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The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Van Goethem E, Silva EA, Xiao H, Franc NC - PLoS ONE (2012)

Bottom Line: As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively.However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes.However, we have not found any genetic interaction between Dmel\ced-12 and simu.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Anatomy and Developmental Biology Department, University College London, London, United Kingdom.

ABSTRACT
Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

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Related in: MedlinePlus

Phenotypic characterization of all three deficiencies and the prd[8] -allele.In A–L embryos were aged to stage 13, fixed, their macrophages immunostained with the CRQ Ab (green) and apoptotic corpses detected with 7-AAD staining (red). Confocal images of twelve focal plans taken through macrophages were merged and projected. A–C are 20× lateral views of a wild-type embryo (A), Df(2L)prd1.7 (B) and Df(2L)Prl (C) homozygous mutant embryos. Homozygous embryos of both Df(2L)prd1.7 (B) and Df(2L)Prl (C) deficiencies show only 6 out of the 12 segments normally observed in wild-type embryos (A), as highlighted by dotted white lines. D–F are respective magnified views of macrophages found in the head regions of these embryos, In both homozygous Df(2L)prd1.7 (E) and Df(2L)Prl (F) mutants, macrophages are able of efficiently engulf multiple apoptotic corpses. Scale bars in panels D–F are 5 µm. G–I are 20× lateral views of a wild-type embryo (G), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos. As expected the deletion of the paired gene causes a segmentation defect in both Df(2L)Esc-P3-0 and prd- mutant embryos. As seen in Df(2L)prd1.7 (B) and Df(2L)Prl (C), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos also have only 6 out of the 12 segments normally observed in wild-type embryos (A and G) due to their loss of prd function. J–L are higher magnification views of macrophages within the head of Df(2L)Esc-P3-0 and prd[8] homozygous mutant embryos, respectively. Mutant macrophages in both Df(2L)Esc-P3-0 (K) and prd- (L) homozygous embryos efficiently engulf multiple apoptotic corpses, with prd- macrophages occasionally engulfing up to 12 corpses. Scale bars in panels J–L are 5 µm. Phagocytic indices for wild-type, the deficiencies, and the prd- mutant embryos have been quantified and are summarised in a graph in M. PIs calculated from three image-stacks taken from 5 to 15 embryos of each genotype were normalized against wild type (with wild-type relative PI set as 100%) and are presented as relative PIs ± standard error of the mean (SEM) for each genotype. In D–F, and J–L, dotted white circles are indicative of individual macrophage cell bodies based on 7-AAD staining of their regular nuclei and CRQ staining.
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pone-0031488-g002: Phenotypic characterization of all three deficiencies and the prd[8] -allele.In A–L embryos were aged to stage 13, fixed, their macrophages immunostained with the CRQ Ab (green) and apoptotic corpses detected with 7-AAD staining (red). Confocal images of twelve focal plans taken through macrophages were merged and projected. A–C are 20× lateral views of a wild-type embryo (A), Df(2L)prd1.7 (B) and Df(2L)Prl (C) homozygous mutant embryos. Homozygous embryos of both Df(2L)prd1.7 (B) and Df(2L)Prl (C) deficiencies show only 6 out of the 12 segments normally observed in wild-type embryos (A), as highlighted by dotted white lines. D–F are respective magnified views of macrophages found in the head regions of these embryos, In both homozygous Df(2L)prd1.7 (E) and Df(2L)Prl (F) mutants, macrophages are able of efficiently engulf multiple apoptotic corpses. Scale bars in panels D–F are 5 µm. G–I are 20× lateral views of a wild-type embryo (G), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos. As expected the deletion of the paired gene causes a segmentation defect in both Df(2L)Esc-P3-0 and prd- mutant embryos. As seen in Df(2L)prd1.7 (B) and Df(2L)Prl (C), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos also have only 6 out of the 12 segments normally observed in wild-type embryos (A and G) due to their loss of prd function. J–L are higher magnification views of macrophages within the head of Df(2L)Esc-P3-0 and prd[8] homozygous mutant embryos, respectively. Mutant macrophages in both Df(2L)Esc-P3-0 (K) and prd- (L) homozygous embryos efficiently engulf multiple apoptotic corpses, with prd- macrophages occasionally engulfing up to 12 corpses. Scale bars in panels J–L are 5 µm. Phagocytic indices for wild-type, the deficiencies, and the prd- mutant embryos have been quantified and are summarised in a graph in M. PIs calculated from three image-stacks taken from 5 to 15 embryos of each genotype were normalized against wild type (with wild-type relative PI set as 100%) and are presented as relative PIs ± standard error of the mean (SEM) for each genotype. In D–F, and J–L, dotted white circles are indicative of individual macrophage cell bodies based on 7-AAD staining of their regular nuclei and CRQ staining.

Mentions: To further characterize the phenotype of the deficiencies, we stained whole-mount homozygous deficient-embryos with the CRQ antibody (CRQ Ab), which labels all embryonic macrophages, and 7-amino actinomycin-D (7-AAD), which brightly stains apoptotic nuclei [34]. 7-AAD also stains the nuclei of all cells of the embryo, including that of macrophages, thus allowing us to monitor their ability to engulf apoptotic cells by confocal microscopy by counting the number of macrophages, as well as the number of apoptotic cells they engulf to establish their phagocytic index (PI) (i.e. the mean number of apoptotic cells per macrophage)[34]. As expected, we observed that homozygous embryos for both Df(2L)prd1.7 and Df(2L)Prl deficiencies had segmentation defects that resulted in elevated levels of apoptosis in the region of the missing segments (compare figures 2B and C with wild-type in figure 2A). The CRQ immunostaining confirmed the presence of macrophages that migrated properly around the brain lobes in the head of wild-type (supplementary figures S1A and S1D), as well as those of Df(2L)prd1.7 and Df(2L)Prl homozygous embryos (supplementary figures S1B and S1C, respectively). In wild-type embryos, individual macrophages were able to engulf up to 4 corpses (figure 2D), with a mean number of apoptotic cells per macrophage, or phagocytic index (PI) of 1.89±0.06 (normalized to 100%±3.3 in figure 2M). Surprisingly, and seemingly in contrast with our previous observation that AO-stained apoptotic cells appeared to mostly fail to cluster in these embryos, arguing that their macrophages may be phagocytosis-defective, we observed that Df(2L)prd1.7 and Df(2L)Prl homozygous macrophages were able to efficiently engulf multiple corpses (figures 2E and F, respectively). When compared to wild type macrophages, for which we normalized the PI to 100%, Df(2L)prd1.7 and Df(2L)Prl homozygous macrophages showed a 58±6 and 55±5% increase in their relative PIs, respectively (p values<0.0001)(figure 2M). As in Df(2L)prd1.7 and Df(2L)Prl, homozygous embryos for Df(2L)esc-P3-0, a third overlapping Dmel\ced-12 deletion, also had a severe segmentation defect associated with the loss of prd, with only six segments present (figure 2H). Df(2L)esc-P3-0 homozygous macrophages also did not appear to show any major defect in migration (Supplementary figure S1E) or phagocytosis of apoptotic cells (compare figure 2K with wild type macrophages in2J), as they engulfed more apoptotic cells than wild-type macrophages with an 80.5±10% increase in PI (p value<0.0001)(figure 2M).


The Drosophila TRPP cation channel, PKD2 and Dmel/Ced-12 act in genetically distinct pathways during apoptotic cell clearance.

Van Goethem E, Silva EA, Xiao H, Franc NC - PLoS ONE (2012)

Phenotypic characterization of all three deficiencies and the prd[8] -allele.In A–L embryos were aged to stage 13, fixed, their macrophages immunostained with the CRQ Ab (green) and apoptotic corpses detected with 7-AAD staining (red). Confocal images of twelve focal plans taken through macrophages were merged and projected. A–C are 20× lateral views of a wild-type embryo (A), Df(2L)prd1.7 (B) and Df(2L)Prl (C) homozygous mutant embryos. Homozygous embryos of both Df(2L)prd1.7 (B) and Df(2L)Prl (C) deficiencies show only 6 out of the 12 segments normally observed in wild-type embryos (A), as highlighted by dotted white lines. D–F are respective magnified views of macrophages found in the head regions of these embryos, In both homozygous Df(2L)prd1.7 (E) and Df(2L)Prl (F) mutants, macrophages are able of efficiently engulf multiple apoptotic corpses. Scale bars in panels D–F are 5 µm. G–I are 20× lateral views of a wild-type embryo (G), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos. As expected the deletion of the paired gene causes a segmentation defect in both Df(2L)Esc-P3-0 and prd- mutant embryos. As seen in Df(2L)prd1.7 (B) and Df(2L)Prl (C), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos also have only 6 out of the 12 segments normally observed in wild-type embryos (A and G) due to their loss of prd function. J–L are higher magnification views of macrophages within the head of Df(2L)Esc-P3-0 and prd[8] homozygous mutant embryos, respectively. Mutant macrophages in both Df(2L)Esc-P3-0 (K) and prd- (L) homozygous embryos efficiently engulf multiple apoptotic corpses, with prd- macrophages occasionally engulfing up to 12 corpses. Scale bars in panels J–L are 5 µm. Phagocytic indices for wild-type, the deficiencies, and the prd- mutant embryos have been quantified and are summarised in a graph in M. PIs calculated from three image-stacks taken from 5 to 15 embryos of each genotype were normalized against wild type (with wild-type relative PI set as 100%) and are presented as relative PIs ± standard error of the mean (SEM) for each genotype. In D–F, and J–L, dotted white circles are indicative of individual macrophage cell bodies based on 7-AAD staining of their regular nuclei and CRQ staining.
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Related In: Results  -  Collection

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pone-0031488-g002: Phenotypic characterization of all three deficiencies and the prd[8] -allele.In A–L embryos were aged to stage 13, fixed, their macrophages immunostained with the CRQ Ab (green) and apoptotic corpses detected with 7-AAD staining (red). Confocal images of twelve focal plans taken through macrophages were merged and projected. A–C are 20× lateral views of a wild-type embryo (A), Df(2L)prd1.7 (B) and Df(2L)Prl (C) homozygous mutant embryos. Homozygous embryos of both Df(2L)prd1.7 (B) and Df(2L)Prl (C) deficiencies show only 6 out of the 12 segments normally observed in wild-type embryos (A), as highlighted by dotted white lines. D–F are respective magnified views of macrophages found in the head regions of these embryos, In both homozygous Df(2L)prd1.7 (E) and Df(2L)Prl (F) mutants, macrophages are able of efficiently engulf multiple apoptotic corpses. Scale bars in panels D–F are 5 µm. G–I are 20× lateral views of a wild-type embryo (G), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos. As expected the deletion of the paired gene causes a segmentation defect in both Df(2L)Esc-P3-0 and prd- mutant embryos. As seen in Df(2L)prd1.7 (B) and Df(2L)Prl (C), Df(2L)Esc-P3-0 (H) and prd[8] (I) homozygous mutant embryos also have only 6 out of the 12 segments normally observed in wild-type embryos (A and G) due to their loss of prd function. J–L are higher magnification views of macrophages within the head of Df(2L)Esc-P3-0 and prd[8] homozygous mutant embryos, respectively. Mutant macrophages in both Df(2L)Esc-P3-0 (K) and prd- (L) homozygous embryos efficiently engulf multiple apoptotic corpses, with prd- macrophages occasionally engulfing up to 12 corpses. Scale bars in panels J–L are 5 µm. Phagocytic indices for wild-type, the deficiencies, and the prd- mutant embryos have been quantified and are summarised in a graph in M. PIs calculated from three image-stacks taken from 5 to 15 embryos of each genotype were normalized against wild type (with wild-type relative PI set as 100%) and are presented as relative PIs ± standard error of the mean (SEM) for each genotype. In D–F, and J–L, dotted white circles are indicative of individual macrophage cell bodies based on 7-AAD staining of their regular nuclei and CRQ staining.
Mentions: To further characterize the phenotype of the deficiencies, we stained whole-mount homozygous deficient-embryos with the CRQ antibody (CRQ Ab), which labels all embryonic macrophages, and 7-amino actinomycin-D (7-AAD), which brightly stains apoptotic nuclei [34]. 7-AAD also stains the nuclei of all cells of the embryo, including that of macrophages, thus allowing us to monitor their ability to engulf apoptotic cells by confocal microscopy by counting the number of macrophages, as well as the number of apoptotic cells they engulf to establish their phagocytic index (PI) (i.e. the mean number of apoptotic cells per macrophage)[34]. As expected, we observed that homozygous embryos for both Df(2L)prd1.7 and Df(2L)Prl deficiencies had segmentation defects that resulted in elevated levels of apoptosis in the region of the missing segments (compare figures 2B and C with wild-type in figure 2A). The CRQ immunostaining confirmed the presence of macrophages that migrated properly around the brain lobes in the head of wild-type (supplementary figures S1A and S1D), as well as those of Df(2L)prd1.7 and Df(2L)Prl homozygous embryos (supplementary figures S1B and S1C, respectively). In wild-type embryos, individual macrophages were able to engulf up to 4 corpses (figure 2D), with a mean number of apoptotic cells per macrophage, or phagocytic index (PI) of 1.89±0.06 (normalized to 100%±3.3 in figure 2M). Surprisingly, and seemingly in contrast with our previous observation that AO-stained apoptotic cells appeared to mostly fail to cluster in these embryos, arguing that their macrophages may be phagocytosis-defective, we observed that Df(2L)prd1.7 and Df(2L)Prl homozygous macrophages were able to efficiently engulf multiple corpses (figures 2E and F, respectively). When compared to wild type macrophages, for which we normalized the PI to 100%, Df(2L)prd1.7 and Df(2L)Prl homozygous macrophages showed a 58±6 and 55±5% increase in their relative PIs, respectively (p values<0.0001)(figure 2M). As in Df(2L)prd1.7 and Df(2L)Prl, homozygous embryos for Df(2L)esc-P3-0, a third overlapping Dmel\ced-12 deletion, also had a severe segmentation defect associated with the loss of prd, with only six segments present (figure 2H). Df(2L)esc-P3-0 homozygous macrophages also did not appear to show any major defect in migration (Supplementary figure S1E) or phagocytosis of apoptotic cells (compare figure 2K with wild type macrophages in2J), as they engulfed more apoptotic cells than wild-type macrophages with an 80.5±10% increase in PI (p value<0.0001)(figure 2M).

Bottom Line: As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively.However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes.However, we have not found any genetic interaction between Dmel\ced-12 and simu.

View Article: PubMed Central - PubMed

Affiliation: Medical Research Council Cell Biology Unit, MRC Laboratory for Molecular Cell Biology and Anatomy and Developmental Biology Department, University College London, London, United Kingdom.

ABSTRACT
Apoptosis, a genetically programmed cell death, allows for homeostasis and tissue remodelling during development of all multi-cellular organisms. Phagocytes swiftly recognize, engulf and digest apoptotic cells. Yet, to date the molecular mechanisms underlying this phagocytic process are still poorly understood. To delineate the molecular mechanisms of apoptotic cell clearance in Drosophila, we have carried out a deficiency screen and have identified three overlapping phagocytosis-defective mutants, which all delete the fly homologue of the ced-12 gene, known as Dmel\ced12. As anticipated, we have found that Dmel\ced-12 is required for apoptotic cell clearance, as for its C. elegans and mammalian homologues, ced-12 and elmo, respectively. However, the loss of Dmel\ced-12 did not solely account for the phenotypes of all three deficiencies, as zygotic mutations and germ line clones of Dmel\ced-12 exhibited weaker phenotypes. Using a nearby genetically interacting deficiency, we have found that the polycystic kidney disease 2 gene, pkd2, which encodes a member of the TRPP channel family, is also required for phagocytosis of apoptotic cells, thereby demonstrating a novel role for PKD2 in this process. We have also observed genetic interactions between pkd2, simu, drpr, rya-r44F, and retinophilin (rtp), also known as undertaker (uta), a gene encoding a MORN-repeat containing molecule, which we have recently found to be implicated in calcium homeostasis during phagocytosis. However, we have not found any genetic interaction between Dmel\ced-12 and simu. Based on these genetic interactions and recent reports demonstrating a role for the mammalian pkd-2 gene product in ER calcium release during store-operated calcium entry, we propose that PKD2 functions in the DRPR/RTP pathway to regulate calcium homeostasis during this process. Similarly to its C. elegans homologue, Dmel\Ced-12 appears to function in a genetically distinct pathway.

Show MeSH
Related in: MedlinePlus