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Riddle DL, Blumenthal T, Meyer BJ, et al., editors. C. elegans II. 2nd edition. Cold Spring Harbor (NY): Cold Spring Harbor Laboratory Press; 1997.

Cover of C. elegans II

C. elegans II. 2nd edition.

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Section VCell Deaths not Mediated by the Common Death Program

Not all developmental cell deaths occur by programmed cell death and not all programmed cell deaths need to be mediated by the same molecular pathway. Examples have been found of both programmed (naturally occurring) and unscheduled (pathological) cell deaths that are not mediated through the classical ced-3 / ced-4 -dependent pathway.

A. Murders

A small number of deaths that occur during normal C. elegans male development appear to occur through a pathway distinct from the “classical” ced-3 / ced-4 -dependent pathway.

The linker cell is required to guide the developing male gonad to the tail, where the reproductive and digestive system fuse (Kimble and Hirsh 1979). Once the destination is reached, the linker cell dies and is engulfed by either U.1p or U.rp, two cells of the proctodeum in an event that has been described as necessary for opening the channel between the vas deferens and the cloaca. If the U progenitor cell is eliminated by laser microsurgery, or if the linker cell is prevented from reaching the proctodeum (by mutation or otherwise), the linker cell fails to die (Sulston et al. 1980). These observations suggest either that U descendants are required to signal the linker cell to commit suicide or that engulfment of the linker cell is required for it to die; in which case, this death would be described more aptly as a murder than as a suicide. In support of the latter interpretation, death of the linker cell still occurs about half the time in ced-3 mutants, suggesting that although the cell death machinery does promote the death of the linker cell, it might not be essential for its demise (Ellis and Horvitz 1986).

The B descendants B.al/rapaav form an equivalence group, with one cell forming part of the vas deferens and the other one being engulfed by P12.pa (Sulston et al. 1980). If the precursor to one of the two cells is ablated, the other invariably survives, suggesting that survival is the primary fate in this group. In P12.pa-ablated animals, and in ced-1 or ced-2 mutants, both B.alapaav and B.arapaav survive, suggesting that engulfment by P12.pa is the cause of death in this case (Sulston et al. 1980; Hedgecock et al. 1983). The effect of mutations in ced-3 , ced-4 , and the other engulfment genes on B.al/rapaav death has not been investigated.

B. lin-24 and lin-33

Gain-of-function mutations in the genes lin-24 and lin-33 can cause the death of the Pn.p cells, resulting, in hermaphrodites, in a Vulvaless phenotype (Ferguson and Horvitz 1985; Ferguson et al. 1987; Kim 1994). The deaths induced by these mutations are morphologically and genetically distinct from programmed cell deaths and appear to be degenerative in nature.

Under Nomarski optics, the Pn.p nuclei in lin-24 and lin-33 mutants increase in refractivity during the late first or early second larval stage of development and form oval bodies that persist from a few minutes to up to 3 hours. These refractile bodies are distinguishable from the refractile corpses formed by programmed cell death because they are larger and less regular, affect the nucleus rather than the whole cell, and often contain an internal “depression.” Once the refractivity decreases, the nucleus becomes granular and then resolves. At this point, one of three outcomes can be observed: (1) The cell dies, (2) the cell survives but the nucleus remains abnormally small, or (3) the cell survives and the nucleus appears to recover completely. Only a minority of the cells actually die; most recover completely. However, even the cells that recover show abnormal division patterns (most Pn.p cells fail to divide altogether). Thus, the Vulvaless phenotype is mostly the result of abnormal cell division patterns rather than of cell death. The refractile Pn.p cells also show ultrastructural features, including swelling of nuclear and mitochondrial membranes followed by karyolysis, that are distinct from the changes observed in programmed cell death and that are more reminiscent of cells undergoing necrosis than apoptosis (Kim 1994).

The mutations that cause the Pn.p deaths result in a gain of lin-24 or lin-33 function, and but for one recessive lin-24 (gf) allele, all are semidominant. Dosage studies for several of these mutations point to the existence of complex interactions between mutant and wild-type alleles, and between different mutant alleles, but in general suggest that the mutant allele does not result in an overexpression of gene product but rather might direct the synthesis of an abnormal, cytotoxic gene product. Loss-of-function mutations have no obvious phenotype on their own, but they act as cis- and trans-dominant suppressors of gain-of-function mutations in the same gene. Furthermore, loss-of-function alleles of one gene can suppress gain-of-function mutations in the other gene, indicating that normal lin-24 function is required for lin-33 to kill cells, and vice versa. This mutual suppression suggests that these two genes function in the same step of a yet to be defined pathway (Kim 1994).

ced-3 , ced-4 , and ced-9 (gf) do not prevent the Pn.p deaths in lin-24 and lin-33 mutants. However, rather intriguingly, mutations in ced-2 , ced-5 , and ced-10 , which constitute one of the partially redundant group of genes required for the engulfment of programmed cell deaths, efficiently prevent the Pn.p deaths and partially suppress the Vulvaless phenotype (Kim 1994). One hypothesis consistent with this observation would be that lin-24 (gf) and lin-33 (gf) do not actually kill the Pn.p cells but make them very sick. The sick cells are then presumably recognized by neighboring cells in a ced-2/ced-5/ced-10-dependent manner and phagocytosed. If phagocytosis is prevented, the cells survive and eventually recover.

C. Necrotic Deaths

Mutations in a number of genes, including but not limited to mec-4 , deg-1 , and deg-3 , lead to the death of particular cells by causing them to swell and lyse. Analysis of these mutations has shown that in most cases, the degeneration-inducing mutation results in a gain of gene function, that the mutated gene encodes an ion channel subunit, and that the affected cells are neuronal. The molecular nature of these mutations and the cells that they affect are discussed in this volume by Driscoll and Kaplan and elsewhere (Ellis et al. 1991b; Driscoll 1992; Driscoll and Chalfie 1992). Reviewed in this chapter are the morphological and genetic features of these deaths that distinguish them from programmed cell deaths.

Swelling deaths induced by gain-of-function mutations in genes of the degenerin family, such as mec-4 , deg-1 , and mec-10 , exhibit morphological features of necrotic cell death. Such deaths are distinct from programmed cell death in several ways: (1) Cells undergoing programmed cell death appear to be compacted and “button-like,” whereas cells undergoing degenerative cell death appear to be swollen and enlarged, (2) distinct ultrastructural changes accompany the two types of death (Robertson and Thomson 1982; M. Driscoll, pers. comm.), (3) programmed cell deaths transpire within the hour, whereas degenerative deaths occur over several hours (Chalfie and Sulston 1981; Robertson and Thompson 1982), and (4) ced-3 and ced-4 , needed for execution of all programmed cell deaths, are not required for degenerin-induced deaths (Hedgecock et al. 1983; Ellis and Horvitz 1986; Chalfie and Wolinsky 1990).

The time of onset of degenerative death correlates with initiation of degenerin gene expression, and the rapidity with which death occurs correlates with dose of the toxic allele (Chalfie and Wolinsky 1990; Hall et al. 1997). These observations are consistent with the hypothesis that a threshold ion influx is needed to initiate the degenerative process. Ultrastructural analysis has established that degeneration initiates with striking infoldings of the plasma membrane (M. Driscoll, pers. comm.). Small tightly wrapped membranous whorls are the first indications of pathology. Subsequently, internalized whorls grow in size and large vacuoles appear. Cell body volume can increase 100-fold during this process. The nucleus becomes distorted and chromatin aggregates. Internal degradation of cell contents then follows shortly. Finally, corpse debris is removed in a process that requires the activities of the engulfment ced genes (Hedgecock et al. 1983; Ellis et al. 1991a), with ced-2 , ced-5 , and ced-10 appearing to be most important (S. Chung and M. Driscoll, pers. comm.). Thus, although mechanisms of killing are distinct in programmed and degenerin-induced cell death, corpse recognition and removal mechanisms share common steps.

Degenerin-poisoned cells share features of subcellular pathology exhibited in disorders caused by mutations in mammalian ion channels (e.g., in the muscle Na+ channel affected in hyperkalemic periodic paralysis; Engel et al. 1970) and under degenerative conditions such as neuronal ceroid lipofuscinosis (March et al. 1995). The dramatic endocytosis observed during neurodegeneration in C. elegans and the implication of altered intracellular membrane trafficking in Alzheimer's disease and Huntington's disease suggest that endocytotic responses provoked by diverse types of damage could be a common element of diverse degenerative conditions.

Additional genes can mutate to induce vacuolar degeneration of various C. elegans cells (M. Chalfie; A. Chisholm and H. R. Horvitz; both pers. comm.). Molecular analysis of one of these, deg-3 , established that degenerin family members are not the only channel genes capable of mutation to toxic forms. deg-3 (u662) is a gain-of-function allele that leads to the degeneration of several neurons, including the touch receptor neurons and the PVC interneurons (Treinin and Chalfie 1995). Loss-of-function deg-3 alleles have no apparent phenotype. DEG-3 exhibits significant similarity to α subunits of the neuronal nicotinic acetylcholine receptor and, in the pore-lining domain TMDII, is most like α7 subunits. deg-3 (u662) encodes an I293N substitution in TMDII at a site equivalent to the chicken α7-4 V251T mutant. Since the mutant chicken subunit exhibits defective desensitization when expressed as a homomeric channel in Xenopus oocytes, the C. elegans channel, which harbors a substitution of a polar for a hydrophobic residue at the equivalent site, is likely to permit excessive ion influx. Consistent with this hypothesis, some nicotinic antagonists partially suppress the deg-3 (u662) Mec phenotype.

Copyright © 1997, Cold Spring Harbor Laboratory Press.
Bookshelf ID: NBK20161

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