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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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TUNEL-staining pattern argue that mutant macrophages in all three dced-12-deficiencies fail to efficiently clear apoptotic cells.A–I are projected confocal images of stage 13 embryos subjected to TUNEL. In A is a wild-type embryo where labelled apoptotic cells are found throughout the embryo in small clusters reflecting their engulfment by macrophages. In B and C, a 20× and enlarged views of a prd[8]- mutant are shown where, as expected, TUNEL-labelled apoptotic cells are present at a higher level, with big clusters of labelled apoptotic cells seen that reflect their engulfment by prd[8] macrophages. In TUNEL of Df(2L)prd1.7 (D), Df(2L)Prl (E) and Df(2L)esc-P3-0 (F) homozygous embryos, a cell death pattern in stripes is observed along the segments, arguing that macrophages in these deficiencies are not able to clear apoptotic cells as efficiently as macrophages do in prd[8]- mutants. G–I are 40× magnified counterparts of D–F, where some clusters of labelled apoptotic cells can still be observed (arrows), although the majority of labelled apoptotic cells remain scattered around the embryo. Scale bars in panels G–I are 50 µm.
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pone-0031488-g003: TUNEL-staining pattern argue that mutant macrophages in all three dced-12-deficiencies fail to efficiently clear apoptotic cells.A–I are projected confocal images of stage 13 embryos subjected to TUNEL. In A is a wild-type embryo where labelled apoptotic cells are found throughout the embryo in small clusters reflecting their engulfment by macrophages. In B and C, a 20× and enlarged views of a prd[8]- mutant are shown where, as expected, TUNEL-labelled apoptotic cells are present at a higher level, with big clusters of labelled apoptotic cells seen that reflect their engulfment by prd[8] macrophages. In TUNEL of Df(2L)prd1.7 (D), Df(2L)Prl (E) and Df(2L)esc-P3-0 (F) homozygous embryos, a cell death pattern in stripes is observed along the segments, arguing that macrophages in these deficiencies are not able to clear apoptotic cells as efficiently as macrophages do in prd[8]- mutants. G–I are 40× magnified counterparts of D–F, where some clusters of labelled apoptotic cells can still be observed (arrows), although the majority of labelled apoptotic cells remain scattered around the embryo. Scale bars in panels G–I are 50 µm.

Mentions: Surprisingly, however, we found that the pattern of distribution of apoptotic cells in prd8 mutant embryos differed from that of each of the three deficiencies, as determined by Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP Nick End Labelling (TUNEL). Indeed, while all mutant embryos had increased levels of apoptosis when compared to wild-type embryos (compare figures 3B and 3D–F with figure 3A), prd8 mutant embryos appeared to have fewer apoptotic cells than each of the three deficiencies. More importantly, we observed that TUNEL-stained apoptotic cells in prd8 LOF were found in large clusters (figure 3C and inset). Instead, and as seen previously in their AO-staining (see figure 1), Df(2L)prd1.7, Df(2L)Prl and Df(2L)esc-P3-0 homozygous mutants had excessive amounts of apoptotic cells in a “zebra-like” distribution pattern (figure 3D, E and F, respectively). Thus, while some clustering of TUNEL-stained apoptotic cells could still be observed in these deficiency-mutant embryos (see inset in figure 3H), and while their PIs indicated that their macrophages were capable of engulfing multiple apoptotic cells, their patterns of distribution of AO or TUNEL stained-apoptotic cells argue that they are less efficient than prd8 macrophages in clearing the vast amount of apoptotic cells generated during defective segmentation.


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)

TUNEL-staining pattern argue that mutant macrophages in all three dced-12-deficiencies fail to efficiently clear apoptotic cells.A–I are projected confocal images of stage 13 embryos subjected to TUNEL. In A is a wild-type embryo where labelled apoptotic cells are found throughout the embryo in small clusters reflecting their engulfment by macrophages. In B and C, a 20× and enlarged views of a prd[8]- mutant are shown where, as expected, TUNEL-labelled apoptotic cells are present at a higher level, with big clusters of labelled apoptotic cells seen that reflect their engulfment by prd[8] macrophages. In TUNEL of Df(2L)prd1.7 (D), Df(2L)Prl (E) and Df(2L)esc-P3-0 (F) homozygous embryos, a cell death pattern in stripes is observed along the segments, arguing that macrophages in these deficiencies are not able to clear apoptotic cells as efficiently as macrophages do in prd[8]- mutants. G–I are 40× magnified counterparts of D–F, where some clusters of labelled apoptotic cells can still be observed (arrows), although the majority of labelled apoptotic cells remain scattered around the embryo. Scale bars in panels G–I are 50 µm.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC3275576&req=5

pone-0031488-g003: TUNEL-staining pattern argue that mutant macrophages in all three dced-12-deficiencies fail to efficiently clear apoptotic cells.A–I are projected confocal images of stage 13 embryos subjected to TUNEL. In A is a wild-type embryo where labelled apoptotic cells are found throughout the embryo in small clusters reflecting their engulfment by macrophages. In B and C, a 20× and enlarged views of a prd[8]- mutant are shown where, as expected, TUNEL-labelled apoptotic cells are present at a higher level, with big clusters of labelled apoptotic cells seen that reflect their engulfment by prd[8] macrophages. In TUNEL of Df(2L)prd1.7 (D), Df(2L)Prl (E) and Df(2L)esc-P3-0 (F) homozygous embryos, a cell death pattern in stripes is observed along the segments, arguing that macrophages in these deficiencies are not able to clear apoptotic cells as efficiently as macrophages do in prd[8]- mutants. G–I are 40× magnified counterparts of D–F, where some clusters of labelled apoptotic cells can still be observed (arrows), although the majority of labelled apoptotic cells remain scattered around the embryo. Scale bars in panels G–I are 50 µm.
Mentions: Surprisingly, however, we found that the pattern of distribution of apoptotic cells in prd8 mutant embryos differed from that of each of the three deficiencies, as determined by Terminal deoxyribonucleotide transferase (TdT)-mediated dUTP Nick End Labelling (TUNEL). Indeed, while all mutant embryos had increased levels of apoptosis when compared to wild-type embryos (compare figures 3B and 3D–F with figure 3A), prd8 mutant embryos appeared to have fewer apoptotic cells than each of the three deficiencies. More importantly, we observed that TUNEL-stained apoptotic cells in prd8 LOF were found in large clusters (figure 3C and inset). Instead, and as seen previously in their AO-staining (see figure 1), Df(2L)prd1.7, Df(2L)Prl and Df(2L)esc-P3-0 homozygous mutants had excessive amounts of apoptotic cells in a “zebra-like” distribution pattern (figure 3D, E and F, respectively). Thus, while some clustering of TUNEL-stained apoptotic cells could still be observed in these deficiency-mutant embryos (see inset in figure 3H), and while their PIs indicated that their macrophages were capable of engulfing multiple apoptotic cells, their patterns of distribution of AO or TUNEL stained-apoptotic cells argue that they are less efficient than prd8 macrophages in clearing the vast amount of apoptotic cells generated during defective segmentation.

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