Search tips
Search criteria 


Logo of nihpaAbout Author manuscriptsSubmit a manuscriptHHS Public Access; Author Manuscript; Accepted for publication in peer reviewed journal;
Curr Biol. Author manuscript; available in PMC 2013 April 7.
Published in final edited form as:
PMCID: PMC3618969

Programmed Cell Death: A New Way Worms Get Rid of Unwanted Cells


The genetics and predictable cell death lineages in Caenorhabditis elegans have been critical for identifying a conserved apoptosis pathway. Yet, cells still die in mutants that disrupt this pathway. A recent study shows that this death occurs by cell shedding.

The simplified genetics of Caenorhabditis elegans was critical for elucidating the programmed cell death pathway conserved throughout most species [13]. Because these worms have a streamlined version of the conserved genetic pathway required for apoptosis, it had confused researchers for some time that, in certain instances, the apoptotic pathway is redundant [1]. A recent paper from Denning, Hatch, and Horvitz [4] demonstrates that cell shedding can compensate for the death pathway in a number of cells targeted to die by developmental programmed cell death. A number of C. elegans mutants in which apoptosis is inhibited at different stages of the pathway shed cells that then eventually die by a caspase-independent type of apoptosis.

One feature of C. elegans development that is critical for defining the apoptotic pathway is the ability to precisely predict which cells die, making it easy to identify those that do not. To investigate what controls cell shedding, the authors screened various mutants engineered to express GFP in cells that consistently shed when cell death is inhibited (i.e. in worms lacking the caspase CED-3). In cases where these GFP-positive cells do not shed, they instead divide, producing two GFP-positive cells. Thus, by screening for mutations that produced two GFP-positive cells, they found that cell shedding requires the genes PIG-1, a serine–threonine kinase related to AMP-activated kinase, and a complex that phosphorylates PIG-1, composed of LKB1, STADα and MO25α. By finding genes required for shedding, they discovered that the apoptotic and shedding pathways act redundantly. GFP-labeled cells would still die by programmed cell death in single ced-3 or pig-1 mutants, but in a double mutant lacking both the apoptotic and shedding pathways these cells instead divide and produce two cells of the same fate — in the example they studied, an excretory cell (Figure 1).

Figure 1
Cell death in developing C. elegans can occur through a canonical apoptosis pathway, cell shedding, or both.

Although these findings suggest that shedding can compensate to promote cell death in cases where the apoptotic pathway is blocked, another possibility is that normally these cells can both die and be shed. Because C. elegans has highly efficient phagocytosis mechanisms, shed cells that are also targeted for cell death may be engulfed so rapidly that they are not apparent. Indeed, mutations in engulfment genes also produce `floaters', suggesting that typically cells are shed but become engulfed so rapidly that they are not noticeable. Blocking the apoptotic pathway reveals shed cells because they persist longer in the embryo without triggering engulfment. It is not clear how the shed cells in ced-3 mutants eventually die; however, they may use a pathway similar to anoikis — a form of apoptosis triggered solely by loss of survival signaling [5]. This raises the question of whether shedding can compensate for apoptosis pathways in all cells programmed to die in the worm, or whether shedding only promotes death of cells derived from epithelial-like tissues, since anoikis has only been associated with epithelia [5].

The idea that the cells investigated in this study might die as a result of shedding is supported by recent findings that epithelia normally extrude cells prior to their death [6,7]. Eisenhoffer et al. [6] found that epithelial cells from a variety of tissues — human colon, developing zebrafish epidermis, and cell culture — all extrude or shed live cells, which later die by anoikis. In all cases observed, extrusions occurred at sites of high cell crowding. Further, experimentally crowding cultured epithelial monolayers in a mechanical device confirmed that crowding alone could induce extrusion of live cells. Live extruded cells, as in vivo, will later die but can also survive and proliferate if given a new substratum. Similarly, Marinari et al. [7] found that during Drosophila pupariation, developmental crowding forces drive live cells to extrude or `delaminate'. While blocking cell death had no impact on live cell extrusion, blocking cell growth elsewhere in the epithelium blocked extrusion, suggesting that cell proliferation produces the crowding forces that then drive extrusion.

By using the cell crowding device and zebrafish genetics, Eisenhoffer et al. [6] identified proteins that mediate live cell extrusion following crowding and determined that extrusion normally drives cell death in epithelia. Although triggering cell death can activate extrusion through the apoptotic pathway [6,8,9], live cell extrusion during homeostasis or following experimental cell crowding requires the stretch-activated channel Piezo-1 [6]. Blocking this channel leads to the formation of cell masses, indicating that live cell extrusion drives epithelial cell death. Denning et al. [4] also point out that mutations in genes that they found to be important for shedding results in greater epithelial defects than those arising from mutations in the apoptotic pathway [1012]. Therefore, cell shedding in C. elegans may also promote apoptosis normally.

Does cell shedding in C. elegans use the same mechanism described to control epithelial cell extrusion? Epithelial cell extrusion occurs within all epithelia during homeostasis or following apoptotic stimuli. The ability to readily observe extrusion in zebrafish epidermis and tissue culture epithelia has enabled dissection of the cytoskeletal mechanics that drive this process. For extrusion, a cell destined to die produces the lipid sphingosine 1-phosphate, which binds to its receptor in the surrounding epithelial cells, resulting in the formation and contraction of an intercellular actomyosin ring that squeezes the cell out from the epithelium [9,13]. Although the prominent muscles just beneath the epidermis make it difficult to follow actomyosin-based processes in the C. elegans epidermis, the Hardin lab has developed tools to follow ventral enclosure of the epidermis during C. elegans development [14,15]. Based on the similarity of dorsal closure in Drosophila melanogaster to ventral enclosure in C. elegans, it is tempting to think that the cells that are shed from the ventral pocket during ventral enclosure in the worm also extrude or delaminate, as they do from the amnioserosa during dorsal closure in Drosophila (Figure 2A).

Figure 2
Possible mechanisms governing cell shedding in C. elegans.

On the other hand, alternative mechanisms may drive cell shedding in C. elegans. While it is not clear whether all shed cells in C. elegans arise from epithelial-like cells, in the cases reported here, cells that are adjacent to shed cells appear to have epithelial adhesion proteins. However, cells shedding from the ventral pocket lack the cell adhesion proteins seen in their neighboring cells. This suggests that shedding occurs by a different mechanism from epithelial cell extrusion because extruding epithelial cells maintain contacts with their neighbors throughout the extrusion process (Figure 2A). Instead, a cell-sorting mechanism, similar to that seen when either oncogenic Ras or Src is induced within a monolayer, may drive cell shedding in C. elegans [16,17]. In these situations, cells with altered adhesion appear to exclude themselves from surrounding wild-type cells. The importance of the endocytic ARF GTPases in shedding [4,18] may indicate that shedding requires endocytosis of adhesion proteins. Loss of adhesion proteins in one cell could act to exclude it from surrounding cells to promote its shedding during C. elegans development (Figure 2B).

Alternatively, cell shedding could occur by asymmetric cell division. PIG-1 is a kinase that is required cell autonomously for many asymmetric neuroblast cell divisions in C. elegans [19,20]. One possibility is that cell shedding in these cases could result from asymmetric divisions during differentiation. The daughter cell that has divided and no longer maintains contacts with the surrounding epidermis could die from lack of attachment to the matrix or other cells (Figure 2C).

Future studies may determine the mechanism by which these cells in developing C. elegans shed and die. Yet, the findings by Denning et al. [4] suggest that a variety of species have evolved ways of removing unwanted cells that can substitute for programmed apoptotic pathways and may even work in concert with them.


1. Metzstein MM, Stanfield GM, Horvitz HR. Genetics of programmed cell death in C. elegans: past, present and future. Trends Genet. 1998;14:410–416. [PubMed]
2. Hengartner MO, Horvitz HR. Programmed cell death in Caenorhabditis elegans. Curr. Opin. Genet. Dev. 1994;4:581–586. [PubMed]
3. Ellis HM, Horvitz HR. Genetic control of programmed cell death in the nematode C. elegans. Cell. 1986;44:817–829. [PubMed]
4. Denning DP, Hatch V, Horvitz HR. Programmed elimination of cells by caspase-independent cell extrusion in C. elegans. Nature. 2012;488:226–230. [PMC free article] [PubMed]
5. Frisch SM, Vuori K, Ruoslahti E, Chan-Hui PY. Control of adhesion-dependent cell survival by focal adhesion kinase. J. Cell Biol. 1996;134:793–799. [PMC free article] [PubMed]
6. Eisenhoffer GT, Loftus PD, Yoshigi M, Otsuna H, Chien CB, Morcos PA, Rosenblatt J. Crowding induces live cell extrusion to maintain homeostatic cell numbers in epithelia. Nature. 2012;484:546–549. [PMC free article] [PubMed]
7. Marinari E, Mehonic A, Curran S, Gale J, Duke T, Baum B. Live-cell delamination counterbalances epithelial growth to limit tissue overcrowding. Nature. 2012;484:542–545. [PubMed]
8. Andrade D, Rosenblatt J. Apoptotic regulation of epithelial cellular extrusion. Apoptosis. 2011;16:491–501. [PMC free article] [PubMed]
9. 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]
10. Hemminki A, Markie D, Tomlinson I, Avizienyte E, Roth S, Loukola A, Bignell G, Warren W, Aminoff M, Hoglund P, et al. A serine/threonine kinase gene defective in Peutz-Jeghers syndrome. Nature. 1998;391:184–187. [PubMed]
11. Yuan J, Kroemer G. Alternative cell death mechanisms in development and beyond. Genes Dev. 2010;24:2592–2602. [PubMed]
12. Coopersmith CM, O'Donnell D, Gordon JI. Bcl-2 inhibits ischemia-reperfusion-induced apoptosis in the intestinal epithelium of transgenic mice. Am. J. Physiol. 1999;276:G677–G686. [PubMed]
13. Gu Y, Forostyan T, Sabbadini R, Rosenblatt J. Epithelial cell extrusion requires the sphingosine-1-phosphate receptor 2 pathway. J. Cell Biol. 2011;193:667–676. [PMC free article] [PubMed]
14. Hardin J. Imaging embryonic morphogenesis in C. elegans. Methods Cell Biol. 2011;106:377–412. [PubMed]
15. Chisholm AD, Hardin J. Epidermal morphogenesis. WormBook; 2005. pp. 1–22. [PubMed]
16. Hogan C, Dupre-Crochet S, Norman M, Kajita M, Zimmermann C, Pelling AE, Piddini E, Baena-Lopez LA, Vincent JP, Itoh Y, et al. Characterization of the interface between normal and transformed epithelial cells. Nat. Cell Biol. 2009;11:460–467. [PubMed]
17. Kajita M, Hogan C, Harris AR, Dupre-Crochet S, Itasaki N, Kawakami K, Charras G, Tada M, Fujita Y. Interaction with surrounding normal epithelial cells influences signalling pathways and behaviour of Src-transformed cells. J. Cell Sci. 2010;123:171–180. [PubMed]
18. Singhvi A, Teuliere J, Talavera K, Cordes S, Ou G, Vale RD, Prasad BC, Clark SG, Garriga G. The Arf GAP CNT-2 regulates the apoptotic fate in C. elegans asymmetric neuroblast divisions. Curr. Biol. 2011;21:948–954. [PMC free article] [PubMed]
19. Ou G, Stuurman N, D'Ambrosio M, Vale RD. Polarized myosin produces unequal-size daughters during asymmetric cell division. Science. 2010;330:677–680. [PMC free article] [PubMed]
20. Cordes S, Frank CA, Garriga G. The C. elegans MELK ortholog PIG-1 regulates cell size asymmetry and daughter cell fate in asymmetric neuroblast divisions. Development. 2006;133:2747–2756. [PubMed]