E Jane Albert Hubbard (2001) International C. elegans Meeting "An Upper-level Undergraduate Project-Based "Laboratory in Genetics" Course using C. elegans"

E Jane Albert Hubbard

[International C. elegans Meeting, 2001]

I designed a new upper-level 14-week undergraduate "Laboratory in Genetics" course using C. elegans, and the course was offered for the first time this spring semester (2001). The course is geared for juniors and seniors with prerequisites of Introductory Biology I and II, Molecular and Cell Biology I and II, and Genetics. The course covers many genetic principles by means of a project-based laboratory in two parts: forward and reverse genetics projects. After familiarizing themselves with a variety of mutant phenotypes, teams of 2-4 students consider a biological process, select mutants that perturb a process, and characterize the mutant phenotype. Next, they begin a classical genetic analysis. First they describe their mutant phenotype in terms of penetrance and expressivity and assess dominance and X-linkage. Then they carry out further linkage tests and three-factor mapping experiments. Finally, they perform complementation tests, deficiency mapping and dosage analysis to further characterize their mutant. Two demonstrations (worms under Nomarski optics in the first class and injections later in the semester) round out the first part of the course. The design of this part of the course was drawn from a similar course offered by Leilani Miller at Santa Clara University. The second part of the course consists of a reverse genetics experiment, made possible by the availability of the full sequence of the C. elegans genome and RNAi. First, students select a gene of interest from a provided list of possibilities (e.g., human disease-related genes). Then, using gene databases and information available on the web, they identify a closely related gene in the C. elegans genome. Finally, they examine the consequences of an RNAi feeding experiment with appropriate controls. The class meets once/week for 4 and one quarter hours and is run with the help of a graduate teaching assistant. Approximately 45 minutes of the class time consists of a lab-lecture. In addition, during the class time teams of students prepare, pour and seed plates. The experiments for the first part of the course are accomplished more or less within the allotted time with occasional follow-up scoring the day or two after the regularly scheduled lab. The second set of experiments requires a more flexible schedule, allowing the students to come in each day for a shorter period of time. Grading for the course is based on (1) four required written lab reports (three of which have a re-write option), (2) a final exam and (3) laboratory conduct. The reports are written individually although the lab work is done in teams. The final examination consists of problems to test students’ knowledge of the concepts applied during the semester. I am deeply grateful to Leilani Miller for sharing material, suggestions and encouragement during the preparation of the course.

Tolkin, T. et al. (2019) International Worm Meeting "Quantifying the relationship between niche contact and the Notch-dependent transcriptional response in C. elegans germline stem cells."

Tolkin, T., Hubbard, E. Jane Albert

[International Worm Meeting, 2019]

Contact between cells is an essential feature of many signaling pathways but the role of cell contact architecture in modulating cell-cell signaling has been largely underappreciated. Stem cells and their niche in the C. elegans germ line provide an excellent in vivo model to investigate how morphology of cell-cell contact affects signaling. In this case, the signal in question is Notch. The Notch pathway is a contact-mediated intercellular signaling mechanism that activates specific gene transcription in the signal-receiving cell. Proper levels of target gene transcription are essential for development and homeostasis, yet how the physical relationship between sending and receiving cells contributes to appropriate levels of target gene transcription is poorly understood. I am investigating how sender-receiver cell contact surface area controls levels of Notch target gene transcription at the single-cell level, using the larval C. elegans germ line. In this system, a unicellular signal-sending niche cell (the DTC) contacts multiple signal-receiving germline stem cells (GSCs). I have found that the contact surface area between the DTC and individual GSCs is highly heterogeneous even in larval stages prior to elaboration of the DTC plexus. Using quantitative image analysis, I am correlating DTC-GSC contact surface area with Notch target gene expression in single cells. This research may reveal general mechanisms by which morphological changes in cell-cell contact modulate Notch signaling.

Qin, Zhao et al. (2011) International Worm Meeting "Maintenance of adult proliferative germ cells."

Qin, Zhao, Hubbard, E. Jane Albert

[International Worm Meeting, 2011]

Failure to maintain stem cells with age is associated with conditions such as tissue degeneration and increased susceptibility to tissue damage in many organisms, including humans. We are using the C. elegans germ line as a general model for stem cell aging because it combines a well-established genetic model for aging studies with a well-defined and accessible stem cell system, providing a unique opportunity to dissect the effects of aging on stem cell dynamics. We hypothesize that aging affects stem cells through regulating interactions between stem cells and their microenvironment, the niche, which has been shown to be required for stem cell maintenance during normal tissue homeostasis.

Qin, Zhao et al. (2015) International Worm Meeting "Non-autonomous DAF-16/FOXO activity antagonizes age-related loss of C. elegans germline stem/progenitor cells."

Qin, Zhao, Hubbard, E. Jane Albert

[International Worm Meeting, 2015]

Stem cells maintain tissues and organs over the lifespan of individuals. How aging influences this process is unclear. Here we use the C. elegans germ line as a model to study stem cell aging. The C. elegans germ line combines a well-established genetic model for aging studies with a well-defined, accessible stem cell system, providing a unique opportunity to dissect the effects of aging on stem cell dynamics.We found that the stem/progenitor cell pool in the C. elegans germ line becomes depleted over time. This depletion is suppressed in mutants with reduced insulin/IGF-like signaling (IIS) via the downstream transcription factor DAF-16/FOXO. DAF-16/FOXO acts neither in the germ line itself nor in the intestine, suggesting that DAF-16/FOXO regulates age-related germline stem/progenitor cell loss through a novel mechanism that is anatomically separable from its previously described roles in larval germline proliferation and lifespan regulation. Further, we found that DAF-16/FOXO activity is required in the proximal region of the somatic gonad, suggesting that the part of the reproductive system that experiences the transit of gametes and/or embryos might signal to and influence the stem/progenitor cell pool. Consistent with this hypothesis, we found that animals with an altered rate of germ cell flux through the reproductive tract lose their germline stem/progenitor cells at a significantly different rate compared with controls and that DAF-16/FOXO partially mediates the effect of germ cell flux on germline stem/progenitor cell maintenance over time. Together, our results represent a novel mechanism of stem cell regulation at the organ level in addition to regulation of stem cells by their niche and the systemic environment of the organism.

Dalfo, Diana et al. (2011) International Worm Meeting "Sensory control of germline differentiation via TGFb."

Hubbard, E. Jane Albert, Dalfo, Diana

[International Worm Meeting, 2011]

The balance between proliferation and differentiation is critical for stem and progenitor cell populations. Defects in this balance during development can be deleterious to the establishment of adult stem cells. However, the mechanisms that influence this balance are poorly understood. GLP-1/Notch signaling is important for maintaining the proliferating population of C. elegans germ cells: loss of glp-1 activity causes all germ cells to differentiate. We have shown that Notch-independent signaling pathways such as insulin/IGF contribute to the robust proliferation of the larval germ line, and that this is required for optimal fecundity. In large-scale RNAi screens for genes affecting early germline proliferation and/or differentiation, we identified genes encoding TGFb "dauer" pathway components. Further analysis indicates a role for this TGFb pathway in the regulation of proliferation versus differentiation in the C. elegans germ line. Like its role in the dauer decision, this role is dependent on daf-3/Co-SMAD and daf-5/SNO/SKI. Unlike dauer, however, it is daf-12/NHR-independent. We find that the TGFb ligand-producing ASI sensory neurons are required for TGFb-mediated germ cell accumulation, and that the TGFb receptor and downstream transcription complex acts in the distal tip cell, in parallel with GLP-1/Notch signaling. Our results implicate the TGFb pathway as a mediator between environmental cues perceived by specific sensory neurons and a stem cell niche to influence the proliferation/differentiation balance.

Amar, Ani et al. (2021) International Worm Meeting "Modeling the C. elegans Germline Stem Cell Genetic Network using Automated Reasoning"

Amar, Ani, Hubbard, E. Jane Albert, Kugler , Hillel

[International Worm Meeting, 2021]

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 model 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 the reasoning engine for interaction networks tool (RE:IN) to derive predictive networks for control of differentiation. Using RE:IN 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. This work lays a foundation for developing realistic whole tissue models of the C.elegans germline where each cell in the model will execute a synthesized genetic network.

Venzon, Mericien et al. (2021) International Worm Meeting "A multi-organism genetic model for microbiota-driven parasite burden"

Belasco, Joel G., Cadwell, Ken, Luciano, Daniel J., Das, Ritika, Venzon, Mericien, Hubbard, E. Jane Albert

[International Worm Meeting, 2021]

Parasitic nematodes infect over one billion people worldwide and although the majority of them live in the digestive tract, a possible role for the microbiota in determining successful host infection is underexplored. The Trichuris nematode parasite lives in the microbiota-rich mammalian cecum for years, and it has an enormous reproductive capacity that contributes to its infectious spread. We previously determined that C. elegans germline development is sensitive to the microbial environment. Though the two nematode species are not closely related, we predicted that some aspects of reproduction in the well-characterized, model organism C. elegans may be conserved in Trichuris. To assess systematically which components of the bacterial diet are most important for C. elegans reproduction, we screened an E. coli library for mutants that, when fed to C. elegans, interfered with timely fertility. We identified ten E. coli mutants. Focusing on two that function in fatty acid biosynthesis (fabF, fabH) and two that function in ethanolamine utilization (eutD, eutN), we found that fabF and eutN delayed C. elegans germline development relative to somatic development, though none of the four reduced germline progenitor numbers and none were dependent upon DAF-2 insulin or DAF-7 TGFB signaling pathways. Metabolomics and functional analysis unexpectedly revealed that fabH E. coli mutants provide insufficient arginine, such that arginine supplementation rescued the C. elegans fertility delay. We further investigated whether E. coli mutants might interfere with development or reproduction of the Trichuris species that infects mice, Trichuris muris. Remarkably, fabH and eutN mutant E. coli were associated with T. muris hatching defects, the former also being arginine-dependent, while eutN likely acts by producing a toxic substance. Both were also associated with aberrant reproduction of T. muris in vivo. Overall, these findings establish C. elegans as a novel system for investigating the effects of specific microbial genes and pathways in supporting the parasitic nematode life cycle.

Korta, Dorota Z. et al. (2011) International Worm Meeting "LET-363/TOR and RSKS-1/S6K promote proper germline development in C. elegans."

Hubbard, E. Jane Albert, Tuck, Simon, Korta, Dorota Z.

[International Worm Meeting, 2011]

It is well established that nutritional status affects many aspects of physiology, including reproduction and cell proliferation. However, the molecular mechanisms by which nutritionally sensitive molecular pathways control cell proliferation are not well understood. Recently, our lab has shown that insulin/IGF-like receptor (IIR) signaling is required for robust larval germline proliferation. The TOR (Target of Rapamycin)-S6K(p70 ribosomal S6 kinase) pathway is responsive to insulin signaling and nutrition in mammals. However, in C. elegans the pathway is not well defined. We have found that let-363/TOR, daf-15/Raptor, and downstream target rsks-1/S6K are required for proper proliferative germ cell accumulation during larval stages, and this effect is germ line-autonomous. We investigated the mechanism by which germ cell numbers were affected in rsks-1 null mutants. We found that loss of rsks-1 leads to general slowing of larval germ cell proliferation and a distal shift in germ cell meiotic entry, suggesting it affects both proliferation and differentiation. Furthermore, rsks-1 likely acts in parallel to both glp-1/Notch and the daf-16/FOXO branch of the insulin pathway. The rsks-1 germline defect is not suppressible by mutations that mediate the role of rsks-1 in longevity, namely pha-4/FOXA and egl-9. Thus the reason rsks-1 animals are long lived cannot simply be due to decreased numbers of proliferating germ cells. We are actively investigating effectors of the rsks-1 germline phenotype.

Korta, Dorota et al. (2009) International Worm Meeting "Insulin Signaling is Required for Robust Larval Germline Proliferation in C. elegans."

Capua, Josef, Michaelson, David, Hubbard, E. Jane Albert, Korta, Dorota

[International Worm Meeting, 2009]

The proper control of cell proliferation is critical for development, and understanding this control at the molecular level in the whole-organism context has broad implications for cancer and stem cell biology. We are using the C. elegans germ line as a model to investigate proliferation control. Notch receptor-mediated activity determines the region within the germ line where mitosis can occur and/or differentiation is inhibited. Our previous studies indicated that the distal-most pair of gonadal sheath cells is required for robust larval germline proliferation but not for the proliferation/differentiation decision per se, suggesting that these controls are separable. Our more recent results indicate that soma-to-germ line signaling through the insulin/IGF-like receptor (IIR) pathway influences the number of undifferentiated germ cells in the distal germ line. Establishment of proper germ cell numbers requires somatic activity of genes encoding putative insulin-like ligands INS-3 and INS-33. The insulin/IGF-like receptor DAF-2 and components of the PI3K branch of this conserved signaling pathway, including DAF-18/PTEN and DAF-16/FOXO are required in the germ line. IIR signaling in C. elegans is well-characterized for the dauer decision: reduction of IIR signaling causes constitutive dauer formation, during which germline proliferation is impaired. However, we find that the requirement for IIR for larval germ cells is anatomically and temporally separable from its role in the dauer decision. We further investigated the mechanism by which germ cell numbers were affected. In mutants defective in IIR signaling, cell death is not elevated, cell size is not reduced, and the distance to the proliferation/differentiation border is only slightly reduced. Rather, the primary effect of reduced IIR signaling is a reduction in the mitotic index. By contrast, reduced Notch signaling does not reduce mitotic index, but greatly reduces the distance to the proliferation/differentiation border. Reducing the activity of both IIR and Notch pathways results in both defects (reduced mitotic index and reduced distance to differentiation) without additional synergistic effects. Taken together, our results suggest that (i) the IIR pathway responds to post-dauer-decision cues, ensuring adequate larval proliferation for adult gametogenesis and (ii) two conserved pathways regulate larval germline development largely independently: Notch signaling sets up the border of differentiation, and insulin signaling stimulates robust mitosis.

Nadarajan, Priah et al. (2009) International Worm Meeting "MSP AND GLP-1/NOTCH SIGNALING COORDINATELY REGULATE OOCYTE GROWTH."

Greenstein, David, Nadarajan, Priah, Hubbard, E. Jane Albert, Govindan, J. Amaranath, McGovern, Marie

[International Worm Meeting, 2009]

Fertility depends on germline stem cell proliferation, meiosis, and gametogenesis, yet how these key transitions are coordinated is unclear. We present a model in which the two major signaling centers in the adult hermaphrodite gonad, GLP-1/Notch distally and major sperm protein (MSP) proximally, while individually functioning in a separate process-germline proliferation and meiotic maturation, respectively-work in concert to regulate the differentiation of female germ cells into functional oocytes. In C. elegans, a sperm-sensing mechanism coordinates oocyte meiotic maturation and fertilization. Sperm release MSP to trigger meiotic resumption (meiotic maturation) and to promote the contraction of the follicle-like gonadal sheath cells. All known MSP-dependent responses in the germline require GSA-1 (Gas)-ACY-4 (adenylate cyclase) signaling in the gonadal sheath cells, including the promotion of actomyosin-dependent cytoplasmic streaming that drives oocyte growth. Thus, MSP signaling via the gonadal sheath cells links oogenesis and embryo production. These findings prompted us to conduct a genetic analysis of the MSP-dependent oocyte growth process. Surprisingly, we found that glp-1 signaling functions in the germline to ensure proper oocyte growth when the MSP hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large in a manner that is dependent on the presence of MSP and Gas-adenylate cyclase signaling in the gonadal sheath cells. By contrast, gain-of-function glp-1 mutations result in the production of small oocytes. Further, MSP-dependent oocyte growth depends on DTC signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1(rf) mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. MSP is sufficient to promote cytoplasmic streaming, and we show that MSP, but not GLP-1, promotes the phosphorylation of the regulatory myosin light chain (rMLC-4) of NMY-2 smooth muscle myosin throughout the germline. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Interestingly, abnormal oocyte growth in glp-1 mutants, but not premature meiotic entry of germline stem cells, requires the function of the cell death pathway. These data illustrate how germline processes are intertwined when MSP is present and the adult gonad is switched into a reproductive mode.