Project description:In all animals examined, somatic cells of the gonad control multiple biological processes essential for germline development. Gap junction channels, composed of connexins in vertebrates and innexins in invertebrates, permit direct intercellular communication between cells and frequently form between somatic gonadal cells and germ cells. Gap junctions comprise hexameric hemichannels in apposing cells that dock to form channels for the exchange of small molecules. Here we report essential roles for two classes of gap junction channels, composed of five innexin proteins, in supporting the proliferation of germline stem cells and gametogenesis in the nematode Caenorhabditis elegans. Transmission electron microscopy of freeze-fracture replicas and fluorescence microscopy show that gap junctions between somatic cells and germ cells are more extensive than previously appreciated and are found throughout the gonad. One class of gap junctions, composed of INX-8 and INX-9 in the soma and INX-14 and INX-21 in the germ line, is required for the proliferation and differentiation of germline stem cells. Genetic epistasis experiments establish a role for these gap junction channels in germline proliferation independent of the glp-1/Notch pathway. A second class of gap junctions, composed of somatic INX-8 and INX-9 and germline INX-14 and INX-22, is required for the negative regulation of oocyte meiotic maturation. Rescue of gap junction channel formation in the stem cell niche rescues germline proliferation and uncovers a later channel requirement for embryonic viability. This analysis reveals gap junctions as a central organizing feature of many soma-germline interactions in C. elegans.
Project description:PGL-1 is a constitutive protein component of C. elegans germ granules, also known as P granules. Maternally supplied PGL-1 is essential for germline development but only at elevated temperature, raising the possibility that redundant factors provide sufficient function at lower temperatures. We have identified two PGL-1-related proteins, PGL-2 and PGL-3, by sequence analysis of the C. elegans genome and by a yeast two-hybrid screen for proteins that interact with PGL-1. PGL-3 is associated with P granules at all stages of development, while PGL-2 is associated with P granules only during postembryonic development. All three PGL proteins interact with each other in vitro. Furthermore, PGL-1 and PGL-3 are co-immunoprecipitated from embryo extracts, indicating that they are indeed in the same protein complex in vivo. Nevertheless, each PGL protein localizes to P granules independently of the other two. pgl-2 or pgl-3 single-mutant worms do not show obvious defects in germline development. However, pgl-1; pgl-3 (but not pgl-2; pgl-1) double-mutant hermaphrodites and males show significantly enhanced sterility at all temperatures, compared to pgl-1 alone. Mutant hermaphrodites show defects in germline proliferation and in production of healthy gametes and viable embryos. Our findings demonstrate that both PGL-2 and PGL-3 are components of P granules, both interact with PGL-1, and at least PGL-3 functions redundantly with PGL-1 to ensure fertility in both sexes of C. elegans.
Project description:To assess the mechanism by which adult germ cells induce cbs-1 expression in the intestine at cold temperature, we performed transcriptome analysis of extruded germ lines from wild-type worms upon iff-1 knockdown or temperature increase Overall design: We extruded germ line of iff-1 RNAi-treated worms at 15°C and empty vector (EV) RNAi-treated worms at 20°C and compared to the germ line of EV RNAi-treated worms at 15°C.
Project description:Here we describe an in vitro primary culture system for Caenorhabditis elegans germline stem cells. This culture system was used to identify a bacterial folate as a positive regulator of germ cell proliferation. Folates are a family of B-complex vitamins that function in one-carbon metabolism to allow the de novo synthesis of amino acids and nucleosides. We show that germ cell proliferation is stimulated by the folate 10-formyl-tetrahydrofolate-Glun both in vitro and in animals. Other folates that can act as vitamins to rescue folate deficiency lack this germ cell stimulatory activity. The bacterial folate precursor dihydropteroate also promotes germ cell proliferation in vitro and in vivo, despite its inability to promote one-carbon metabolism. The folate receptor homolog FOLR-1 is required for the stimulation of germ cells by 10-formyl-tetrahydrofolate-Glun and dihydropteroate. This work defines a folate and folate-related compound as exogenous signals to modulate germ cell proliferation.
Project description:Specialized ribonucleoprotein organelles collectively known as germ granules are found in the germline cytoplasm from worms to humans . In Drosophila, germ granules have been implicated in germline determination . C. elegans germ granules, known as P granules, do not appear to be required for primordial germ cell (PGC) determination , but their components are still needed for fertility [4-6]. One potential role for P granules is to maintain germline fate and totipotency. This is suggested by the loss of P granules from germ cells that transform into somatic cell types, e.g., in germlines lacking MEX-3 and GLD-1 or upon neuronal induction by CHE-1 [7, 8]. However, it has not been established whether loss of P granules is the cause or effect of cell fate transformation. To test cause and effect, we severely compromised P granules by simultaneously knocking down factors that nucleate granule formation (PGL-1 and PGL-3) and promote their perinuclear localization (GLH-1 and GLH-4)  and investigated whether this causes germ cells to lose totipotency and initiate somatic reprogramming. We found that compromising P granules causes germ cells to express neuronal and muscle markers and send out neurite-like projections, suggesting that P granules maintain totipotency and germline identity by antagonizing somatic fate.
Project description: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.
Project description:A number of environmental factors (e.g. toxicants) have been shown to promote the epigenetic transgenerational inheritance of disease and phenotypic variation. Transgenerational inheritance requires the germline transmission of altered epigenetic information between generations in the absence of direct environmental exposures. The primary periods for epigenetic programming of the germline is associated with primordial germ cell development and during fetal gonadal sex determination. The current study examined the actions of an agricultural fungicide vinclozolin on gestating female (F0 generation) progeny in regards to the primordial germ cell (PGC) epigenetic reprogramming of the F3 generation (i.e. great-grandchildren). The F3 generation primordial germ cell transcriptome and epigenome (DNA methylation) was altered transgenerationally. Interestingly, the differential DNA methylation regions (DMR) and altered transcriptomes were distinct between the onset of gonadal sex determination at embryonic day 13 (E13) and after cord formation in the testis at embryonic day 16 (E16). A larger number of DMR and transcriptional alterations were observed in the E13 PGC than E16 germ cells. Observations demonstrate an altered transgenerational epigenetic reprogramming and function of the primordial germ cells and subsequent male germline is a component of vinclozolin induced epigenetic transgenerational inheritance of disease. Insights into the molecular control of germline transmitted epigenetic inheritance are provided. The combined observations demonstrate ancestral exposure of a gestating female during fetal gonadal sex determination can promote transgenerational alterations in the primordial germ cell and subsequent male germline epigenetic and transcriptional programming. This altered germline programming leads to the epigenetic transgenerational inheritance of disease and phenotypic variation. Observations support the role of the primordial germ cell programming in the molecular mechanism involved and provides insights into the molecular mechanisms that control the epigenetic transgenerational inheritance phenomena. Results suggest a cascade of epigenetic and transcriptional events during germ cell development is needed to obtain the mature germline epigenome that is then transmitted transgenerationally. RNA samples from PGC of 2 F3-control lineage groups were compared to PGC of 2 F3-vinclozolin lineage groups for two embryonic age E13 and E16
Project description:The ubiquitin-proteolytic system controls the stability of proteins in space and time. In this study, using a temperature-sensitive mutant allele of the cul-2 gene, we show that CRL2(LRR-1) (CUL-2 RING E3 ubiquitin-ligase and the Leucine Rich Repeat 1 substrate recognition subunit) acts at multiple levels to control germline development. CRL2(LRR-1) promotes germ cell proliferation by counteracting the DNA replication ATL-1 checkpoint pathway. CRL2(LRR-1) also participates in the mitotic proliferation/meiotic entry decision, presumably controlling the stability of meiotic promoting factors in the mitotic zone of the germline. Finally, CRL2(LRR-1) inhibits the first steps of meiotic prophase by targeting in mitotic germ cells degradation of the HORMA domain-containing protein HTP-3, required for loading synaptonemal complex components onto meiotic chromosomes. Given its widespread evolutionary conservation, CUL-2 may similarly regulate germline development in other organisms as well.
Project description:Purpose:Pediatric germ cell tumors are rare, representing about 3% of childhood malignancies in children less than 15 years of age, presenting in neonates or adolescents with a greater incidence noted in older adolescents. Aberrations in primordial germ cell proliferation/differentiation can lead to a variety of neoplasms, including teratomas, embryonal carcinoma, choriocarcinoma, and yolk sac tumors. Patients and Methods:Three Finnish families with varying familial germ cell tumors were identified, and whole-genome sequencing was performed using an Illumina sequencing platform. In total, 22 unique subjects across the three families were sequenced. Family 1 proband (female) was affected by malignant ovarian teratoma, Family 2 proband (female) was affected by sacrococcygeal teratoma with yolk sac tumor in the setting of Cornelia de Lange syndrome, and Family 3 proband (male) was affected by malignant testicular teratoma. Rare variants were identified using an autosomal recessive or de novo model of inheritance. Results:For family 1 proband (female), an autosomal recessive or de novo model of inheritance identified variants of interest in the following genes: CD109, IKBKB, and CTNNA3, SUPT6H, MUC5AC, and FRG1. Family 2 proband (female) analysis identified gene variants of interest in the following genes: LONRF2, ANO7, HS6ST1, PRB2, and DNM2. Family 3 proband (male) analysis identified the following potential genes: CRIPAK, KRTAP5-7, and CACNA1B. Conclusion:Leveraging deep pedigrees and next-generation sequencing, rare germline variants were identified that were enriched in three families from Finland with a history of familial germ cell tumors. The data presented support the importance of germline mutations when analyzing complex cancers with a low somatic mutation landscape.
Project description:Proliferating germ cells in Caenorhabditiselegans provide a useful model system for deciphering fundamental mechanisms underlying the balance between proliferation and differentiation. Using gene expression profiling, we identified approximately 200 genes upregulated in the proliferating germ cells of C. elegans. Functional characterization using RNA-mediated interference demonstrated that over forty of these factors are required for normal germline proliferation and development. Detailed analysis of two of these factors defined an important regulatory relationship controlling germ cell proliferation. We established that the kinase VRK-1 is required for normal germ cell proliferation, and that it acts in part to regulate CEP-1(p53) activity. Loss of cep-1 significantly rescued the proliferation defects of vrk-1 mutants. We suggest that VRK-1 prevents CEP-1 from triggering an inappropriate cell cycle arrest, thereby promoting germ cell proliferation. This finding reveals a previously unsuspected mechanism for negative regulation of p53 activity in germ cells to control proliferation.