Deficiency of Bloom's syndrome protein causes hypersensitivity of C. elegans to ionizing radiation but not to UV radiation, and induces p53-dependent physiological apoptosis

Caenorhabditis elegans him-6 mutants, which show a high incidence of males and partial embryonic lethality, are defective in the orthologue of human Bloom's syndrome protein (BLM). When strain him-6(e1104) containing a missense him-6 mutation was irradiated with gamma-rays during germ cell development or embryogenesis, embryonic lethality was higher than in the wild type, suggesting a critical function of the wild type gene in mitotic and pachytene stage germ cells as well as in early embryos. Even in the absence of gamma-irradiation, apoptosis was elevated in the germ cells of the him-6 strain and this increase was dependent on a functional p53 homologue (CEP-1), suggesting that spontaneous DNA damage accumulates due to him-6 deficiency. However, induction of germline apoptosis by ionizing radiation was not significantly affected by the deficiency, indicating that HIM-6 has no role in the induction of apoptosis by exogenous DNA damage. We conclude that the C. elegans BLM orthologue is involved in DNA repair in promeiotic cells undergoing homologous recombination, as well as in actively dividing germline and somatic cells.     

Caenorhabditis elegans HIM-18/SLX-4 Interacts with SLX-1 and XPF-1 and Maintains Genomic Integrity in the Germline by Processing Recombination Intermediates

Homologous recombination (HR) is essential for the repair of blocked or collapsed replication forks and for the production of crossovers between homologs that promote accurate meiotic chromosome segregation. Here, we identify HIM-18, an ortholog of MUS312/Slx4, as a critical player required in vivo for processing late HR intermediates in Caenorhabditis elegans. DNA damage sensitivity and an accumulation of HR intermediates (RAD-51 foci) during premeiotic entry suggest that HIM-18 is required for HR–mediated repair at stalled replication forks. A reduction in crossover recombination frequencies—accompanied by an increase in HR intermediates during meiosis, germ cell apoptosis, unstable bivalent attachments, and subsequent chromosome nondisjunction—support a role for HIM-18 in converting HR intermediates into crossover products. Such a role is suggested by physical interaction of HIM-18 with the nucleases SLX-1 and XPF-1 and by the synthetic lethality of him-18 with him-6, the C. elegans BLM homolog. We propose that HIM-18 facilitates processing of HR intermediates resulting from replication fork collapse and programmed meiotic DSBs in the C. elegans germline.     

The C. elegans TPR Containing Protein,TRD-1, Regulates Cell Fate Choice in the Developing Germ Line and Epidermis

Correct cell fate choice is crucial in development. In post-embryonic development of the hermaphroditic Caenorhabitis elegans, distinct cell fates must be adopted in two diverse tissues. In the germline, stem cells adopt one of three possible fates: mitotic cell cycle, or gamete formation via meiosis, producing either sperm or oocytes. In the epidermis, the stem cell-like seam cells divide asymmetrically, with the daughters taking on either a proliferative (seam) or differentiated (hypodermal or neuronal) fate. We have isolated a novel conserved C. elegans tetratricopeptide repeat containing protein, TRD-1, which is essential for cell fate determination in both the germline and the developing epidermis and has homologs in other species, including humans (TTC27). We show that trd-1(RNAi) and mutant animals have fewer seam cells as a result of inappropriate differentiation towards the hypodermal fate. In the germline, trd-1 RNAi results in a strong masculinization phenotype, as well as defects in the mitosis to meiosis switch. Our data suggests that trd-1 acts downstream of tra-2 but upstream of fem-3 in the germline sex determination pathway, and exhibits a constellation of phenotypes in common with other Mog (masculinization of germline) mutants. Thus, trd-1 is a new player in both the somatic and germline cell fate determination machinery, suggestive of a novel molecular connection between the development of these two diverse tissues.     

A role for Caenorhabditis elegans chromatin-associated protein HIM-17 in the proliferation vs. meiotic entry decision

Chromatin-associated protein HIM-17 was previously shown to function in the chromosomal events of meiotic prophase. Here we report an additional role for HIM-17 in regulating the balance between germ cell proliferation and meiotic development. A cryptic function for HIM-17 in promoting meiotic entry and/or inhibiting proliferation was revealed by defects in germline organization in him-17 mutants grown at high temperature (25°) and by a synthetic tumorous germline phenotype in glp-1(ar202); him-17 mutants at 15°.     

C. elegans HIM-8 functions outside of meiosis to antagonize EGL-13 Sox protein function

egl-13 encodes a Sox domain protein that is required for proper uterine seam cell development in Caenorhabditis elegans. We demonstrate that mutations of the C2H2 zinc fingers encoded by the him-8 (high incidence of males) gene partially suppress the egg-laying and connection-of-gonad morphology defects caused by incompletely penetrant alleles of egl-13. him-8 alleles have previously characterized recessive effects on recombination and segregation of the X chromosome during meiosis due to failure of X chromosome homolog pairing and subsequent synapsis. However, we show that him-8 alleles are semi-dominant suppressors of egl-13, and the semi-dominant effect is due to haplo-insufficiency of the him-8 locus. Thus, we conclude that the wild-type him-8 gene product acts antagonistically to EGL-13. Null alleles of egl-13 cannot be suppressed, suggesting that this antagonistic interaction most likely occurs either upstream of or in parallel with EGL-13. Moreover, we conclude that suppression of egl-13 is due to a meiosis-independent function of him-8 because suppression is observed in mutants that have severely reduced meiotic germ cell populations and suppression does not depend on the function of him-8 in the maternal germ line. We also show that the chromosomal context of egl-13 seems important in the him-8 suppression mechanism. Interactions between these genes can give insight into function of Sox family members, which are important in many aspects of metazoan development, and into functions of him-8 outside of meiosis.     

An extracellular matrix protein prevents cytokinesis failure and aneuploidy in the C. elegans germline

Interactions between extracellular matrix (ECM) proteins and their transmembrane receptors mediate cytoskeletal reorganization and corresponding changes in cell shape during cell migration, adhesion, differentiation and polarization. Cytokinesis is the final step in cell division as cells employ a contractile ring composed of actin and myosin to partition one cell into two. Cells undergo dramatic changes in cell shape during the division process, creating new membrane and forming an extracellular invagination called the cleavage furrow. However, existing models of cytokinesis include no role for the ECM. In a recent paper, we demonstrate that depletion of a large secreted protein, hemicentin, results in membrane destabilization, cleavage furrow retraction and cytokinesis failure in C. elegans germ cells and in preimplantation mouse embryos.Here, we demonstrate that cytokinesis failure produces tetraploid intermediate cells with multipolar spindles, providing a potential explanation for the large number of aneuploid progeny observed among C. elegans hemicentin mutant hermaphrodites.Key words: aneuploidy, cytokinesis, extracellular matrix, C. elegans, cleavage furrow, hemicentin, tetraploid intermediateThe karyotype of C. elegans has five autosomes and one or two X chromosomes in males and hermaphrodites, respectively. The majority of self-progeny produced by wild-type hermaphrodites are hermaphrodites (∼99.8%), while rare meiotic nondisjunction of the X chromosome produces nullo-X gametes and 0.2% males. Mutations in over 30 genes result in a 10–150-fold increase in the frequency of males among hermaphrodite self-progeny, due to increases in defects in X chromosome segregation.¹ The majority of these ‘him’ (high incidence of males) loci are genes that encode proteins associated with the intracellular machinery of meiotic chromosome segregation.^2,3 Unique among him genes, the him-4 locus encodes hemicentin, a large, highly conserved component of the extracellular matrix (ECM).⁴ In addition to defects in germline chromosome segregation, him-4 mutants have pleiotropic defects in somatic cell adhesion and migration.^1,4 The extracellular distribution of hemicentin at cell junctions that are defective in him-4 mutants dovetails with current models of cell adhesion and migration.⁵ However, it leaves unexplained several questions about how a secreted ECM component promotes correct chromosome segregation in the C. elegans germline.C. elegans hermaphrodite gonads are composed of two U-shaped tubes, and gametogenesis proceeds sequentially from the distal to the proximal end of each tube. Germ cells in C. elegans have incomplete cleavage furrows that connect them to a central cytoplasmic core, allowing distal cells to act as “nurses” while allowing more mature proximal oocytes to fill with bulk cytoplasm.^6–8 Several genetic and cytogenetic observations suggest a mitotic rather than a meiotic origin for germline chromosome segregation defects observed in the absence of hemicentin.⁴ For example, jackpots of male progeny from individual hermaphrodites and nullisomy of primary meiocytes in him-4 mutants suggest a defect in a mitotic germline stem cell rather than in a post-mitotic process. Our recent work describing hemicentin localization at the cleavage furrows of dividing cells in the early mouse embryo and C. elegans germline, in addition to membrane destabilization, cleavage furrow retraction and cytokinesis failure in the absence of hemicentin, suggests that hemicentin has an evolutionarily conserved role in stabilizing and preventing retraction of nascent cleavage furrows.⁹Aneuploid cells are frequently observed in, and may be associated with the generation of, human tumor cells. Recent work from several laboratories suggests that cytokinesis failure is one of several mechanisms whereby tumor cells generate tetraploid intermediates that result in the production of aneuploid daughter cells in subsequent cell divisions. One proposed mechanism for the generation of aneuploid daughter cells from a tetraploid intermediate is thought to involve multipolar mitotic spindles that result in asymmetric mitoses.^10–13To determine whether a similar mechanism might be responsible for the aneuploidy observed in the absence of hemicentin, him-4 (rh319) animals were examined for multipolar mitotic spindles. A significant fraction (14%) of mitotic germ cells have multipolar spindles that are not observed in a wild-type background (Fig. 1 and Fig. 1).Open in a separate windowFigure 1Multinucleate germ cells and multipolar germ cells observed in the mitotic zone of him-4 mutant hermaphrodites. (A) PH::RFP and histone::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. Large numbers of multinucleate cells are observed among mitotic germ cells in mutant gonads (arrows). (B) PH::RFP and tubulin::GFP in the mitotic region of wild-type (left) and him-4 (rh319) hermaphrodite gonads. A significant fraction (14^–16.

Table 1

Severity and types of defective germ cells in him-4 gonads

Crossover distribution and frequency are regulated by him-5 in Caenorhabditis elegans

Mutations in the him-5 gene in Caenorhabditis elegans strongly reduce the frequency of crossovers on the X chromosome, with lesser effects on the autosomes. him-5 mutants also show a change in crossover distribution on both the X and autosomes. These phenotypes are accompanied by a delayed entry into pachytene and premature desynapsis of the X chromosome. The nondisjunction, progression defects and desynapsis can be rescued by an exogenous source of double strand breaks (DSBs), indicating that the role of HIM-5 is to promote the formation of meiotic DSBs. Molecular cloning of the gene shows that the inferred HIM-5 product is a highly basic protein of 252 amino acids with no clear orthologs in other species, including other Caenorhabditis species. Although him-5 mutants are defective in segregation of the X chromosome, HIM-5 protein localizes preferentially to the autosomes. The mutant phenotypes and localization of him-5 are similar but not identical to the results seen with xnd-1, although unlike xnd-1, him-5 has no apparent effect on the acetylation of histone H2A on lysine 5 (H2AacK5). The localization of HIM-5 to the autosomes depends on the activities of both xnd-1 and him-17 allowing us to begin to establish pathways for the control of crossover distribution and frequency.     

HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.     

Mutant Rec-1 Eliminates the Meiotic Pattern of Crossing over in Caenorhabditis Elegans

Meiotic crossovers are not randomly distributed along the chromosome. In Caenorhabditis elegans the central portions of the autosomes have relatively few crossovers compared to the flanking regions. We have measured the frequency of crossing over for several intervals across chromosome I in strains mutant for rec-1. The chromosome is ~50 map units in both wild-type and rec-1 homozygotes, however, the distribution of exchanges is very different in rec-1. Map distances expand across the gene cluster and contract near the right end of the chromosome, resulting in a genetic map more consistent with the physical map. Mutations in two other genes, him-6 and him-14, also disrupted the distribution of exchanges. Unlike rec-1, individuals homozygous for him-6 and him-14 had an overall reduction in the amount of crossing over accompanied by a high frequency of nondisjunction and reduced egg hatching. In rec-1; him-6 and rec-1; him-14 homozygotes the frequency of crossing over was characteristic of the Him mutant phenotype, whereas the distribution of the reduced number of exchanges was characteristic of the Rec-1 pattern. It appears that these gene products play a role in establishing the meiotic pattern of exchange events.     

The phenotype of mes-2, mes-3, mes-4 and mes-6, maternal-effect genes required for survival of the germline in Caenorhabditis elegans,is sensitive to chromosome dosage.

Mutations in mes-2, mes-3, mes-4, and mes-6 result in maternal-effect sterility: hermaphrodite offspring of mes/mes mothers are sterile because of underproliferation and death of the germ cells, as well as an absence of gametes. Mutant germ cells do not undergo programmed cell death, but instead undergo a necrotic-type death, and their general poor health apparently prevents surviving germ cells from forming gametes. Male offspring of mes mothers display a significantly less severe germline phenotype than their hermaphrodite siblings, and males are often fertile. This differential response of hermaphrodite and male offspring to the absence of mes+ product is a result of their different X chromosome compositions; regardless of their sexual phenotype, XX worms display a more severe germline phenotype than XO worms, and XXX worms display the most severe phenotype. The sensitivity of the mutant phenotype to chromosome dosage, along with the similarity of two MES proteins to chromatin-associated regulators of gene expression in Drosophila, suggest that the essential role of the mes genes is in control of gene expression in the germline. An additional, nonessential role of the mes genes in the soma is suggested by the surprising finding that mutations in the mes genes, like mutations in dosage compensation genes, feminize animals whose male sexual identity is somewhat ambiguous. We hypothesize that the mes genes encode maternally supplied regulators of chromatin structure and gene expression in the germline and perhaps in somatic cells of the early embryo, and that at least some of their targets are on the X chromosomes.     

Meiotic Mutants That Cause a Polar Decrease in Recombination on the X Chromosome in Caenorhabditis Elegans

Recessive mutations in three autosomal genes, him-1, him-5 and him-8, cause high levels of X chromosome nondisjunction in hermaphrodites of Caenorhabditis elegans, with no comparable effect on autosomal disjunction. Each of the mutants has reduced levels of X chromosome recombination, correlating with the increase in nondisjunction. However, normal or elevated levels of recombination occur at the end of the X chromosome hypothesized to contain the pairing region (the left end), with recombination levels decreasing in regions approaching the right end. Thus, both the number and the distribution of X chromosome exchange events are altered in these mutants. As a result, the genetic map of the X chromosome in the him mutants exhibits a clustering of genes due to reduced recombination, a feature characteristic of the genetic map of the autosomes in non-mutant animals. We hypothesize that these him genes are needed for some processive event that initiates near the left end of the X chromosome.     

A Highlights from MBoC Selection: Mutations in Caenorhabditis elegans him-19 Show Meiotic Defects That Worsen with Age

From a screen for meiotic Caenorhabditis elegans mutants based on high incidence of males, we identified a novel gene, him-19, with multiple functions in prophase of meiosis I. Mutant him-19(jf6) animals show a reduction in pairing of homologous chromosomes and subsequent bivalent formation. Consistently, synaptonemal complex formation is spatially restricted and possibly involves nonhomologous chromosomes. Also, foci of the recombination protein RAD-51 occur delayed or cease altogether. Ultimately, mutation of him-19 leads to chromosome missegregation and reduced offspring viability. The observed defects suggest that HIM-19 is important for both homology recognition and formation of meiotic DNA double-strand breaks. It therefore seems to be engaged in an early meiotic event, resembling in this respect the regulator kinase CHK-2. Most astonishingly, him-19(jf6) hermaphrodites display worsening of phenotypes with increasing age, whereas defects are more severe in female than in male meiosis. This finding is consistent with depletion of a him-19-dependent factor during the production of oocytes. Further characterization of him-19 could contribute to our understanding of age-dependent meiotic defects in humans.     

Somatic signaling mediated by fs(1)Yb is essential for germline stem cell maintenance during Drosophila oogenesis

Drosophila oogenesis starts when a germline stem cell divides asymmetrically to generate a daughter germline stem cell and a cystoblast that will develop into a mature egg. We show that the fs(1)Yb gene is essential for the maintenance of germline stem cells during oogenesis. We delineate fs(1)Yb within a 6.4 kb genomic region by transgenic rescue experiments. fs(1)Yb encodes a 4.1 kb RNA that is present in the third instar larval, pupal and adult stages, consistent with its role in regulating germline stem cells during oogenesis. Germline clonal analysis shows that all fs(1)Yb mutations are soma-dependent. In the adult ovary, fs(1)Yb is specifically expressed in the terminal filament cells, suggesting that fs(1)Yb acts in these signaling cells to maintain germline stem cells. fs(1)Yb encodes a novel hydrophilic protein with no potential signal peptide or transmembrane domains, suggesting that this protein is not itself a signal but a key component of the signaling machinery for germline stem cell maintenance.     

Identification of novel pro-migratory, cancer-associated genes using quantitative, microscopy-based screening

Background

Cell migration is a highly complex process, regulated by multiple genes, signaling pathways and external stimuli. To discover genes or pharmacological agents that can modulate the migratory activity of cells, screening strategies that enable the monitoring of diverse migratory parameters in a large number of samples are necessary.

Methodology

In the present study, we describe the development of a quantitative, high-throughput cell migration assay, based on a modified phagokinetic tracks (PKT) procedure, and apply it for identifying novel pro-migratory genes in a cancer-related gene library. In brief, cells are seeded on fibronectin-coated 96-well plates, covered with a monolayer of carboxylated latex beads. Motile cells clear the beads, located along their migratory paths, forming tracks that are visualized using an automated, transmitted-light screening microscope. The tracks are then segmented and characterized by multi-parametric, morphometric analysis, resolving a variety of morphological and kinetic features.

Conclusions

In this screen we identified 4 novel genes derived from breast carcinoma related cDNA library, whose over-expression induces major alteration in the migration of the stationary MCF7 cells. This approach can serve for high throughput screening for novel ways to modulate cellular migration in pathological states such as tumor metastasis and invasion.     

Production of knockout mice by gene targeting in multipotent germline stem cells

Spermatogonial stem cells can convert into embryonic stem (ES) cell-like multipotent germline stem (mGS) cells in vitro and produce germline chimeras by blastocyst microinjection. Although homologous recombination was previously demonstrated in mGS cells, spermatogenesis was not found in chimeras, suggesting that they are not competent for germline modification. Here we conducted detailed analysis of chimeric animals to determine whether mGS cells retain germline potential after genetic manipulation. Spermatozoa that were deficient in the occludin gene could be recovered from animals that were chimeric with mGS cells that underwent homologous recombination. The phenotypes of the occludin knockout (KO) mice were similar to those reported for KO mice produced using ES cells, and the animals showed growth retardation, gastritis and male infertility. Furthermore, we found that heterozygous mGS cells acquire two copies of the G418-resistant genes and become homozygous for the targeted allele by culturing at high concentrations of G418. Cytogenetic analysis showed that the aneuploid mGS cells observed during genetic manipulation were trisomic for chromosome 8 or 11, which is a common chromosomal abnormality in ES cells. Thus, mGS cells can be used to produce KO animals, and this novel method of germline manipulation may prove useful in diverse mammalian species.     

piwi encodes a nucleoplasmic factor whose activity modulates the number and division rate of germline stem cells

piwi represents the first class of genes known to be required for stem cell self-renewal in diverse organisms. In the Drosophila ovary, piwi is required in somatic signaling cells to maintain germline stem cells. Here we show that piwi encodes a novel nucleoplasmic protein present in both somatic and germline cells, with the highly conserved C-terminal region essential for its function. Removing PIWI protein from single germline stem cells significantly decreases the rate of their division. This suggests that PIWI has a second role as a cell-autonomous promoter of germline stem cell division. Consistent with its dual function, over-expression of piwi in somatic cells causes an increase both in the number of germline stem cells and the rate of their division. Thus, PIWI is a key regulator of stem cell division - its somatic expression modulates the number of germline stem cells and the rate of their division, while its germline expression also contributes to promoting stem cell division in a cell-autonomous manner.     

C. elegans MCM-4 is a general DNA replication and checkpoint component with an epidermis-specific requirement for growth and viability

DNA replication and its connection to M phase restraint are studied extensively at the level of single cells but rarely in the context of a developing animal. C. elegans lin-6 mutants lack DNA synthesis in postembryonic somatic cell lineages, while entry into mitosis continues. These mutants grow slowly and either die during larval development or develop into sterile adults. We found that lin-6 corresponds to mcm-4 and encodes an evolutionarily conserved component of the MCM2-7 pre-RC and replicative helicase complex. The MCM-4 protein is expressed in all dividing cells during embryonic and postembryonic development and associates with chromatin in late anaphase. Induction of cell cycle entry and differentiation continues in developing mcm-4 larvae, even in cells that went through abortive division. In contrast to somatic cells in mcm-4 mutants, the gonad continues DNA replication and cell division until late larval development. Expression of MCM-4 in the epidermis (also known as hypodermis) is sufficient to rescue the growth retardation and lethality of mcm-4 mutants. While the somatic gonad and germline show substantial ability to cope with lack of zygotic mcm-4 function, mcm-4 is specifically required in the epidermis for growth and survival of the whole organism. Thus, C. elegans mcm-4 has conserved functions in DNA replication and replication checkpoint control but also shows unexpected tissue-specific requirements.