aa injection. Brains were dissected 3 h post-injection and then stained with PI. A single focal plane from the middle and surface regions are shown. d Schematic view of selective cell ablation by the Cry1Aa/CryR system. Cry1Aa specifically induces cell swelling and necrosis in CryR-expressing cells by pore formation in the plasma membrane
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Fig7: In vivo cell ablation of peripheral or central nervous system by Cry1Aa injection. a Adult flies injected with Cry1Aa during third instar larval stage lost their bristles. Arrows in the left panel (negative control, without Neur-Gal4) indicate bristle positions (pDC, posterior Dorsocentral; aSC, anterior Scuteller; pSC, posteior Scuteller) and arrows in the right panel (Neur > CryR) indicate the presence (aSC) or absence (pDC, pSC) of bristles. b Survival curve of male flies with CryR expression throughout the whole body (da) or only in neurons (Elav) with and without Cry1Aa; n = 50 for each condition. c Confocal images of Propidium iodide (PI) staining of larval brain from Elav > CryR, with and without Cry1Aa injection. Brains were dissected 3 h post-injection and then stained with PI. A single focal plane from the middle and surface regions are shown. d Schematic view of selective cell ablation by the Cry1Aa/CryR system. Cry1Aa specifically induces cell swelling and necrosis in CryR-expressing cells by pore formation in the plasma membrane

Mentions: We also tested whether our system is applicable to the developmental study of sensory organs by injecting Cry1Aa toxin into Neur >CryR third-instar larvae. Neur-Gal4 is expressed in sensory organ precursors (SOPs) in wing discs that become adult bristles. Cry1Aa injection during late-third instar larvae (6–12 h before pupal formation) resulted in a loss of bristles from epithelia (Fig. 7a). Although not all bristles are lost, the loss of bristles is probably due to differences in developmental timing as some SOPs such as the anterior scuteller bristle, which arise during later stages of development (0–6 h before pupal formation). Indeed, ablated macrocheates such as posterior scuteller bristle, or posterior drosocentral bristle are generated in early to middle-third larval stage (12–30 h before pupal formation) [26]. This suggested that Cry1Aa kills cells rapidly upon injection into larvae, and that Cry1Aa can be inactivated or removed from the hemolymph within a relatively short period after injection. Therefore, the Cry1Aa/CryR system could be useful for spatiotemporal cell ablation. Furthermore, a lack of melanization in adult epithelia at the original SOP positions implied that dying SOPs are eventually eliminated from the tissue rather than remaining in the epithelial sheet.Fig. 7


Induction of rapid and selective cell necrosis in Drosophila using Bacillus thuringiensis Cry toxin and its silkworm receptor.

Obata F, Tanaka S, Kashio S, Tsujimura H, Sato R, Miura M - BMC Biol. (2015)

In vivo cell ablation of peripheral or central nervous system by Cry1Aa injection. a Adult flies injected with Cry1Aa during third instar larval stage lost their bristles. Arrows in the left panel (negative control, without Neur-Gal4) indicate bristle positions (pDC, posterior Dorsocentral; aSC, anterior Scuteller; pSC, posteior Scuteller) and arrows in the right panel (Neur > CryR) indicate the presence (aSC) or absence (pDC, pSC) of bristles. b Survival curve of male flies with CryR expression throughout the whole body (da) or only in neurons (Elav) with and without Cry1Aa; n = 50 for each condition. c Confocal images of Propidium iodide (PI) staining of larval brain from Elav > CryR, with and without Cry1Aa injection. Brains were dissected 3 h post-injection and then stained with PI. A single focal plane from the middle and surface regions are shown. d Schematic view of selective cell ablation by the Cry1Aa/CryR system. Cry1Aa specifically induces cell swelling and necrosis in CryR-expressing cells by pore formation in the plasma membrane
© Copyright Policy - open-access
Related In: Results  -  Collection

License 1 - License 2
Show All Figures
getmorefigures.php?uid=PMC4495774&req=5

Fig7: In vivo cell ablation of peripheral or central nervous system by Cry1Aa injection. a Adult flies injected with Cry1Aa during third instar larval stage lost their bristles. Arrows in the left panel (negative control, without Neur-Gal4) indicate bristle positions (pDC, posterior Dorsocentral; aSC, anterior Scuteller; pSC, posteior Scuteller) and arrows in the right panel (Neur > CryR) indicate the presence (aSC) or absence (pDC, pSC) of bristles. b Survival curve of male flies with CryR expression throughout the whole body (da) or only in neurons (Elav) with and without Cry1Aa; n = 50 for each condition. c Confocal images of Propidium iodide (PI) staining of larval brain from Elav > CryR, with and without Cry1Aa injection. Brains were dissected 3 h post-injection and then stained with PI. A single focal plane from the middle and surface regions are shown. d Schematic view of selective cell ablation by the Cry1Aa/CryR system. Cry1Aa specifically induces cell swelling and necrosis in CryR-expressing cells by pore formation in the plasma membrane
Mentions: We also tested whether our system is applicable to the developmental study of sensory organs by injecting Cry1Aa toxin into Neur >CryR third-instar larvae. Neur-Gal4 is expressed in sensory organ precursors (SOPs) in wing discs that become adult bristles. Cry1Aa injection during late-third instar larvae (6–12 h before pupal formation) resulted in a loss of bristles from epithelia (Fig. 7a). Although not all bristles are lost, the loss of bristles is probably due to differences in developmental timing as some SOPs such as the anterior scuteller bristle, which arise during later stages of development (0–6 h before pupal formation). Indeed, ablated macrocheates such as posterior scuteller bristle, or posterior drosocentral bristle are generated in early to middle-third larval stage (12–30 h before pupal formation) [26]. This suggested that Cry1Aa kills cells rapidly upon injection into larvae, and that Cry1Aa can be inactivated or removed from the hemolymph within a relatively short period after injection. Therefore, the Cry1Aa/CryR system could be useful for spatiotemporal cell ablation. Furthermore, a lack of melanization in adult epithelia at the original SOP positions implied that dying SOPs are eventually eliminated from the tissue rather than remaining in the epithelial sheet.Fig. 7

Bottom Line: Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body.With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis.Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

View Article: PubMed Central - PubMed

Affiliation: Department of Genetics, Graduate School of Pharmaceutical Sciences, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo, 113-0033, Japan. obataf@mol.f.u-tokyo.ac.jp.

ABSTRACT

Background: Genetic ablation of target cells is a powerful tool to study the origins and functions of cells, tissue regeneration, or pathophysiology in a human disease model in vivo. Several methods for selective cell ablation by inducing apoptosis have been established, using exogenous toxins or endogenous proapoptotic genes. However, their application is limited to cells with intact apoptotic machinery.

Results: Herein, we established a method for inducing rapid and selective cell necrosis by the pore-forming bacterial toxin Cry1Aa, which is specifically active in cells expressing the Cry1Aa receptor (CryR) derived from the silkworm Bombyx mori. We demonstrated that overexpressing CryR in Drosophila melanogaster tissues induced rapid cell death of CryR-expressing cells only, in the presence of Cry1Aa toxin. Cry/CryR system was effective against both proliferating cells in imaginal discs and polyploid postmitotic cells in the fat body. Live imaging analysis of cell ablation revealed swelling and subsequent osmotic lysis of CryR-positive cells after 30 min of incubation with Cry1Aa toxin. Osmotic cell lysis was still triggered when apoptosis, JNK activation, or autophagy was inhibited, suggesting that Cry1Aa-induced necrotic cell death occurred independently of these cellular signaling pathways. Injection of Cry1Aa into the body cavity resulted in specific ablation of CryR-expressing cells, indicating the usefulness of this method for in vivo cell ablation.

Conclusions: With Cry toxins from Bacillus thuringiensis, we developed a novel method for genetic induction of cell necrosis. Our system provides a "proteinous drill" for killing target cells through physical injury of the cell membrane, which can potentially be used to ablate any cell type in any organisms, even those that are resistant to apoptosis or JNK-dependent programmed cell death.

No MeSH data available.


Related in: MedlinePlus