DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence
Quiescence, an actively-maintained reversible state of cell cycle arrest, is not well understood. PTEN is one of the most frequently lost tumor suppressors in human cancers and regulates quiescence of stem cells and cancer cells. The sole PTEN ortholog in Caenorhabditis elegans is daf-18. In a C. elegans loss-of-function mutant for daf-18, primordial germ cells (PGCs) divide inappropriately in L1 larvae hatched into starvation conditions, in a TOR-dependent manner. Here, we further investigated the role of daf-18 in maintaining PGC quiescence in L1 starvation. We found that maternal or zygotic daf-18 is sufficient to maintain cell cycle quiescence, that daf-18 acts in the germ line and soma, and that daf-18 affects timing of PGC divisions in fed animals. Importantly, our results also implicate daf-18 in repression of germline zygotic gene activation, though not in germline fate specification. However, TOR is less important to germline zygotic gene expression, suggesting that in the absence of food, daf-18/PTEN prevents inappropriate germline zygotic gene activation and cell division by distinct mechanisms.
Germline Stem and Progenitor Cell Aging in C. elegans
Like many animals and humans, reproduction in the nematode C. elegans declines with age. This decline is the cumulative result of age-related changes in several steps of germline function, many of which are highly accessible for experimental investigation in this short-lived model organism. Here we review recent work showing that a very early and major contributing step to reproductive decline is the depletion of the germline stem and progenitor cell pool. Since many cellular and molecular aspects of stem cell biology and aging are conserved across animals, understanding mechanisms of age-related decline of germline stem and progenitor cells in C. elegans has broad implications for aging stem cells, germline stem cells, and reproductive aging.
A Genome-Wide RNAi Screen for Enhancers of a Germline Tumor Phenotype Caused by Elevated GLP-1/Notch Signaling in Caenorhabditis elegans
Stem cells are tightly controlled in vivo Both the balance between self-renewal and differentiation and the rate of proliferation are often regulated by multiple factors. The Caenorhabditis elegans hermaphrodite germ line provides a simple and accessible system for studying stem cells in vivo In this system, GLP-1/Notch activity prevents the differentiation of distal germ cells in response to ligand production from the nearby distal tip cell, thereby supporting a stem cell pool. However, a delay in germline development relative to somatic gonad development can cause a pool of undifferentiated germ cells to persist in response to alternate Notch ligands expressed in the proximal somatic gonad. This pool of undifferentiated germ cells forms a proximal tumor that, in adulthood, blocks the oviduct. This type of "latent niche"-driven proximal tumor is highly penetrant in worms bearing the temperature-sensitive weak gain-of-function mutation glp-1(ar202) at the restrictive temperature. At the permissive temperature, few worms develop tumors. Nevertheless, several interventions elevate the penetrance of proximal tumor formation at the permissive temperature, including reduced insulin signaling or the ablation of distal-most sheath cells. To systematically identify genetic perturbations that enhance proximal tumor formation, we sought genes that, upon RNAi depletion, elevate the percentage of worms bearing proximal germline tumors in glp-1(ar202) at the permissive temperature. We identified 43 genes representing a variety of functional classes, the most enriched of which is "translation". Some of these genes also influence the distal germ line, and some are conserved genes for which genetic interactions with Notch were not previously known in this system.
Biology of the Caenorhabditis elegans Germline Stem Cell System
Stem cell systems regulate tissue development and maintenance. The germline stem cell system is essential for animal reproduction, controlling both the timing and number of progeny through its influence on gamete production. In this review, we first draw general comparisons to stem cell systems in other organisms, and then present our current understanding of the germline stem cell system in Caenorhabditis elegans In contrast to stereotypic somatic development and cell number stasis of adult somatic cells in C. elegans, the germline stem cell system has a variable division pattern, and the system differs between larval development, early adult peak reproduction and age-related decline. We discuss the cell and developmental biology of the stem cell system and the Notch regulated genetic network that controls the key decision between the stem cell fate and meiotic development, as it occurs under optimal laboratory conditions in adult and larval stages. We then discuss alterations of the stem cell system in response to environmental perturbations and aging. A recurring distinction is between processes that control stem cell fate and those that control cell cycle regulation. C. elegans is a powerful model for understanding germline stem cells and stem cell biology.
Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress
The roundworm C.Â elegans reversibly arrests larval development during starvation , but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success . However, the developmental basis of reduced fertility following early-life starvation is unknown, and it is unclear how maternal diet modifies developmental physiology in progeny. We show here that extended starvation in first-stage (L1) larvae followed by unrestricted feeding results in a variety of developmental abnormalities in the reproductive system, including proliferative germ-cell tumors and uterine masses that express neuronal and epidermal cell fate markers. We found that maternal DR and reduced maternal insulin/insulin-like growth factor (IGF) signaling (IIS) increase oocyte provisioning ofÂ vitellogenin lipoprotein, reducing penetrance of starvation-induced abnormalities in progeny, including tumors. Furthermore, we show that maternal DR and reduced maternal IIS reduce IIS in progeny. daf-16/FoxO and skn-1/Nrf, transcriptional effectors of IIS, are required in progeny for maternal DR and increased vitellogenin provisioning to suppress starvation-induced abnormalities. daf-16/FoxO activity in somatic tissues is sufficient to suppress starvation-induced abnormalities, suggesting cell-nonautonomous regulation of reproductive system development. This work reveals that early-life starvation compromises reproductive development and that vitellogenin-mediated intergenerational insulin/IGF-to-insulin/IGF signaling mediates adaptation to nutrient availability.
Ectopic Germ Cells Can Induce Niche-like Enwrapment by Neighboring Body Wall Muscle
Niche cell enwrapment of stem cells and their differentiating progeny is common and provides a specialized signaling and protective environment. Elucidating the mechanisms underlying enwrapment behavior has important basic and clinical significance in not only understanding how niches are formed and maintained but also how they can be engineered and how they are misregulated in human pathologies, such as cancer. Previous work in C.Â elegans found that, when germ cells, which are enwrapped by somatic gonadal niche cells, are freed into the body cavity, they embed into other tissues. We investigated this phenomenon using live-cell imaging and discovered that ectopic germ cells preferentially induce body-wall muscle to extend cellular processes that enwrap the germ cells, the extent of which was strikingly similar to the distal tip cell (DTC)-germ stem cell niche. Enwrapment was specific for escaped germ cells, and genetic analysis revealed it did not depend on pathways that control cell death and engulfment or muscle arm extension. Instead, using a large-scale RNAi screen and GFPÂ knockin strains, we discovered that the enwrapping behavior of muscle relied upon the same suite ofÂ cell-cell adhesion molecules that functioned inÂ theÂ endogenous niche: the C.Â elegans E-cadherin HMR-1, its intracellular associates Î±-catenin (HMP-1) and Î²-catenin (HMP-2), and the L1CAM protein SAX-7. This ectopic niche-like behavior resembles the seed-and-soil model of cancer metastasis and offers a new model to understand factors regulating ectopic niche formation.
Functional Interactions Between rsks-1/S6K, glp-1/Notch, and Regulators of Caenorhabditis elegans Fertility and Germline Stem Cell Maintenance
The proper accumulation and maintenance of stem cells is critical for organ development and homeostasis. The Notch signaling pathway maintains stem cells in diverse organisms and organ systems. In Caenorhabditis elegans, GLP-1/Notch activity prevents germline stem cell (GSC) differentiation. Other signaling mechanisms also influence the maintenance of GSCs, including the highly-conserved TOR substrate ribosomal protein S6 kinase. Although C. elegans bearing either a null mutation in rsks-1/S6K or a reduction-of-function (rf) mutation in glp-1/Notch produce half the normal number of adult germline progenitors, virtually all these single mutant animals are fertile. However, glp-1(rf)rsks-1(null) double mutant animals are all sterile, and in about half of their gonads, all GSCs differentiate, a distinctive phenotype associated with a significant reduction or loss of GLP-1 signaling. How rsks-1/S6K promotes GSC fate is unknown. Here, we determine that rsks-1/S6K acts germline-autonomously to maintain GSCs, and that it does not act through Cyclin-E or MAP kinase in this role. We found that interfering with translation also enhances glp-1(rf), but that regulation through rsks-1 cannot fully account for this effect. In a genome-scale RNAi screen for genes that act similarly to rsks-1/S6K, we identified 56 RNAi enhancers of glp-1/Notch sterility, many of which were previously not known to interact functionally with Notch. Further investigation revealed six candidates that, by genetic criteria, act linearly with rsks-1/S6K. These include genes encoding translation-related proteins, cacn-1/Cactin, an RNA exosome component and a Hedgehog-related ligand. We found that additional Hedgehog-related ligands may share functional relationships with glp-1/Notch and rsks-1/S6K in maintaining germline progenitors.
The DSL ligand APX-1 is required for normal ovulation in C. elegans
DSL ligands activate the Notch receptor in many cellular contexts across metazoa to specify cell fate. In addition, Notch receptor activity is implicated in post-mitotic morphogenesis and neuronal function. In C. elegans, the DSL family ligand APX-1 is expressed in a subset of cells of the proximal gonad lineage, where it can act as a latent proliferation-promoting signal to maintain proximal germline tumors. Here we examine apx-1 in the proximal gonad and uncover a role in the maintenance of normal ovulation. Depletion of apx-1 causes an endomitotic oocyte (Emo) phenotype and ovulation defects. We find that lag-2 can substitute for apx-1 in this role, that the ovulation defect is partially suppressed by loss of ipp-5, and that lin-12 depletion causes a similar phenotype. In addition, we find that the ovulation defects are often accompanied by a delay of spermathecal distal neck closure after oocyte entry. Although calcium oscillations occur in the spermatheca, calcium signals are abnormal when the distal neck does not close completely. Moreover, oocytes sometimes cannot properly transit through the spermatheca, leading to fragmentation of oocytes once the neck closes. Finally, abnormal oocytes and neck closure defects are seen occasionally when apx-1 or lin-12 activity is reduced in adult animals, suggesting a possible post-developmental role for APX-1 and LIN-12 signaling in ovulation.
Linking the environment, DAF-7/TGFbeta signaling and LAG-2/DSL ligand expression in the germline stem cell niche
The developmental accumulation of proliferative germ cells in the C. elegans hermaphrodite is sensitive to the organismal environment. Previously, we found that the TGFbeta signaling pathway links the environment and proliferative germ cell accumulation. Neuronal DAF-7/TGFbeta causes a DAF-1/TGFbetaR signaling cascade in the gonadal distal tip cell (DTC), the germline stem cell niche, where it negatively regulates a DAF-3 SMAD and DAF-5 Sno-Ski. LAG-2, a founding DSL ligand family member, is produced in the DTC and activates the GLP-1/Notch receptor on adjacent germ cells to maintain germline stem cell fate. Here, we show that DAF-7/TGFbeta signaling promotes expression of lag-2 in the DTC in a daf-3-dependent manner. Using ChIP and one-hybrid assays, we find evidence for direct interaction between DAF-3 and the lag-2 promoter. We further identify a 25 bp DAF-3 binding element required for the DTC lag-2 reporter response to the environment and to DAF-7/TGFbeta signaling. Our results implicate DAF-3 repressor complex activity as a key molecular mechanism whereby the environment influences DSL ligand expression in the niche to modulate developmental expansion of the germline stem cell pool.
How computational models contribute to our understanding of the germ line
Computational models are an invaluable tool in modern biology. They provide a framework within which to summarize existing knowledge, enable competing hypotheses to be compared qualitatively and quantitatively, and to facilitate the interpretation of complex data. Moreover, models allow questions to be investigated that are difficult to approach experimentally. Theories can be tested in context, identifying the gaps in our understanding and potentially leading to new hypotheses. Models can be developed on a variety of scales and with different levels of mechanistic detail, depending on the available data, the biological questions of interest, and the available mathematical and computational tools. The goal of this review is to provide a broad picture of how modeling has been applied to reproductive biology. Specifically, we look at four uses of modeling: i) comparing hypotheses, ii) interpreting data, iii) exploring experimentally challenging questions, and iv) hypothesis evaluation and generation. We present examples of each of these applications in reproductive biology, drawing from a range of organisms - including Drosophila, Caenorhabditis elegans, mouse, and humans. We aim to describe the data and techniques used to construct each model, and to highlight the benefits of modeling to the field, as complementary to experimental work