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.     

MES-1, a protein required for unequal divisions of the germline in early C. elegans embryos, resembles receptor tyrosine kinases and is localized to the boundary between the germline and gut cells

During Caenorhabditis elegans embryogenesis the primordial germ cell, P(4), is generated via a series of unequal divisions. These divisions produce germline blastomeres (P(1), P(2), P(3), P(4)) that differ from their somatic sisters in their size, fate and cytoplasmic content (e.g. germ granules). mes-1 mutant embryos display the striking phenotype of transformation of P(4) into a muscle precursor, like its somatic sister. A loss of polarity in P(2) and P(3) cell-specific events underlies the Mes-1 phenotype. In mes-1 embryos, P(2) and P(3) undergo symmetric divisions and partition germ granules to both daughters. This paper shows that mes-1 encodes a receptor tyrosine kinase-like protein, though it lacks several residues conserved in all kinases and therefore is predicted not to have kinase activity. Immunolocalization analysis shows that MES-1 is present in four- to 24-cell embryos, where it is localized in a crescent at the junction between the germline cell and its neighboring gut cell. This is the region of P(2) and P(3) to which the spindle and P granules must move to ensure normal division asymmetry and cytoplasmic partitioning. Indeed, during early stages of mitosis in P(2) and P(3), one centrosome is positioned adjacent to the MES-1 crescent. Staining of isolated blastomeres demonstrated that MES-1 was present in the membrane of the germline blastomeres, consistent with a cell-autonomous function. Analysis of MES-1 distribution in various cell-fate and patterning mutants suggests that its localization is not dependent on the correct fate of either the germline or the gut blastomere but is dependent upon correct spatial organization of the embryo. Our results suggest that MES-1 directly positions the developing mitotic spindle and its associated P granules within P(2) and P(3), or provides an orientation signal for P(2)- and P(3)-specific events.     

The MES-2/MES-3/MES-6 complex and regulation of histone H3 methylation in C. elegans

The C. elegans proteins MES-2 and MES-6, orthologs of the Polycomb group (PcG) chromatin repressors E(Z) and ESC, exist in a complex with their novel partner MES-3. The MES system participates in silencing the X chromosomes in the hermaphrodite germline. Loss of maternal MES function leads to germline degeneration and sterility. We report here that the MES complex is responsible for di- and trimethylation of histone H3 Lys27 (H3-K27) in the adult germline and in early embryos and that MES-dependent H3-K27 marks are concentrated on the X's. Another H3-K27 HMT functions in adult somatic cells, oocytes, and the PGCs of embryos. In PGCs, the MES complex may specifically convert dimethyl to trimethyl H3-K27. The HMT activity of the MES complex appears to be dependent on the SET domain of MES-2. MES-2 thus joins its orthologs Drosophila E(Z) and human EZH2 among SET domain proteins known to function as HMTs (reviewed in ). Methylation of histones is important for long-term epigenetic regulation of chromatin and plays a key role in diverse processes such as X inactivation and oncogenesis. Our results contribute to understanding the composition and roles of E(Z)/MES-2 complexes across species.     

Cyclin E and Cdk2 control GLD-1, the mitosis/meiosis decision, and germline stem cells in Caenorhabditis elegans

Coordination of the cell cycle with developmental events is crucial for generation of tissues during development and their maintenance in adults. Defects in that coordination can shift the balance of cell fates with devastating clinical effects. Yet our understanding of the molecular mechanisms integrating core cell cycle regulators with developmental regulators remains in its infancy. This work focuses on the interplay between cell cycle and developmental regulators in the Caenorhabditis elegans germline. Key developmental regulators control germline stem cells (GSCs) to self-renew or begin differentiation: FBF RNA-binding proteins promote self-renewal, while GLD RNA regulatory proteins promote meiotic entry. We first discovered that many but not all germ cells switch from the mitotic into the meiotic cell cycle after RNAi depletion of CYE-1 (C. elegans cyclin E) or CDK-2 (C. elegans Cdk2) in wild-type adults. Therefore, CYE-1/CDK-2 influences the mitosis/meiosis balance. We next found that GLD-1 is expressed ectopically in GSCs after CYE-1 or CDK-2 depletion and that GLD-1 removal can rescue cye-1/cdk-2 defects. Therefore, GLD-1 is crucial for the CYE-1/CDK-2 mitosis/meiosis control. Indeed, GLD-1 appears to be a direct substrate of CYE-1/CDK-2: GLD-1 is a phosphoprotein; CYE-1/CDK-2 regulates its phosphorylation in vivo; and human cyclin E/Cdk2 phosphorylates GLD-1 in vitro. Transgenic GLD-1(AAA) harbors alanine substitutions at three consensus CDK phosphorylation sites. GLD-1(AAA) is expressed ectopically in GSCs, and GLD-1(AAA) transgenic germlines have a smaller than normal mitotic zone. Together these findings forge a regulatory link between CYE-1/CDK-2 and GLD-1. Finally, we find that CYE-1/CDK-2 works with FBF-1 to maintain GSCs and prevent their meiotic entry, at least in part, by lowering GLD-1 abundance. Therefore, CYE-1/CDK-2 emerges as a critical regulator of stem cell maintenance. We suggest that cyclin E and Cdk-2 may be used broadly to control developmental regulators.     

Translational repression of a C. elegans Notch mRNA by the STAR/KH domain protein GLD-1

In C. elegans, the Notch receptor GLP-1 is localized within the germline and early embryo by translational control of glp-1 mRNA. RNA elements in the glp-1 3'untranslated region (3' UTR) are necessary for repression of glp-1 translation in germ cells, and for localization of translation to anterior cells of the early embryo. The direct regulators of glp-1 mRNA are not known. Here, we show that a 34 nucleotide region of the glp-1 3' UTR contains two regulatory elements, an element that represses translation in germ cells and posterior cells of the early embryo, and an element that inhibits repressor activity to promote translation in the embryo. Furthermore, we show that the STAR/KH domain protein GLD-1 binds directly and specifically to the repressor element. Depletion of GLD-1 activity by RNA interference causes loss of endogenous glp-1 mRNA repression in early meiotic germ cells, and in posterior cells of the early embryo. Therefore, GLD-1 is a direct repressor of glp-1 translation at two developmental stages. These results suggest a new function for GLD-1 in regulating early embryonic asymmetry. Furthermore, these observations indicate that precise control of GLD-1 activity by other regulatory factors is important to localize this Notch receptor, and contributes to the spatial organization of Notch signaling.     

Linker histone HIS-24 (H1.1) cytoplasmic retention promotes germ line development and influences histone H3 methylation in Caenorhabditis elegans

RNA interference with one of the eight Caenorhabditis elegans linker histone genes triggers desilencing of a repetitive transgene and developmental defects in the hermaphrodite germ line. These characteristics are similar to the phenotype of the C. elegans Polycomb group genes mes-2, mes-3, mes-4, and mes-6 (M. A. Jedrusik and E. Schulze, Development 128:1069-1080, 2001; I. Korf, Y. Fan, and S. Strome, Development 125:2469-2478, 1998). These Polycomb group proteins contribute to germ line-specific chromatin modifications. Using a his-24 deletion mutant and an isoform-specific antibody, we characterized the role of his-24 in C. elegans germ line development. We describe an unexpected cytoplasmic retention of HIS-24 in peculiar granular structures. This phenomenon is confined to the developing germ lines of both sexes. It is strictly dependent on the activities of the chromatin-modifying genes mes-2, mes-3, mes-4, and mes-6, as well as on the C. elegans sirtuin gene sir-2.1. A temperature shift experiment with a mes-3(ts) mutant revealed that mes gene activity is required in a time window ranging from L3 to the early L4 stage before the onset of meiosis. We find that the his-24(ok1024) mutant germ line is characterized by an increased level of the activating H3K4 methylation mark concomitant with a decrease of the repressive H3K9 methylation. In the germ line of his-24(ok1024) mes-3(bn35) double mutant animals, the repressive H3K27 methylation is more reduced than in the respective mes single mutant. These observations distinguish his-24 as an unusual element in the developmental regulation of germ line chromatin structure in C. elegans.     

synMuv B proteins antagonize germline fate in the intestine and ensure C. elegans survival

Previous studies demonstrated that a subset of synMuv B mutants ectopically misexpress germline-specific P-granule proteins in their somatic cells, suggesting a failure to properly orchestrate a soma/germline fate decision. Surprisingly, this fate confusion does not affect viability at low to ambient temperatures. Here, we show that, when grown at high temperature, a majority of synMuv B mutants irreversibly arrest at the L1 stage. High temperature arrest (HTA) is accompanied by upregulation of many genes characteristic of germ line, including genes encoding components of the synaptonemal complex and other meiosis proteins. HTA is suppressed by loss of global regulators of germline chromatin, including MES-4, MRG-1, ISW-1 and the MES-2/3/6 complex, revealing that arrest is caused by somatic cells possessing a germline-like chromatin state. Germline genes are preferentially misregulated in the intestine, and necessity and sufficiency tests demonstrate that the intestine is the tissue responsible for HTA. We propose that synMuv B mutants fail to erase or antagonize an inherited germline chromatin state in somatic cells during embryonic and early larval development. As a consequence, somatic cells gain a germline program of gene expression in addition to their somatic program, leading to a mixed fate. Somatic expression of germline genes is enhanced at elevated temperature, leading to developmentally compromised somatic cells and arrest of newly hatched larvae.     

MEG-1 and MEG-2 are embryo-specific P-granule components required for germline development in Caenorhabditis elegans

In Caenorhabditis elegans, germ granules called P granules are directly inherited from mother to daughter and segregate with the germ lineage as it separates from the soma during initial embryonic cell divisions. Here we define meg-1 and meg-2 (maternal-effect germ-cell defective), which are expressed in the maternal germline and encode proteins that localize exclusively to P granules during embryonic germline segregation. Localization of MEG-1 to P granules depends upon the membrane-bound protein MES-1. meg-1 mutants exhibit multiple germline defects: P-granule mis-segregation in embryos, underproliferation and aberrant P-granule morphology in larval germ cells, and ultimately, sterility as adults. The penetrance of meg-1 phenotypes increases when meg-2 is also absent. Loss of the P-granule component pgl-1 in meg-1 mutants increases germ-cell proliferation, while loss of glh-1 decreases proliferation. Because meg-1 is provided maternally but its action is required in the embryonic germ lineage during segregation from somatic lineages, it provides a critical link for ensuring the continuity of germline development from one generation to the next.     

EGO-1, a putative RNA-directed RNA polymerase, promotes germline proliferation in parallel with GLP-1/notch signaling and regulates the spatial organization of nuclear pore complexes and germline P granules in Caenorhabditis elegans

Caenorhabditis elegans EGO-1, a putative cellular RNA-directed RNA polymerase, promotes several aspects of germline development, including proliferation, meiosis, and gametogenesis, and ensures a robust response to RNA interference. In C. elegans, GLP-1/Notch signaling from the somatic gonad maintains a population of proliferating germ cells, while entry of germ cells into meiosis is triggered by the GLD-1 and GLD-2 pathways. GLP-1 signaling prevents germ cells from entering meiosis by inhibiting GLD-1 and GLD-2 activity. We originally identified the ego-1 gene on the basis of a genetic interaction with glp-1. Here, we investigate the role of ego-1 in germline proliferation. Our data indicate that EGO-1 does not positively regulate GLP-1 protein levels or GLP-1 signaling activity. Moreover, GLP-1 signaling does not positively regulate EGO-1 activity. EGO-1 does not inhibit expression of GLD-1 protein in the distal germline. Instead, EGO-1 acts in parallel with GLP-1 signaling to influence the proliferation vs. meiosis fate choice. Moreover, EGO-1 and GLD-1 act in parallel to ensure germline health. Finally, the size and distribution of nuclear pore complexes and perinuclear P granules are altered in the absence of EGO-1, effects that disrupt germ cell biology per se and probably limit germline growth.     

The STAR/Maxi-KH domain protein GLD-1 mediates a developmental switch in the translational control of C. elegans PAL-1

Translational control is an essential mechanism of gene control utilized throughout development, yet the molecular mechanisms underlying translational activation and repression are poorly understood. We have investigated the translational control of the C. elegans caudal homolog, pal-1, and found that GLD-1, a member of the evolutionarily conserved STAR/Maxi-KH domain family, acts through a minimal pal-1 3' UTR element to repress pal-1 translation in the distal germline. We also provide data suggesting that GLD-1 may repress pal-1 translation after initiation. Finally, we show that GLD-1 represses the distal germline expression of the KH domain protein MEX-3, which was previously shown to repress PAL-1 expression in the proximal germline and which appears specialized to control PAL-1 expression patterns in the embryo. Hence, GLD-1 mediates a developmental switch in the control of PAL-1 repression, allowing MEX-3 to accumulate and take over the task of PAL-1 repression in the proximal germline, where GLD-1 protein levels decline.     

Caenorhabditis elegans atx-2 promotes germline proliferation and the oocyte fate

In the Caenorhabditis elegans germline, proliferation is induced by Notch-type signaling. Entry of germ cells into meiosis is triggered by activity of the GLD-1 and GLD-2 pathways, which function redundantly to promote meiosis and/or inhibit proliferation. Activation of the germline Notch-type receptor, GLP-1, ultimately inhibits the activities of the GLD-1 and GLD-2 pathways. We previously identified several ego (enhancer of glp-1) genes that promote germline proliferation and interact genetically with the GLP-1 signaling pathway. Here, we show that atx-2 is an ego gene. Our data suggest that ATX-2 is not a positive regulator of the GLP-1 signaling pathway and GLP-1 signaling is not the sole positive regulator of ATX-2 activity. Moreover, our data indicate that GLP-1 must have an additional function, which may be to repress activity of a third meiotic entry pathway that would work in parallel with the GLD-1 and GLD-2 pathways. In addition to its role in proliferation, ATX-2 acts downstream of FOG-2 to promote the female germline fate.