Genetic control of sex determination in the germ line of Caenorhabditis elegans

The nematode Caenorhabditis elegans normally exists as one of two sexes: self-fertilizing hermaphrodite or male. Development as hermaphrodite or male requires the differentiation of each tissue in a sex-specific way. In this review, I discuss the genetic control of sex determination in a single tissue of C. elegans: the germ line. Sex determination in the germ line depends on the action of two types of genes:--those that act globally in all tissues to direct male or female development and those that act only in the germ line to specify either spermatogenesis or oogenesis. First, I consider a tissue-specific sex-determining gene, fog-1, which promotes spermatogenesis in the germ line. Second, I consider the regulation of the hermaphrodite pattern of germ-line gametogenesis where first sperm and then oocytes are produced.     

The role of cell-cell interactions in postembryonic development of the Caenorhabditis elegans germ line.

This review addresses the role of cell-cell interactions in the development of the Caenorhabditis elegans germ line: specifically, the relative contributions of germ-line-soma interactions versus autonomous processes are considered. Current knowledge of the interacting cell types and the genes essential for various aspects of germ-line development is discussed.     

Dynamic regulation of caveolin-1 trafficking in the germ line and embryo of Caenorhabditis elegans

Caveolin is the major protein component required for the formation of caveolae on the plasma membrane. Here we show that trafficking of Caenorhabditis elegans caveolin-1 (CAV-1) is dynamically regulated during development of the germ line and embryo. In oocytes a CAV-1-green fluorescent protein (GFP) fusion protein is found on the plasma membrane and in large vesicles (CAV-1 bodies). After ovulation and fertilization the CAV-1 bodies fuse with the plasma membrane in a manner reminiscent of cortical granule exocytosis as described in other species. Fusion of CAV-1 bodies with the plasma membrane appears to be regulated by the advancing cell cycle, and not fertilization per se, because fusion can proceed in spe-9 fertilization mutants but is blocked by RNA interference-mediated knockdown of an anaphase-promoting complex component (EMB-27). After exocytosis, most CAV-1-GFP is rapidly endocytosed and degraded within one cell cycle. CAV-1 bodies in oocytes appear to be produced by the Golgi apparatus in an ARF-1-dependent, clathrin-independent, mechanism. Conversely endocytosis and degradation of CAV-1-GFP in embryos requires clathrin, dynamin, and RAB-5. Our results demonstrate that the distribution of CAV-1 is highly dynamic during development and provides new insights into the sorting mechanisms that regulate CAV-1 localization.     

Proteasomal regulation of the proliferation vs. meiotic entry decision in the Caenorhabditis elegans germ line

Reproductive fitness in many animals relies upon a tight balance between the number of cells that proliferate in the germ line and the number of cells that enter meiosis and differentiate as gametes. In the Caenorhabditis elegans germ line, the GLP-1/Notch signaling pathway controls this balance between proliferation and meiotic entry. Here we describe the identification of the proteasome as an additional regulator of this balance. We show that a decrease in proteasome activity, through either genetic mutation or RNAi to core components of the proteasome, shifts this balance toward excess germ-line proliferation. We further demonstrate that there are likely two or more proteasome targets that contribute to excess germ-line proliferation when proteasome activity is reduced. One of these targets is likely a component or regulator of the Notch-signaling pathway, while the other functions on one of the two major redundant genetic pathways downstream of GLP-1/Notch signaling. We propose a model in which the proteasome degrades proteins that are necessary for proliferation as cells switch from proliferation to meiotic entry.     

A role for Caenorhabditis elegans importin IMA-2 in germ line and embryonic mitosis

The importin alpha family of nuclear-cytoplasmic transport factors mediates the nuclear localization of proteins containing classical nuclear localization signals. Metazoan animals express multiple importin alpha proteins, suggesting their possible roles in cell differentiation and development. Adult Caenorhabditis elegans hermaphrodites express three importin alpha proteins, IMA-1, IMA-2, and IMA-3, each with a distinct expression and localization pattern. IMA-2 was expressed exclusively in germ line cells from the early embryonic through adult stages. The protein has a dynamic pattern of localization dependent on the stage of the cell cycle. In interphase germ cells and embryonic cells, IMA-2 is cytoplasmic and nuclear envelope associated, whereas in developing oocytes, the protein is cytoplasmic and intranuclear. During mitosis in germ line cells and embryos, IMA-2 surrounded the condensed chromosomes but was not directly associated with the mitotic spindle. The timing of IMA-2 nuclear localization suggested that the protein surrounded the chromosomes after fenestration of the nuclear envelope in prometaphase. Depletion of IMA-2 by RNA-mediated gene interference (RNAi) resulted in embryonic lethality and a terminal aneuploid phenotype. ima-2(RNAi) embryos have severe defects in nuclear envelope formation, accumulating nucleoporins and lamin in the cytoplasm. We conclude that IMA-2 is required for proper chromosome dynamics in germ line and early embryonic mitosis and is involved in nuclear envelope assembly at the conclusion of mitosis.     

Progression from mitotic catastrophe to germ cell death in Caenorhabditis elegans lis-1 mutants requires the spindle checkpoint

Deletion of the lissencephaly disease gene LIS-1 in humans causes an extreme disorganization of the brain associated with significant reduction in cortical neurons. Here we show that deletion or RNA interference (RNAi) of Caenorhabditis elegans lis-1 results in a reduction in germline nuclei and causes a variety of cellular, developmental, and neurological defects throughout development. Our analysis of the germline defects suggests that the reduction in nuclei number stems from dysfunctional mitotic spindles resulting in cell cycle arrest and eventually programmed cell death (apoptosis). Deletion of the spindle checkpoint gene mdf-1 blocks lis-1(lf)-induced cell cycle arrest and germline apoptosis, placing the spindle checkpoint pathway upstream of the programmed cell death pathway. These results suggest that apoptosis may contribute to the cell-sparse pathology of lissencephaly.     

On the control of germ cell development in Caenorhabditis elegans

After hatching, the germ line progenitor cells in C. elegans begin to divide mitotically; later, some of the germ line cells enter meiosis and differentiate into gametes. In the adult, mitotic germ cells, or stem cells, are found at one end (the distal end) and meiotic cells occupy the rest of the elongate gonad. Removal of two somatic gonadal cells, the distal tip cells, by laser microsurgery has a dramatic effect on germ cell development. In either sex, this operation leads to the arrest of mitosis and the initiation of meiosis in germ cells. The function of the distal tip cell in the intact animal appears to be the inhibition of meiosis (or stimulation of mitosis) in nearby germ cells. During development, this permits growth and, in the adult, it maintains the germ line stem cell population. A change in the position of the distal tip cell in the gonad at an early point in development is correlated with a change in the axial polarity of the germ line tissue. This suggests that the localization of the distal tip cell's inhibitory activity at the distal end of the gonad establishes the axial polarity of the germ line tissue in the intact animal.     

Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line

Interactions between the somatic gonad and the germ line influence the amplification, maintenance, and differentiation of germ cells. In Caenorhabditis elegans, the distal tip cell/germline interaction promotes a mitotic fate and/or inhibits meiosis through GLP-1/Notch signaling. However, GLP-1-mediated signaling alone is not sufficient for a wild-type level of germline proliferation. Here, we provide evidence that specific cells of the somatic gonadal sheath lineage influence amplification, differentiation, and the potential for tumorigenesis of the germ line. First, an interaction between the distal-most pair of sheath cells and the proliferation zone of the germ line is required for larval germline amplification. Second, we show that insufficient larval germline amplification retards gonad elongation and thus delays meiotic entry. Third, a more severe delay in meiotic entry, as is exhibited in certain mutant backgrounds, inappropriately juxtaposes undifferentiated germ cells with cells of the proximal sheath lineage, leading to the formation of a proximal germline tumor derived from undifferentiated germ cells. Tumors derived from dedifferentiated germ cells, however, respond to the proximal interaction differently depending on the mutant background. Our study underscores the importance of strict developmental coordination between neighboring tissues. We discuss these results in the context of mechanisms that may underlie tumorigenesis.     

Cellular Symmetry Breaking during Caenorhabditis elegans Development

The nematode worm Caenorhabditis elegans has produced a wellspring of insights into mechanisms that govern cellular symmetry breaking during animal development. Here we focus on two highly conserved systems that underlie many of the key symmetry-breaking events that occur during embryonic and larval development in the worm. One involves the interplay between Par proteins, Rho GTPases, and the actomyosin cytoskeleton and mediates asymmetric cell divisions that establish the germline. The other uses elements of the Wnt signaling pathway and a highly reiterative mechanism that distinguishes anterior from posterior daughter cell fates. Much of what we know about these systems comes from intensive study of a few key events—Par/Rho/actomyosin-mediated polarization of the zygote in response to a sperm-derived cue and the Wnt-mediated induction of endoderm at the four-cell stage. However, a growing body of work is revealing how C. elegans exploits elements/variants of these systems to accomplish a diversity of symmetry-breaking tasks throughout embryonic and larval development.Over the past few decades, the C. elegans embryo has become a premiere system for studying cellular symmetry breaking in a developmental context. During C. elegans development, nearly every division produces daughter cells with different developmental trajectories. In some cases, these differences are imposed on daughters before or after division through inductive signals, but many of these divisions are intrinsically asymmetric—an initial symmetry-breaking step creates polarized distributions or activities of factors that control developmental potential. Registration of the cleavage plane with the axis of polarity then ensures differential inheritance of these potentials. With respect to cell fates, the output of these asymmetric divisions is amazingly diverse, yet the embryo seems to accomplish this diversity through variants of a few conserved symmetry-breaking systems. Thus the C. elegans embryo provides an exceptional opportunity to explore not only the core mechanisms underlying cellular symmetry breaking, but also how evolution can reconfigure these mechanisms to do different but related jobs in multiple contexts.In this review, we focus most of our attention on two conserved systems that together account for much of the cellular asymmetry observed during C. elegans embryogenesis. The first, which is best known for its role in the early asymmetric cell divisions that segregate germline from the soma, involves a complex interplay between Par proteins, Rho-family GTPases, and the actomyosin cytoskeleton. Interestingly, the embryo exploits elements of this same system to break symmetry during cleavage furrow specification and to establish apicobasal polarity in early embryonic cells and in the first true embryonic epithelia. The second system we focus on involves an unusual application of WNT signaling pathway components and is used reiteratively throughout embryonic and larval development to distinguish anterior and posterior daughter cell fates. Rather than comprehensively review these systems, we highlight topics not extensively covered in other reviews.     

Morphologically distinct microtubule ends in the mitotic centrosome of Caenorhabditis elegans

During mitosis, the connections of microtubules (MTs) to centrosomes and kinetochores are dynamic. From in vitro studies, it is known that the dynamic behavior of MTs is related to the structure of their ends, but we know little about the structure of MT ends in spindles. Here, we use high-voltage electron tomography to study the centrosome- and kinetochore-associated ends of spindle MTs in embryonic cells of the nematode, Caenorhabditis elegans. Centrosome-associated MT ends are either closed or open. Closed MT ends are more numerous and are uniformly distributed around the centrosome, but open ends are found preferentially on kinetochore-attached MTs. These results have structural implications for models of MT interactions with centrosomes.     

Circadian stress tolerance in adult Caenorhabditis elegans

Circadian rhythms control several behaviors through neural networks, hormones and gene expression. One of these outputs in invertebrates, vertebrates and plants is the stress resistance behavior. In this work, we studied the circadian variation in abiotic stress resistance of adult C. elegans as well as the genetic mechanisms that underlie such behavior. Measuring the stress resistance by tap response behavior we found a rhythm in response to osmotic (NaCl LC(50) = 340 mM) and oxidative (H(2)O(2) LC(50) = 50 mM) shocks, with a minimum at ZT0 (i.e., lights off) and ZT12 (lights on), respectively. In addition, the expression of glutathione peroxidase (C11E4.1) and glycerol-3-phosphate dehydrogenase (gpdh-1) (genes related to the control of stress responses) also showed a circadian fluctuation in basal levels with a peak at night. Moreover, in the mutant osr-1 (AM1 strain), a negative regulator of the gpdh-1 pathway, the osmotic resistance rhythms were masked at 350 mM but reappeared when the strain was treated with a higher NaCl concentration. This work demonstrates for the first time that in the adult nematode, C. elegans stress responses vary daily, and provides evidence of an underlying rhythmic gene expression that governs these behaviors.     

Differential proteome analysis and mass spectrometric characterization of germ line development-related proteins of Caenorhabditis elegans

Proteome maps and differences of protein patterns of the synchronized larval stage L4 of the temperature-sensitive Caenorhabditis elegans (C. elegans) glp-1 mutant (e2144ts) were investigated after cultivation at 15 degrees C (developing a normal phenotype) or 25 degrees C (developing a mutated phenotype) by two-dimensional gel electrophoresis (2-DE) and matrix-assisted laser desorption/ionization (MALDI)-mass spectrometry. From the 183 identified protein spots six proteins were found differently expressed. The Vit-6 vitellogenin (CE28594), the hypothetical 17.2 protein (CE25224), the hypothetical 17.4 protein (CE16999), and the heat shock protein 16 kDa (CE14249) were more abundant when growing worm cultures at 25 degrees C. By contrast, the nucleoside diphosphate kinase (CE09650) was found increased at 15 degrees C. Most notably, the eukariotic initiation factor 5A-1 (CE00503), highly abundant at 15 degrees C, was not present in cultures grown at 25 degrees C. Its absence at 25 degrees C can not be attributed to lack of the enzymatic machinery that is necessary for hypusinylation. Instead, a direct downstream effect of the lack of functionality of GLP-1 may cause the expression of this protein. The yolk proteins 115 kDa and 88 kDa were attributed by mass spectrometric protein structure analysis as C-terminal and N-terminal fragments of the Vit-6 vitellogin protein (CE28594), respectively. The cleavage site between both derivatives was located between R764 and A768. A conflict in the database sequences at amino acid positions 1622 and 1623 of vitellogenin-6 was solved by mass spectrometric sequence analysis. The combination of 2-DE with mass spectrometry enabled the identification of mutation-associated differences on somatic gonadal cell and germ line cell development-associated proteins.     

More mog genes that influence the switch from spermatogenesis to oogenesis in the hermaphrodite germ line of Caenorhabditis elegans

The Caenorhobditis elegans XX animal possesses a hermaphrodite germ line, producing first sperm, then oocytes. In this paper, we report the genetic identification of five genes, mog-2, mog-3, mog-4, mog-5, and mog-6, that influence the hermaphrodite switch from sper-matogenesis to oogenesis. In mcg-2-mog-6 mutants, spermatogenesis continues past the time at which hermaphrodites normally switch into oogenesis and no oocytes are observed. Therefore, in these mutants, germ cells are transformed from a female fate (oocyte) to a male fate (sperm). The fem-3 gene is one of five genes that acts at the end of the germline sex determination pathway to direct spermatogenesis. Analyses of mog;fem-3 double mutants suggest that the mog-2-mog-6 genes act before fem-3; thus these genes may be in a position to negatively regulate fem-3 or one of the other terminal regulators of germline sex determination. Double mutants of fem-3 and any one of the mog mutations make oocytes. Using these double mutants, we show that oocytes from any mog;fem-3 double mutant are defective in their ability to support embryogenesis. This maternal effect lethality indicates that each of the mog genes is required for embryogenesis. The two defects in mog-2-mog-6 mutants are similar to those of mog-1: all six mog genes eliminate the sperm/oocyte switch in hermaphrodites and cause maternal effect lethality. We propose that the mog-2-mog-6 mutations identify genes that act with mog-1 to effect the sperm/oocyte switch. We further speculate that the mog-1-mog-6 mutations all interfere with translational controls of fem-3 and other maternal mRNAs. © 1993 Wiley-Liss, Inc.     

In vivo construction of recombinant molecules within the Caenorhabditis elegans germ line using short regions of terminal homology

Homologous recombination provides a means for the in vivo construction of recombinant DNA molecules that may be problematic to assemble in vitro. We have investigated the efficiency of recombination within the Caenorhabditis elegans germ line as a function of the length of homology between recombining molecules. Our findings indicate that recombination can occur between molecules that share only 10 bp of terminal homology, and that 25 bp is sufficient to mediate relatively high levels of recombination. Recombination occurs with lower efficiency when the location of the homologous segment is subterminal or internal. As in yeast, recombination can also be mediated by either single- or double-stranded bridging oligonucleotides. We find that ligation between cohesive ends is highly efficient and does not require that the ends be phosphorylated; furthermore, precise intermolecular ligation between injected molecules that have blunt ends can also occur within the germ line.     

Phosphorylation of the N-terminal region of Caenorhabditis elegans paramyosin

Paramyosin from Caenorhabditis elegans was examined for post-translational modification by phosphorylation. Paramyosin purified from populations of mixed-age animals contained 0.7 to 2.0 moles of phosphate per mole of paramyosin. Paramyosin was also phosphorylated in vitro by an endogenous kinase in the particulate fraction. Analysis of the in vitro phosphorylated paramyosin in comparison with the DNA sequence of the unc-15 paramyosin gene of C. elegans shows that serine residues in the non-alpha-helical N-terminal region are the targets of the kinase. The N-terminal region of paramyosin has significant similarity to the non-helical C-terminal region of the two body wall myosin heavy chains of C. elegans. All three regions contain three copies of a Ser-*-Ser-*-Ala motif, the most likely target for phosphorylation in paramyosin, suggesting that these regions may be modified by the same kinase.     

Characterization of wheat germ agglutinin ligand on soluble glycoproteins in Caenorhabditis elegans

Some mutants of Caenorhabditis elegans show altered patterns of ectopic binding with wheat germ agglutinin (WGA). Some of these mutants also have defects of morphogenesis and movement during development. To clarify the structures of WGA-ligands in C. elegans that may be involved in developmental events, we have analyzed glycan structures capable of binding WGA. We isolated glycoproteins from wild-type C. elegans by WGA-affinity chromatography, and analyzed their glycan structures by a combination of hydrazine degradation and fluorescent labeling. The glycoproteins had oligomannose-type and complex-type N-glycans that included agalacto-biantenna and agalacto-tetraantenna glycans. Although the complex-type glycans carried beta-GlcNAc residues at their non-reducing ends, they did not bind to the WGA-agarose-resin. Thus, it was suggested that these N-glycans were not responsible for WGA-binding of the isolated glycoproteins. Hydrazinolysis of the glycoproteins also released a considerable amount of GalNAc monosaccharide. It was surmised that N-acetylgalactosamine was derived from mucin-type O-glycans with the Tn-antigen structure (GalNAcalpha1-O-Ser/Thr). WGA-blotting assay of neoglycoproteins revealed that a cluster of Tn-antigens was a good ligand for WGA. These results suggested that the WGA-ligand in C. elegans is a cluster of alpha-GalNAc monosaccharides linked to mucin-like glycoprotein(s). The observations reported in this paper emphasize the possible significance of mucin-type O-glycans in the development of a multicellular organism.     

MRG-1 is required for genomic integrity in Caenorhabditis elegans germ cells

During meiotic cell division, proper chromosome synapsis and accurate repair of DNA double strand breaks (DSBs) are required to maintain genomic integrity, loss of which leads to apoptosis or meiotic defects. The mechanisms underlying meiotic chromosome synapsis, DSB repair and apoptosis are not fully understood. Here, we report that the chromodomain-containing protein MRG-1 is an important factor for genomic integrity in meiosis in Caenorhabditis elegans. Loss of mrg-1 function resulted in a significant increase in germ cell apoptosis that was partially inhibited by mutations affecting DNA damage checkpoint genes. Consistently, mrg-1 mutant germ lines exhibited SPO-11-generated DSBs and elevated exogenous DNA damage-induced chromosome fragmentation at diakinesis. In addition, the excessive apoptosis in mrg-1 mutants was partially suppressed by loss of the synapsis checkpoint gene pch-2, and a significant number of meiotic nuclei accumulated at the leptotene/zygotene stages with an elevated level of H3K9me2 on the chromatin, which was similarly observed in mutants deficient in the synaptonemal complex, suggesting that the proper progression of chromosome synapsis is likely impaired in the absence of mrg-1. Altogether, these findings suggest that MRG-1 is critical for genomic integrity by promoting meiotic DSB repair and synapsis progression in meiosis.