Faculty Bibliography

Coupling of stem/progenitor cell proliferation and differentiation to organismal physiological demands ensures the proper growth and homeostasis of tissues. However, in vivo mechanisms underlying this control are poorly characterized. We investigated the role of ribosomal protein S6 kinase (S6K) at the intersection of nutrition and the establishment of a stem/progenitor cell population using the C. elegans germ line as a model. We find that rsks-1 (which encodes the worm homolog of mammalian p70S6K) is required germline-autonomously for proper establishment of the germline progenitor pool. In the germ line, rsks-1 promotes cell cycle progression and inhibits larval progenitor differentiation, promotes growth of adult tumors and requires a conserved TOR phosphorylation site. Loss of rsks-1 and ife-1 (eIF4E) together reduces the germline progenitor pool more severely than either single mutant and similarly to reducing the activity of let-363 (TOR) or daf-15 (RAPTOR). Moreover, rsks-1 acts in parallel with the glp-1 (Notch) and daf-2 (insulin-IGF receptor) pathways, and does not share the same genetic dependencies with its role in lifespan control. We show that overall dietary restriction and amino acid deprivation cause germline defects similar to a subset of rsks-1 mutant phenotypes. Consistent with a link between diet and germline proliferation via rsks-1, loss of rsks-1 renders the germ line largely insensitive to the effects of dietary restriction. Our studies establish the C. elegans germ line as an in vivo model to understand TOR-S6K signaling in proliferation and differentiation and suggest that this pathway is a key nutrient-responsive regulator of germline progenitors.

The proper renewal and maintenance of tissues by stem cell populations is simultaneously influenced by anatomical constraints, cell proliferation dynamics and cell fate specification. However, their relative influence is difficult to examine in vivo. To address this difficulty we built, as a test case, a cell-centered state-based computational model of key behaviors that govern germline development in C. elegans, and used it to drive simulations of cell population dynamics under a variety of perturbations. Our analysis provided unexpected possible explanations for laboratory observations, including certain 'all-or-none' phenotypes and complex differentiation patterns. The simulations also offered insights into niche-association dynamics and the interplay between cell cycle and cell fate. Subsequent experiments validated several predictions generated by the simulations. Notably, we found that early cell cycle defects influence later maintenance of the progenitor cell population. This general modeling approach is potentially applicable to other stem cell systems.

Germline proliferation in Caenorhabditis elegans is emerging as a compelling model system for understanding the molecular basis for the developmental and physiological control of cell proliferation. This review covers the discovery and implications of the role of the insulin/IGF-like signaling pathway in germline proliferation during germline development. This pathway plays a host of important roles in C. elegans biology. Its role in germline proliferation is important to generate the proper adult stem/progenitor population and to ensure optimal fecundity. Moreover, in this role, it is restricted to reproductive (as opposed to dauer) larval stages and impinges on the G2 of the cell cycle. Two putative insulin ligands are especially important for the germline role but do not mediate signaling in other tissues. A picture is emerging of a complex web of developmentally and temporally restricted, ligand- and tissue-specific responses to insulin signaling. Avenues for future studies include the regulation of specific insulin-like ligands and the mechanisms for tissue-specific responses to them

Caenorhabditis elegans boasts a short lifecycle and high fecundity, two features that make it an attractive and powerful genetic model organism. Several recent studies indicate that germline proliferation, a prerequisite to optimal fecundity, is tightly controlled over the course of development. Cell proliferation control includes regulation of competence to proliferate, a poorly understood aspect of cell fate specification, as well as cell-cycle control. Furthermore, dynamic regulation of cell proliferation occurs in response to multiple external signals. The C. elegans germ line is proving a valuable model for linking genetic, developmental, systemic, and environmental control of cell proliferation. Here, we consider recent studies that contribute to our understanding of germ cell proliferation in C. elegans. We focus primarily on somatic control of germline proliferation, how it differs at different life stages, and how it can be altered in the context of the life cycle and changes in environmental status

Cell proliferation must be coordinated with cell fate specification during development, yet interactions among pathways that control these two critical aspects of development are not well understood. The coordination of cell fate specification and proliferation is particularly crucial during early germline development, when it impacts the establishment of stem/progenitor cell populations and ultimately the production of gametes. In C. elegans, insulin/IGF-like receptor (IIR) signaling has been implicated in fertility, but the basis for the fertility defect had not been previously characterized. We found that IIR signaling is required for robust larval germline proliferation, separate from its well-characterized role in preventing dauer entry. IIR signaling stimulates the larval germline cell cycle. This activity is distinct from Notch signaling, occurs in a predominantly germline-autonomous manner, and responds to somatic activity of ins-3 and ins-33, genes that encode putative insulin-like ligands. IIR signaling in this role acts through the canonical PI3K pathway, inhibiting DAF-16/FOXO. However, signaling from these ligands does not inhibit daf-16 in neurons nor in the intestine, two tissues previously implicated in other IIR roles. Our data are consistent with a model in which: (1) under replete reproductive conditions, the larval germline responds to insulin signaling to ensure robust germline proliferation that builds up the germline stem cell population; and (2) distinct insulin-like ligands contribute to different phenotypes by acting on IIR signaling in different tissues

Stem cells, their niches, and their relationship to cancer are under intense investigation. Because tumors and metastases acquire self-renewing capacity, mechanisms for their establishment may involve cell-cell interactions similar to those between stem cells and stem cell niches. On the basis of our studies in Caenorhabditis elegans, we introduce the concept of a 'latent niche' as a differentiated cell type that does not normally contact stem cells nor act as a niche but that can, under certain conditions, promote the ectopic self-renewal, proliferation, or survival of competent cells that it inappropriately contacts. Here, we show that ectopic germ-line stem cell proliferation in C. elegans is driven by a latent niche mechanism and that the molecular basis for this mechanism is inappropriate Notch activation. Furthermore, we show that continuous Notch signaling is required to maintain ectopic germ-line proliferation. We highlight the latent niche concept by distinguishing it from a normal stem cell niche, a premetastatic niche and an ectopic niche. One of the important distinguishing features of this mechanism for tumor initiation is that it could operate in the absence of genetic changes to the tumor cell or the tumor-promoting cell. We propose that a latent niche mechanism may underlie tumorigenesis and metastasis in humans

Fertility depends on germline stem cell proliferation, meiosis and gametogenesis, yet how these key transitions are coordinated is unclear. In C. elegans, we show that GLP-1/Notch signaling functions in the germline to modulate oocyte growth when sperm are available for fertilization and the major sperm protein (MSP) hormone is present. Reduction-of-function mutations in glp-1 cause oocytes to grow abnormally large when MSP is present and Galpha(s)-adenylate cyclase signaling in the gonadal sheath cells is active. By contrast, gain-of-function glp-1 mutations lead to the production of small oocytes. Surprisingly, proper oocyte growth depends on distal tip cell signaling involving the redundant function of GLP-1 ligands LAG-2 and APX-1. GLP-1 signaling also affects two cellular oocyte growth processes, actomyosin-dependent cytoplasmic streaming and oocyte cellularization. glp-1 reduction-of-function mutants exhibit elevated rates of cytoplasmic streaming and delayed cellularization. GLP-1 signaling in oocyte growth depends in part on the downstream function of the FBF-1/2 PUF RNA-binding proteins. Furthermore, abnormal oocyte growth in glp-1 mutants, but not the inappropriate differentiation of germline stem cells, requires the function of the cell death pathway. The data support a model in which GLP-1 function in MSP-dependent oocyte growth is separable from its role in the proliferation versus meiotic entry decision. Thus, two major germline signaling centers, distal GLP-1 activation and proximal MSP signaling, coordinate several spatially and temporally distinct processes by which germline stem cells differentiate into functional oocytes

lim-7 is one of seven Caenorhabditis elegans LIM-homeodomain (LIM-HD)-encoding genes and the sole Islet ortholog. LIM-HD transcription factors, including Islets, function in neuronal and non-neuronal development across diverse phyla. Our results show that a lim-7 deletion allele causes early larval lethality with terminal phenotypes including uncoordination, detached pharynx, constipation and morphological defects. A lim-7(+) transgene rescues lethality but not adult sterility. A lim-7(+) reporter in the full genomic context is expressed in all gonadal sheath cells, URA neurons, and additional cells in the pharyngeal region. Finally, we identify a 45-bp regulatory element in the first intron that is necessary and sufficient for lim-7 gonadal sheath expression.

Studies of developmental biology are often facilitated by diagram 'models' that summarize the current understanding of underlying mechanisms. The increasing complexity of our understanding of development necessitates computational models that can extend these representations to include their dynamic behavior. Here we present a prototype model of Caenorhabditis elegans vulval precursor cell fate specification that represents many processes crucial for this developmental event but that are hard to integrate using other modeling methodologies. We demonstrate the integrative capabilities of our methodology by comprehensively incorporating the contents of three seminal papers, showing that this methodology can lead to comprehensive models of developmental biology. The prototype computational model was built and is run using a language (Live Sequence Charts) and tool (the Play-Engine) that facilitate the same conceptual processes biologists use to construct and probe diagram-type models. We demonstrate that this modeling approach permits rigorous tests of mutual consistency between experimental data and mechanistic hypotheses and can identify specific conflicting results, providing a useful approach to probe developmental systems

We present a two-part system for conditional FLP-out of FRT-flanked sequences in Caenorhabditis elegans to control gene activity in a spatially and/or temporally regulated manner. Using reporters, we assess the system for efficacy and demonstrate its use as a cell lineage marking tool. In addition, we construct and test a dominant-negative form of hlh-12, a gene that encodes a basic helix-loop-helix (bHLH) transcription factor required for proper distal tip cell (DTC) migration. We show that this allele can be conditionally expressed from a heat-inducible FLP recombinase and can interfere with DTC migration. Using the same DTC assay, we conditionally express an hlh-12 RNAi-hairpin and induce the DTC migration defect. Finally, we introduce a set of traditional and Gateway-compatible vectors to facilitate construction of plasmids for this technology using any promoter, reporter, and gene/hairpin of interest