C. elegans cell cycles: invariance and stem cell divisions

The adult Caenorhabditis elegans nematode, a small roundworm, has a precisely defined number of somatic cells that create organs that are also found in larger animals, including intestine, muscles, skin, an excretory system and a primitive brain. Every cell has a defined role in this sophisticated, but tiny animal. Therefore, stringent control of the cell cycle is required to produce the almost invariant cell lineage that generates the C. elegans somatic body plan. The proliferation of germ cells is regulated differently, and occurs within a stem cell niche.     

MOM-5 frizzled regulates the distribution of DSH-2 to control C. elegans asymmetric neuroblast divisions

Asymmetric cell divisions produce all 302 neurons of the C. elegans hermaphrodite. Here, we describe a role for a C. elegans Dishevelled homolog, DSH-2, in an asymmetric neuroblast division. In dsh-2 mutants, neurons normally descended from the anterior neuroblast daughter of the ABpl/rpppa blast cell were frequently duplicated, while non-neuronal cells produced by the posterior daughter cell were often missing. These observations indicate that in the absence of dsh-2 function, the posterior daughter cell was transformed into a second anterior-like cell. Loss of mom-5, a C. elegans frizzled homolog, produced a similar phenotype. We also show that the DSH-2 protein localized to the cell cortex in most cells of the embryo. In the absence of MOM-5/Fz, DSH-2 was localized to the cytoplasm, suggesting that MOM-5 regulates asymmetric cell division by controlling the localization of DSH-2. Although all neurons in C. elegans are produced by an invariant pattern of cell divisions, our results indicate that cell signaling may contribute to asymmetric neuroblast division during embryogenesis.     

Conditional dominant mutations in the Caenorhabditis elegans gene act-2 identify cytoplasmic and muscle roles for a redundant actin isoform

Animal genomes each encode multiple highly conserved actin isoforms that polymerize to form the microfilament cytoskeleton. Previous studies of vertebrates and invertebrates have shown that many actin isoforms are restricted to either nonmuscle (cytoplasmic) functions, or to myofibril force generation in muscle cells. We have identified two temperature-sensitive and semidominant embryonic-lethal Caenorhabditis elegans mutants, each with a single mis-sense mutation in act-2, one of five C. elegans genes that encode actin isoforms. These mutations alter conserved and adjacent amino acids predicted to form part of the ATP binding pocket of actin. At the restrictive temperature, both mutations resulted in aberrant distributions of cortical microfilaments associated with abnormal and striking membrane ingressions and protrusions. In contrast to the defects caused by these dominant mis-sense mutations, an act-2 deletion did not result in early embryonic cell division defects, suggesting that additional and redundant actin isoforms are involved. Accordingly, we found that two additional actin isoforms, act-1 and act-3, were required redundantly with act-2 for cytoplasmic function in early embryonic cells. The act-1 and -3 genes also have been implicated previously in muscle function. We found that an ACT-2::GFP reporter was expressed cytoplasmically in embryonic cells and also was incorporated into contractile filaments in adult muscle cells. Furthermore, one of the dominant act-2 mutations resulted in uncoordinated adult movement. We conclude that redundant C. elegans actin isoforms function in both muscle and nonmuscle contractile processes.     

Genotoxic effects of radiotherapy and chemotherapy on circulating lymphocytes in patients with Hodgkin's disease

Chemical mutagenesis of Caenorhabditis elegans has relied primarily on EMS to produce missense mutations. The drawback of EMS mutagenesis is that the molecular lesions are primarily G/C --> A/T transitions. ENU has been shown to produce a different spectrum of mutations, but its greater toxicity to C. elegans makes it a difficult mutagen to use. We describe here methods for minimizing ENU toxicity in C. elegans. Methods include preparing ENU stocks in absolute ethanol and storing stock solutions for not more than 2 weeks at -20 degrees C. To maintain reasonable brood sizes of mutagenized animals, mutagenic solutions should not exceed 1.0mM ENU. We provide data which suggest ENU is degraded or altered to more toxic products in aqueous solution, but less so in solvents such as absolute ethanol.     

Developmental modulation of nonhomologous end joining in Caenorhabditis elegans

Homologous recombination and nonhomologous end joining (NHEJ) are important DNA double-strand break repair pathways in many organisms. C. elegans strains harboring mutations in the cku-70, cku-80, or lig-4 NHEJ genes displayed multiple developmental abnormalities in response to radiation-induced DNA damage in noncycling somatic cells. These phenotypes did not result from S-phase, DNA damage, or mitotic checkpoints, apoptosis, or stress response pathways that regulate dauer formation. However, an additional defect in him-10, a kinetochore component, synergized with NHEJ mutations for the radiation-induced developmental phenotypes, suggesting that they may be triggered by mis-segregation of chromosome fragments. Although NHEJ was an important DNA repair pathway for noncycling somatic cells in C. elegans, homologous recombination was used to repair radiation-induced DNA damage in cycling somatic cells and in germ cells at all times. Noncycling germ cells that depended on homologous recombination underwent cell cycle arrest in G2, whereas noncycling somatic cells that depended on NHEJ arrested in G1, suggesting that cell cycle phase may modulate DNA repair during development. We conclude that error-prone NHEJ plays little or no role in DNA repair in C. elegans germ cells, possibly ensuring homology-based double-strand break repair and transmission of a stable genome from one generation to the next.     

The C. elegans par-4 gene encodes a putative serine-threonine kinase required for establishing embryonic asymmetry

During the first cell cycle of Caenorhabditis elegans embryogenesis, asymmetries are established that are essential for determining the subsequent developmental fates of the daughter cells. The maternally expressed par genes are required for establishing this polarity. The products of several of the par genes have been found to be themselves asymmetrically distributed in the first cell cycle. We have identified the par-4 gene of C. elegans, and find that it encodes a putative serine-threonine kinase with similarity to a human kinase associated with Peutz-Jeghers Syndrome, LKB1 (STK11), and a Xenopus egg and embryo kinase, XEEK1. Several strong par-4 mutant alleles are missense mutations that alter conserved residues within the kinase domain, suggesting that kinase activity is essential for PAR-4 function. We find that the PAR-4 protein is present in the gonads, oocytes and early embryos of C. elegans, and is both cytoplasmically and cortically distributed. The cortical distribution begins at the late 1-cell stage, is more pronounced at the 2- and 4-cell stages and is reduced at late stages of embryonic development. We find no asymmetry in the distribution of PAR-4 protein in C. elegans embryos. The distribution of PAR-4 protein in early embryos is unaffected by mutations in the other par genes.     

Caenorhabditis elegans genes required for the engulfment of apoptotic corpses function in the cytotoxic cell deaths induced by mutations in lin-24 and lin-33

Two types of cell death have been studied extensively in Caenorhabditis elegans, programmed cell death and necrosis. We describe a novel type of cell death that occurs in animals containing mutations in either of two genes, lin-24 and lin-33. Gain-of-function mutations in lin-24 and lin-33 cause the inappropriate deaths of many of the Pn.p hypodermal blast cells and prevent the surviving Pn.p cells from expressing their normal developmental fates. The abnormal Pn.p cells in lin-24 and lin-33 mutant animals are morphologically distinct from the dying cells characteristic of C. elegans programmed cell deaths and necrotic cell deaths. lin-24 encodes a protein with homology to bacterial toxins. lin-33 encodes a novel protein. The cytotoxicity caused by mutation of either gene requires the function of the other. An evolutionarily conserved set of genes required for the efficient engulfment and removal of both apoptotic and necrotic cell corpses is required for the full cell-killing effect of mutant lin-24 and lin-33 genes, suggesting that engulfment promotes these cytotoxic cell deaths.     

Most ultraviolet irradiation induced mutations in the nematode Caenorhabditis elegans are chromosomal rearrangements

In this study we have determined the utility of 254-nm ultraviolet light (UV) as a mutagenic tool in C. elegans. We have demonstrated that irradiation of adult hermaphrodites provides a simple method for the induction of heritable chromosomal rearrangements. A screening protocol was employed that identifies either recessive lethal mutations in the 40 map unit region balanced by the translocation eT1(III;V), or unc-36(III) duplications. Mutations were recovered in 3% of the chromosomes screened after a dose of 120 J/m2. This rate resembles that for 1500 R gamma-ray-induced mutations selected in a similar manner. The mutations were classified either as lethals [mapping to Linkage Group (LG)III or LGV] or as putative unc-36 duplications. In contrast to the majority of UV-induced mutations analysed in microorganisms, we found that a large fraction of the C. elegans UV-induced mutations are not simple intragenic lesions, but are deficiencies for more than one adjacent gene or more complex events. Preliminary evidence for this conclusion came from the high frequency of mutations that had a dominant effect causing reduced numbers of adult progeny. Subsequently 6 out of 9 analysed LGV mutations were found to be deficiencies. Other specific rearrangements also identified were: one translocation, sT5(II;III), and two unc-36 duplications, sDp8 and sDp9. It was concluded that UV irradiation can easily be used as an additional tool for the analysis of C. elegans chromosomes, and that C. elegans should prove to be a useful organism in which to study the mechanisms whereby UV acts as a mutagen in cells of complex eukaryotes.     

The Caenorhabditis elegans pvl-5 gene protects hypodermal cells from ced-3-dependent, ced-4-independent cell death

Programmed cell death (PCD) is regulated by multiple evolutionarily conserved mechanisms to ensure the survival of the cell. Here we describe pvl-5, a gene that likely regulates PCD in Caenorhabditis elegans. In wild-type hermaphrodites at the L2 stage there are 11 Pn.p hypodermal cells in the ventral midline arrayed along the anterior-posterior axis and 6 of these cells become the vulval precursor cells. In pvl-5(ga87) animals there are fewer Pn.p cells (average of 7.0) present at this time. Lineage analysis reveals that the missing Pn.p cells die around the time of the L1 molt in a manner that often resembles the programmed cell deaths that occur normally in C. elegans development. This Pn.p cell death is suppressed by mutations in the caspase gene ced-3 and in the bcl-2 homolog ced-9, suggesting that the Pn.p cells are dying by PCD in pvl-5 mutants. Surprisingly, the Pn.p cell death is not suppressed by loss of ced-4 function. ced-4 (Apaf-1) is required for all previously known apoptotic cell deaths in C. elegans. This suggests that loss of pvl-5 function leads to the activation of a ced-3-dependent, ced-4-independent form of PCD and that pvl-5 may normally function to protect cells from inappropriate activation of the apoptotic pathway.     

Synthetic lethality of cohesins with PARPs and replication fork mediators

Synthetic lethality has been proposed as a way to leverage the genetic differences found in tumor cells to affect their selective killing. Cohesins, which tether sister chromatids together until anaphase onset, are mutated in a variety of tumor types. The elucidation of synthetic lethal interactions with cohesin mutants therefore identifies potential therapeutic targets. We used a cross-species approach to identify robust negative genetic interactions with cohesin mutants. Utilizing essential and non-essential mutant synthetic genetic arrays in Saccharomyces cerevisiae, we screened genome-wide for genetic interactions with hypomorphic mutations in cohesin genes. A somatic cell proliferation assay in Caenorhabditis elegans demonstrated that the majority of interactions were conserved. Analysis of the interactions found that cohesin mutants require the function of genes that mediate replication fork progression. Conservation of these interactions between replication fork mediators and cohesin in both yeast and C. elegans prompted us to test whether other replication fork mediators not found in the yeast were required for viability in cohesin mutants. PARP1 has roles in the DNA damage response but also in the restart of stalled replication forks. We found that a hypomorphic allele of the C. elegans SMC1 orthologue, him-1(e879), genetically interacted with mutations in the orthologues of PAR metabolism genes resulting in a reduced brood size and somatic cell defects. We then demonstrated that this interaction is conserved in human cells by showing that PARP inhibitors reduce the viability of cultured human cells depleted for cohesin components. This work demonstrates that large-scale genetic interaction screening in yeast can identify clinically relevant genetic interactions and suggests that PARP inhibitors, which are currently undergoing clinical trials as a treatment of homologous recombination-deficient cancers, may be effective in treating cancers that harbor cohesin mutations.     

Genetic analysis of dauer formation in Caenorhabditis briggsae

Molecular changes that underlie evolutionary changes in behavior and physiology are not well understood. Dauer formation in Caenorhabditis elegans is a temperature-sensitive process controlled through a network of signaling pathways associated with sensory neurons and is potentially an excellent system in which to investigate molecular changes in neuronal function during evolution. To begin to investigate the evolution of dauer formation in the genus Caenorhabditis at the molecular level, we isolated dauer-formation mutations in C. briggsae, a species closely related to the model organism C. elegans. We identified mutations in orthologs of C. elegans genes daf-2 (insulin receptor), daf-3 (Smad), and daf-4 (TGF-beta type 2 receptor), as well as genes required for formation of sensory cilia. Phenotypic analyses revealed that functions of these genes are conserved between C. elegans and C. briggsae. Analysis of C. briggsae mutations also revealed a significant difference between the two species in their responses to high temperatures (>26 degrees). C. elegans is strongly induced to form dauers at temperatures above 26 degrees, near the upper limit for growth of C. elegans. In contrast, C. briggsae, which is capable of growth at higher temperatures than C. elegans, lacks this response.     

Transposable elements, mutational correlations, and population divergence in Caenorhabditis elegans

Transposable element activity is thought to be responsible for a large portion of all mutations, but its influence on the evolution of populations has not been well studied. Using mutation accumulation experiments with the nematode Caenorhabditis elegans, we investigated the impact of transposable element activity on the production of mutational variances and covariances. The experiments involved the use of two mutator strains (RNAi-deficient mutants) that are characterized by high levels of germline transposition, as well as the Bristol N2 strain, which lacks germline transposition. We found that transposition led to an increase in mutational heritabilities, as well as to the intensification of correlation patterns observed in the absence of transposition. No mutational trade-offs were detected and mutations generally had a deleterious effect on components of fitness. We also tested whether the pattern of mutational covariation could be used to predict observed patterns of population divergence in this species. Using 15 natural populations, we found that population divergence of C. elegans in multivariate phenotypic space occurred in directions only partially concordant with mutation, and thus other evolutionary factors, such as natural selection and genetic drift, must be acting to produce divergence within this species. Our results suggest that mutations induced by mobile elements in C. elegans are similar to other spontaneous mutations with respect to their contribution to the microevolution of quantitative traits.     

The lin-12 locus specifies cell fates in Caenorhabditis elegans

We describe two classes of mutations in the lin-12 locus of the nematode Caenorhabditis elegans. Ten semidominant mutations (lin-12[d]) appear to elevate the level of lin-12 activity. Thirty-two recessive alleles (lin-12[0]), including two amber mutations, appear to eliminate gene activity. The lin-12(d) and lin-12(0) mutations result in reciprocal homeotic transformations in the fates of defined cells in several different tissues. Gene dosage studies suggest that a high level of lin-12 activity specifies one cell fate and a low level specifies an alternative fate. Temperature-shift experiments indicate that lin-12 acts at the time cell fate is determined in wild type. We propose that lin-12 functions as a binary switch to control decisions between alternative cell fates during C. elegans development.     

Autophagy protects against hypoxic injury in C. elegans

Macroautophagy has been implicated in a variety of pathological processes. Hypoxic/ischemic cellular injury is one such process in which autophagy has emerged as an important regulator. In general, autophagy is induced after a hypoxic/ischemic insult; however, whether the induction of autophagy promotes cell death or recovery is controversial and appears to be context dependent. We have developed C. elegans as a genetically tractable model for the study of hypoxic cell injury. Both necrosis and apoptosis are mechanisms of cell death following hypoxia in C. elegans. However, the role of autophagy in hypoxic injury in C. elegans has not been examined. Here, we found that RNAi knockdown of the C. elegans homologs of beclin 1/Atg6 (bec-1) and LC3/Atg8 (lgg-1, lgg-2), and mutation of Atg1 (unc-51) decreased animal survival after a severe hypoxic insult. Acute inhibition of autophagy by the type III phosphatidylinositol 3-kinase inhibitors, 3-methyladenine and Wortmannin, also sensitized animals to hypoxic death. Hypoxia-induced neuronal and myocyte injury as well as necrotic cellular morphology were increased by RNAi knockdown of BEC-1. Hypoxia increased the expression of a marker of autophagosomes in a bec-1-dependent manner. Finally, we found that the hypoxia hypersensitive phenotype of bec-1(RNAi) animals could be blocked by loss-of-function mutations in either the apoptosis or necrosis pathway. These results argue that inhibition of autophagy sensitizes C. elegans and its cells to hypoxic injury and that this sensitization is blocked or circumvented when either of the two major cell-death mechanisms is inhibited.     

DYC-1, a protein functionally linked to dystrophin in Caenorhabditis elegans is associated with the dense body, where it interacts with the muscle LIM domain protein ZYX-1

In Caenorhabditis elegans, mutations of the dystrophin homologue, dys-1, produce a peculiar behavioral phenotype (hyperactivity and a tendency to hypercontract). In a sensitized genetic background, dys-1 mutations also lead to muscle necrosis. The dyc-1 gene was previously identified in a genetic screen because its mutation leads to the same phenotype as dys-1, suggesting that the two genes are functionally linked. Here, we report the detailed characterization of the dyc-1 gene. dyc-1 encodes two isoforms, which are expressed in neurons and muscles. Isoform-specific RNAi experiments show that the absence of the muscle isoform, and not that of the neuronal isoform, is responsible for the dyc-1 mutant phenotype. In the sarcomere, the DYC-1 protein is localized at the edges of the dense body, the nematode muscle adhesion structure where actin filaments are anchored and linked to the sarcolemma. In yeast two-hybrid assays, DYC-1 interacts with ZYX-1, the homologue of the vertebrate focal adhesion LIM domain protein zyxin. ZYX-1 localizes at dense bodies and M-lines as well as in the nucleus of C. elegans striated muscles. The DYC-1 protein possesses a highly conserved 19 amino acid sequence, which is involved in the interaction with ZYX-1 and which is sufficient for addressing DYC-1 to the dense body. Altogether our findings indicate that DYC-1 may be involved in dense body function and stability. This, taken together with the functional link between the C. elegans DYC-1 and DYS-1 proteins, furthermore suggests a requirement of dystrophin function at this structure. As the dense body shares functional similarity with both the vertebrate Z-disk and the costamere, we therefore postulate that disruption of muscle cell adhesion structures might be the primary event of muscle degeneration occurring in the absence of dystrophin, in C. elegans as well as vertebrates.     

Hypoxic preconditioning requires the apoptosis protein CED-4 in C. elegans

Hypoxic preconditioning (HP) is a rapid and reversible proadaptive response to mild hypoxic exposure with such a response protecting cells from subsequent hypoxic or ischemic insult. HP mechanisms are of great interest because of their therapeutic potential and insight into metabolic adaptation and cell death. HP has been widely demonstrated in the vertebrate subphylum but not in invertebrates. Here, we report that the nematode Caenorhabditis elegans has a potent HP mechanism that protects the organism as well as its neurons and myocytes from hypoxic injury. The time course of C. elegans HP was consistent with vertebrate-delayed HP, appearing 16 hr after preconditioning and lasting at least 36 hr. The apoptosis pathway has been proposed as either a trigger or target of HP. Testing of mutations in the canonical C. elegans apoptosis pathway showed that in general, genes in this pathway are not required for HP. However, loss-of-function mutations in ced-4, which encodes an Apaf-1 homolog, completely blocked HP. RNAi silencing of ced-4 in adult animals immediately preceding preconditioning blocked HP, indicating that CED-4 is required in adults during or after preconditioning. CED-4/Apaf-1 is essential for HP in C. elegans and acts through a mechanism independent of the classical apoptosis pathway.     

C. elegans HAM-1 positions the cleavage plane and regulates apoptosis in asymmetric neuroblast divisions

Asymmetric cell division occurs when a mother cell divides to generate two distinct daughter cells, a process that promotes the generation of cellular diversity in metazoans. During Caenorhabditis elegans development, the asymmetric divisions of neural progenitors generate neurons, neural support cells and apoptotic cells. C. elegans HAM-1 is an asymmetrically distributed cortical protein that regulates several of these asymmetric neuroblast divisions. Here, we show that HAM-1 is a novel protein and define residues important for HAM-1 function and distribution to the cell cortex. Our phenotypic analysis of ham-1 mutant embryos suggests that HAM-1 controls only neuroblast divisions that produce apoptotic cells. Moreover, ham-1 mutant embryos contain many unusually large cell-death corpses. An investigation of this corpse phenotype revealed that it results from a reversal of neuroblast polarity. A misplacement of the neuroblast cleavage plane generates daughter cells of abnormal size, with the apoptotic daughters larger than normal. Thus, HAM-1 regulates the position of the cleavage plane, apoptosis and mitotic potential in C. elegans asymmetric cell divisions.     

Eggs to die for: cell death during Drosophila oogenesis

Extensive programmed cell death occurs in the female germline of many species ranging from C. elegans to humans. One purpose for germline apoptosis is to remove defective cells unable to develop into fertile eggs. In addition, recent work suggests that the death of specific germline cells may also play a vital role by providing essential nutrients to the surviving oocytes. In Drosophila, the genetic control of germline apoptosis and the proteins that carry out the death sentences are beginning to emerge from studies of female sterile mutations. These studies suggest that the morphological changes that occur during the late stages of Drosophila oogenesis may be initiated and driven by a modified form of programmed cell death.     

Characterization of seven genes affecting Caenorhabditis elegans hindgut development.

We have identified and characterized 12 mutations in seven genes that affect the development of the Caenorhabditis elegans hindgut. We find that the mutations can disrupt the postembryonic development of the male-specific blast cells within the hindgut, the hindgut morphology in both males and hermaphrodites, and in some cases, the expression of a hindgut marker in hermaphrodite animals. Mutations in several of the genes also affect viability. On the basis of their mutant phenotypes, we propose that the genes fall into four distinct classes: (1) egl-5 is required for regional identity of the tail; (2) sem-4 is required for a variety of ectodermal and mesodermal cell types, including cells in the hindgut; (3) two genes, lin-49 and lin-59, affect development of many cells, including hindgut; and (4) three genes, mab-9, egl-38, and lin-48, are required for patterning fates within the hindgut, making certain hindgut cells different from others. We also describe a new allele of the Pax gene egl-38 that is temperature sensitive and affects the conserved beta-hairpin of the EGL-38 paired domain. Our results suggest that a combination of different factors contribute to normal C. elegans hindgut development.