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Innexin function dictates the spatial relationship between distal somatic cells in the Caenorhabditis elegans gonad without impacting the germline stem cell pool

Tolkin, Theadora; Mohammad, Ariz; Starich, Todd A; Nguyen, Ken C Q; Hall, David H; Schedl, Tim; Hubbard, E Jane Albert; Greenstein, David
Gap-junctional signaling mediates myriad cellular interactions in metazoans. Yet, how gap junctions control the positioning of cells in organs is not well understood. Innexins compose gap junctions in invertebrates and affect organ architecture. Here, we investigate the roles of gap-junctions in controlling distal somatic gonad architecture and its relationship to underlying germline stem cells in Caenorhabditis elegans. We show that a reduction of soma-germline gap-junctional activity causes displacement of distal sheath cells (Sh1) towards the distal end of the gonad. We confirm, by live imaging, transmission electron microscopy, and antibody staining, that bare regions-lacking somatic gonadal cell coverage of germ cells-are present between the distal tip cell (DTC) and Sh1, and we show that an innexin fusion protein used in a prior study encodes an antimorphic gap junction subunit that mispositions Sh1. We determine that, contrary to the model put forth in the prior study based on this fusion protein, Sh1 mispositioning does not markedly alter the position of the borders of the stem cell pool nor of the progenitor cell pool. Together, these results demonstrate that gap junctions can control the position of Sh1, but that Sh1 position is neither relevant for GLP-1/Notch signaling nor for the exit of germ cells from the stem cell pool.
PMID: 36098634
ISSN: 2050-084x
CID: 5335042

Modeling the C. elegans germline stem cell genetic network using automated reasoning

Amar, Ani; Hubbard, E Jane Albert; Kugler, Hillel
Computational methods and tools are a powerful complementary approach to experimental work for studying regulatory interactions in living cells and systems. We demonstrate the use of formal reasoning methods as applied to the Caenorhabditis elegans germ line, which is an accessible system for stem cell research. The dynamics of the underlying genetic networks and their potential regulatory interactions are key for understanding mechanisms that control cellular decision-making between stem cells and differentiation. We model the "stem cell fate" versus entry into the "meiotic development" pathway decision circuit in the young adult germ line based on an extensive study of published experimental data and known/hypothesized genetic interactions. We apply a formal reasoning framework to derive predictive networks for control of differentiation. Using this approach we simultaneously specify many possible scenarios and experiments together with potential genetic interactions, and synthesize genetic networks consistent with all encoded experimental observations. In silico analysis of knock-down and overexpression experiments within our model recapitulate published phenotypes of mutant animals and can be applied to make predictions on cellular decision-making. A methodological contribution of this work is demonstrating how to effectively model within a formal reasoning framework a complex genetic network with a wealth of known experimental data and constraints. We provide a summary of the steps we have found useful for the development and analysis of this model and can potentially be applicable to other genetic networks. This work also lays a foundation for developing realistic whole tissue models of the C. elegans germ line where each cell in the model will execute a synthesized genetic network.
PMID: 35469833
ISSN: 1872-8324
CID: 5264332

Microbial byproducts determine reproductive fitness of free-living and parasitic nematodes

Venzon, Mericien; Das, Ritika; Luciano, Daniel J; Burnett, Julia; Park, Hyun Shin; Devlin, Joseph Cooper; Kool, Eric T; Belasco, Joel G; Hubbard, E Jane Albert; Cadwell, Ken
Trichuris nematodes reproduce within the microbiota-rich mammalian intestine and lay thousands of eggs daily, facilitating their sustained presence in the environment and hampering eradication efforts. Here, we show that bacterial byproducts facilitate the reproductive development of nematodes. First, we employed a pipeline using the well-characterized, free-living nematode C. elegans to identify microbial factors with conserved roles in nematode reproduction. A screen for E. coli mutants that impair C. elegans fertility identified genes in fatty acid biosynthesis and ethanolamine utilization pathways, including fabH and eutN. Additionally, Trichuris muris eggs displayed defective hatching in the presence of fabH- or eutN-deficient E. coli due to reduced arginine or elevated aldehydes, respectively. T. muris reared in gnotobiotic mice colonized with these E. coli mutants displayed morphological defects and failed to lay viable eggs. These findings indicate that microbial byproducts mediate evolutionarily conserved transkingdom interactions that impact the reproductive fitness of distantly related nematodes.
PMID: 35413267
ISSN: 1934-6069
CID: 5219002

Model organism databases are in jeopardy

Bellen, Hugo J; Hubbard, E J A; Lehmann, Ruth; Madhani, Hiten D; Solnica-Krezel, Lila; Southard-Smith, E Michelle
PMID: 35231122
ISSN: 1477-9129
CID: 5174352

DAF-18/PTEN inhibits germline zygotic gene activation during primordial germ cell quiescence

Fry, Amanda L; Webster, Amy K; Burnett, Julia; Chitrakar, Rojin; Baugh, L Ryan; Hubbard, E Jane Albert
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.
PMID: 34288923
ISSN: 1553-7404
CID: 4969012

Germline Stem and Progenitor Cell Aging in C. elegans

Tolkin, Theadora; Hubbard, E Jane Albert
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.
PMID: 34307379
ISSN: 2296-634x
CID: 4969022

A Genome-Wide RNAi Screen for Enhancers of a Germline Tumor Phenotype Caused by Elevated GLP-1/Notch Signaling in Caenorhabditis elegans

Dalfo, Diana; Ding, Yanhui; Liang, Qifei; Fong, Alex; Cipriani, Patricia Giselle; Piano, Fabio; Zheng, Jialin C; Qin, Zhao; Hubbard, E Jane Albert
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.
PMID: 33077477
ISSN: 2160-1836
CID: 4642062

Biology of the Caenorhabditis elegans Germline Stem Cell System

Hubbard, E Jane Albert; Schedl, Tim
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.
PMID: 31796552
ISSN: 1943-2631
CID: 4240772

Insulin/IGF Signaling and Vitellogenin Provisioning Mediate Intergenerational Adaptation to Nutrient Stress

Jordan, James M; Hibshman, Jonathan D; Webster, Amy K; Kaplan, Rebecca E W; Leinroth, Abigail; Guzman, Ryan; Maxwell, Colin S; Chitrakar, Rojin; Bowman, Elizabeth Anne; Fry, Amanda L; Hubbard, E Jane Albert; Baugh, L Ryan
The roundworm C. elegans reversibly arrests larval development during starvation [1], but extended early-life starvation reduces reproductive success [2, 3]. Maternal dietary restriction (DR) buffers progeny from starvation as young larvae, preserving reproductive success [4]. 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 of 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.
PMID: 31280992
ISSN: 1879-0445
CID: 4538472

Ectopic Germ Cells Can Induce Niche-like Enwrapment by Neighboring Body Wall Muscle

Gordon, Kacy L; Payne, Sara G; Linden-High, Lara M; Pani, Ariel M; Goldstein, Bob; Hubbard, E Jane Albert; Sherwood, David R
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. 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 GFP knockin strains, we discovered that the enwrapping behavior of muscle relied upon the same suite of cell-cell adhesion molecules that functioned in the endogenous niche: the C. elegans E-cadherin HMR-1, its intracellular associates α-catenin (HMP-1) and β-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.
PMID: 30799241
ISSN: 1879-0445
CID: 3752002