Skip to main page content
U.S. flag

An official website of the United States government

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2012 Aug 9;488(7410):226-30.
doi: 10.1038/nature11240.

Programmed elimination of cells by caspase-independent cell extrusion in C. elegans

Daniel P Denning  1 , Victoria Hatch, H Robert Horvitz
Affiliations

Programmed elimination of cells by caspase-independent cell extrusion in C. elegans

Daniel P Denning et al. Nature. .

Abstract

The elimination of unnecessary or defective cells from metazoans occurs during normal development and tissue homeostasis, as well as in response to infection or cellular damage. Although many cells are removed through caspase-mediated apoptosis followed by phagocytosis by engulfing cells, other mechanisms of cell elimination occur, including the extrusion of cells from epithelia through a poorly understood, possibly caspase-independent, process. Here we identify a mechanism of cell extrusion that is caspase independent and that can eliminate a subset of the Caenorhabditis elegans cells programmed to die during embryonic development. In wild-type animals, these cells die soon after their generation through caspase-mediated apoptosis. However, in mutants lacking all four C. elegans caspase genes, these cells are eliminated by being extruded from the developing embryo into the extra-embryonic space of the egg. The shed cells show apoptosis-like cytological and morphological characteristics, indicating that apoptosis can occur in the absence of caspases in C. elegans. We describe a kinase pathway required for cell extrusion involving PAR-4, STRD-1 and MOP-25.1/-25.2, the C. elegans homologues of the mammalian tumour-suppressor kinase LKB1 and its binding partners STRADα and MO25α. The AMPK-related kinase PIG-1, a possible target of the PAR-4–STRD-1–MOP-25 kinase complex, is also required for cell shedding. PIG-1 promotes shed-cell detachment by preventing the cell-surface expression of cell-adhesion molecules. Our findings reveal a mechanism for apoptotic cell elimination that is fundamentally distinct from that of canonical programmed cell death.

PubMed Disclaimer

Figures

Figure 1
Figure 1
Cells with apoptotic morphology are shed from C. elegans embryos lacking caspase activity. (a, b) (a) Low and (b) high magnification differential interference contrast (DIC) images of a ced-3(n3692) egg with a cluster of six cells that detached from the embryo. Asterisks, individual shed cells in (b). (c) Quantification of shed cells or floaters in mutants defective in programmed cell death and/or corpse engulfment. Error bars, s.d. (d) A ced-5(n1812) egg with two floaters. (e) A csp-3; csp-1; csp-2 ced-3 egg with a cluster of five shed cells. (f, g) Transmission electron micrographs of shed cells from (f) ced-3(n717) and (g) ced-5(n1812) embryos. Arrows, shed cells; arrowhead, an embryonic cell in (g); n, nucleus. (h) The phosphatidylserine binding protein MFG-e8 expressed from the transgene nIs398[Pdyn-1::mfge8::Venus] associates with the surface of csp-Δ shed cells. (i) The shed cells of csp-Δ; nuc-1(e1392) eggs are TUNEL-reactive. Scale bars, 10 µm.
Figure 2
Figure 2
The cells that are shed from ced-3 embryos are normally fated to die early during wild-type embryogenesis. (a, b) DIC micrographs of ced-3(n3692) embryos showing (a) ABplpappap and (b) ABaraaaapp 5 min after generation and shortly after shedding from the embryo (85 and 70 min later, respectively); vp, ventral pocket; asd, anterior sensory depression. (c) Cells that can be shed from ced-3(n3692) embryos, their locations when extruded, and the timings of their deaths in wild-type embryos. (d) DIC and fluorescence micrographs of shed cells from a ced-3(n717) embryo containing the nIs342[Pegl-1::gfp] transgene, which expresses GFP from the egl-1 promoter. Scale bars, 10 µm.
Figure 3
Figure 3
The LKB1 homolog PAR-4 and the AMPK-related kinase PIG-1 are required for cell shedding from ced-3 embryos. (a) The sublineage that produces the shed cell ABplpappap, which is the lineal aunt of the neuron RMEV and the excretory cell (exc cell). (b, c) Merged DIC and fluorescence micrographs of (b) wild-type and (c) pig-1(gm344) ced-3(n3692) larvae containing the transgene nIs434[Ppgp-12::gfp], which expresses GFP in the excretory cell. Arrowheads, excretory and ectopic excretory-like cells. (d) Percentage of L3 larvae with ectopic excretory cells. (e) The head of a larval pig-1(gm344) ced-3(n3692) animal containing large cysts (asterisks). (f, g) The fate of the cell ABplpappap in pig-1(gm344) and pig-1(gm344) ced-3(n3692) embryos. (f) ABplpappap in a pig-1 embryo shown 5 min after its generation and shortly after it underwent programmed cell death 45 min later. (g) ABplpappap in a pig-1 ced-3 embryo shown 5 min after its generation and immediately after it divided 115 min later. (h) pig-1 and strd-1 are required for cell shedding. Mutation of pig-1 of strd-1 reduced the number of shed cells in ced-3, ced-4 or ced-9(gf) eggs. Error bars, s.d.; asterisk, p < 5×10−7 (Student’s t-test). (i) The T-Loop threonine (T169) of PIG-1 is required for the elimination of ABplpappap. Average percentage of larvae with ectopic excretory cells from multiple pig-1(gm344) ced-3(n3692) lines carrying the following pig-1 transgenes: pig-1(wt), the wild-type pig-1 genomic locus (three lines, n = 42, 47 and 58); pig-1(null), two STOP codons in the first exon (three lines, n = 40, 43 and 45); pig-1(T169A), threonine 169 changed to alanine (three lines, n = 40, 41 and 48) ; and, pig-1(T169D), threonine 169 changed to aspartic acid (five lines, n = 31, 32, 43, 48 and 50); error bars, s.e.m.; asterisk, p < 10−3; n.s., p > 0.05 (Student’s t-test). (j, k, l) Merged DIC and fluorescence micrographs of (j) ced-3(n3692); par-4(RNAi), (k) ced-3(n3692); strd-1(RNAi), and (l) mop-25.2(ok2073); ced-3(n3692); mop-25.1(RNAi) larvae carrying nIs434[Ppgp-12::gfp]. Arrowheads, excretory and ectopic excretory-like cells. (m) Redundant pathways mediate the elimination of ABplpappap and other cells shed from ced-3 embryos. Scale bars, 10 µm.
Figure 4
Figure 4
Shed cells lack cell-adhesion molecules that are inappropriately expressed in pig-1 mutants, possibly because of impaired endocytosis. (a, b, c) Merged DIC and confocal fluorescence micrographs of shed cells from (a, b) ced-4(n1162); jcIs1[ajm-1::gfp] and (c) ced-3(n3692); jcIs17[hmp-1::gfp] eggs. Arrowheads, detached shed cells. (d, e, f, g) DIC and epifluorescence micrographs of (d, e) ced-3(n3692); jcIs17[hmp-1::gfp] and (f, g) pig-1(gm344) ced-3(n3692); jcIs17[hmp-1::gfp] embryos just prior to the completion of ventral enclosure. Black arrowheads, ABplpappap (d, e) or its descendant (f, g); white arrowhead, excretory cell (ABplpappaap); insets, magnification of ABplpappap or the excretory cell and the ABplpappap descendant in (f, g). (h) Number of persistent cell corpses in wild-type, pig-1(gm344), strd-1(ok2283) and strd-1(ok2283); pig-1(gm344) embryos. n values are provided in Supplemental Table S11; error bars, s.e.m.; asterisk, p < 0.002 for pair-wise comparison with the wild type (Student’s t-test). (i) Time required for engulfment and degradation of ABplpappap cell corpses in wild-type, pig-1(gm344) or strd-1(ok2283) embryos. (j) DIC and fluorescence micrographs of cell corpses from “bean” stage pig-1(gm344) embryos either carrying the nIs400[Pced-1::ced-1ΔC::gfp] transgene or stained with acridine orange. (k) Percentage of L3 larvae with ectopic excretory cells.
See this image and copyright information in PMC

Comment in

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Fuchs Y, Steller H. Programmed cell death in animal development and disease. Cell. 2011;147:742–758. - PMC - PubMed
    1. Reddien PW, Horvitz HR. The engulfment process of programmed cell death in Caenorhabditis elegans. Annu. Rev. Cell Dev. Biol. 2004;20:193–221. - PubMed
    1. Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Genes Dev. 2010;24:2592–2602. - PMC - PubMed
    1. Rosenblatt J, Raff MC, Cramer LP. An epithelial cell destined for apoptosis signals its neighbors to extrude it by an actin- and myosin-dependent mechanism. Curr. Biol. 2001;11:1847–1857. - PubMed
    1. Ellis HM, Horvitz HR. Genetic control of programmed cell death in the nematode C. elegans. Cell. 1986;44:817–829. - PubMed

Methods References

    1. Brenner S. The genetics of Caenorhabditis elegans. Genetics. 1974;77:71–94. - PMC - PubMed
    1. Hirose T, Galvin BD, Horvitz HR. Six and Eya promote apoptosis through direct transcriptional activation of the proapoptotic BH3-only gene egl-1 in Caenorhabditis elegans. Proc. Natl. Acad. Sci. U.S.A. 2010;107:15479–15484. - PMC - PubMed
    1. Venegas V, Zhou Z. Two alternative mechanisms that regulate the presentation of apoptotic cell engulfment signal in Caenorhabditis elegans. Mol. Biol. Cell. 2007;18:3180–3192. - PMC - PubMed
    1. Schwartz HT. A protocol describing pharynx counts and a review of other assays of apoptotic cell death in the nematode worm Caenorhabditis elegans. Nat Protoc. 2007;2:705–714. - PubMed
    1. Fraser AG, et al. Functional genomic analysis of C. elegans chromosome I by systematic RNA interference. Nature. 2000;408:325–330. - PubMed

Publication types

MeSH terms