BIBLIOGRAPHY

1. McGovern, Marie, Voutev, Roumen, Maciejowski, John, Corsi, Ann K, Hubbard, E Jane Albert. "A "latent niche" mechanism for tumor initiation," Proceedings of the National Academy of Sciences of the United States of America 2009 Jul 14;106(28):11617-22.   (MEDL:19564624 PMID: 19564624 #J0174544)    

   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.

2. Nadarajan, Saravanapriah, Govindan, J Amaranath, McGovern, Marie, Hubbard, E Jane Albert, Greenstein, David. "MSP and GLP-1/Notch signaling coordinately regulate actomyosin-dependent cytoplasmic streaming and oocyte growth in C. elegans," Development 2009 Jul;136(13):2223-34.   (MEDL:19502484 PMID: 19502484 #J0180757)    

   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.

3. Voutev, Roumen, Keating, Ryan, Jane Albert Hubbard, E, Vallier, Laura G. "Characterization of the Caenorhabditis elegans Islet LIM-homeodomain ortholog, lim-7," FEBS letters 2009 Jan 22;583(2):456-64.   (MEDL:19116151 PMID: 19116151 #J0164242)    

   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.

4. Kam, Na'aman, Kugler, Hillel, Marelly, Rami, Appleby, Lara, Fisher, Jasmin, Pnueli, Amir, Harel, David, Stern, Michael J, Hubbard, E Jane Albert. "A scenario-based approach to modeling development: a prototype model of C. elegans vulval fate specification," Developmental biology (Orlando) 2008 Nov 1;323(1):1-5.   (MEDL:18706404 PMID: 18706404 #J0155951)    

   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.

5. Sadot, Avital, Fisher, Jasmin, Barak, Dan, Admanit, Yishai, Stern, Michael J, Hubbard, E Jane Albert, Harel, David. "Toward verified biological models," IEEE/ACM transactions on computational biology & bioinformatics 2008 Apr-Jun;5(2):223-34.   (MEDL:18451431 PMID: 18451431 #J0155952)    

   The last several decades have witnessed a vast accumulation of biological data and data analysis. Many of these data sets represent only a small fraction of the system's behavior, making the visualization of full system behavior difficult. A more complete understanding of a biological system is gained when different types of data (and/or conclusions drawn from the data) are integrated into a larger-scale representation or model of the system. Ideally, this type of model is consistent with all available data about the system, and it is then used to generate additional hypotheses to be tested. Computer-based methods intended to formulate models that integrate various events and to test the consistency of these models with respect to the laboratory-based observations on which they are based are potentially very useful. In addition, in contrast to informal models, the consistency of such formal computer-based models with laboratory data can be tested rigorously by methods of formal verification. We combined two formal modeling approaches in computer science that were originally developed for non-biological system design. One is the inter-object approach using the language of live sequence charts (LSCs) with the Play-Engine tool, and the other is the intra-object approach using the language of statecharts and Rhapsody as the tool. Integration is carried out using InterPlay, a simulation engine coordinator. Using these tools, we constructed a combined model comprising three modules. One module represents the early lineage of the somatic gonad of C. elegans in LSCs, while a second more detailed module in statecharts represents an interaction between two cells within this lineage that determine their developmental outcome. Using the advantages of the tools, we created a third module representing a set of key experimental data using LSCs. We tested the combined statechart-LSC model by showing that the simulations were consistent with the set of experimental LSCs. This small-scale modular example demonstrates the potential for using similar approaches for verification by exhaustive testing of models by LSCs. It also shows the advantages of these approaches for modeling biology.

6. Voutev, Roumen, Hubbard, E Jane Albert. "A "FLP-Out" system for controlled gene expression in Caenorhabditis elegans," Genetics 2008 Sep;180(1):103-19.   (MEDL:18723890 PMID: 18723890 #J0155950)    

   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.

7. Maciejowski, John, Ugel, Nadia, Mishra, Bud, Isopi, Marco, Hubbard, E Jane Albert. "Quantitative analysis of germline mitosis in adult C. elegans," Developmental biology (Orlando) 2006 Apr 1;292(1):142-51.   (MEDL:16480707 PMID: 16480707 #J0125773)    

   Certain aspects of the distal gonad of C. elegans are comparable to niche/stem cell systems in other organisms. The distal tip cell (DTC) caps a blind-ended tube; only the distal germ cells maintain proliferation in response to signaling from the DTC via the GLP-1/Notch signaling pathway in the germ line. Fruitful comparison between this system and other stem cell systems is limited by a lack of basic information regarding germ cell division behavior in C. elegans. Here, we explore the spatial pattern of cell division frequency in the adult C. elegans germ line relative to distance from the distal tip. We mapped the positions of actively dividing germline nuclei in over 600 fixed gonad preparations including the wild type and a gain-of-function ligand-responsive GLP-1 receptor mutant with an extended mitotic zone. One particularly surprising observation from these data is that the frequency of cell divisions is lower in distal-most cells-cells that directly contact the distal tip cell body-relative to cells further proximal, a difference that persists in the gain-of-function GLP-1 mutant. These results suggest that cell division frequency in the distal-most cells may be suppressed or otherwise controlled in a complex manner. Further, our data suggest that the presence of an active cell division influences the probability of observing simultaneous cell divisions in the same gonad arm, and that simultaneous divisions tend to cluster spatially. We speculate that this system behaves similarly to niche/stem cell/transit amplifying cell systems in other organisms.

8. Voutev, Roumen, Killian, Darrell J, Ahn, James Hyungsoo, Hubbard, E Jane Albert. "Alterations in ribosome biogenesis cause specific defects in C. elegans hermaphrodite gonadogenesis," Developmental biology (Orlando) 2006 Oct 1;298(1):45-58.   (MEDL:16876152 PMID: 16876152 #J0126775)    

   Ribosome biogenesis is a cell-essential process that influences cell growth, proliferation, and differentiation. How ribosome biogenesis impacts development, however, is poorly understood. Here, we establish a link between ribosome biogenesis and gonadogenesis in Caenorhabditis elegans that affects germline proliferation and patterning. Previously, we determined that pro-1(+)activity is required in the soma--specifically, the sheath/spermatheca sublineage--to promote normal proliferation and prevent germline tumor formation. Here, we report that PRO-1, like its yeast ortholog IPI3, influences rRNA processing. pro-1 tumors are suppressed by mutations in ncl-1 or lin-35/Rb, both of which elevate pre-rRNA levels. Thus, in this context, lin-35/Rb acts as a soma-autonomous germline tumor promoter. We further report the characterization of two additional genes identified for their germline tumor phenotype, pro-2 and pro-3, and find that they, too, encode orthologs of proteins involved in ribosome biogenesis in yeast (NOC2 and SDA1, respectively). Finally, we demonstrate that depletion of additional C. elegans orthologs of yeast ribosome biogenesis factors display phenotypes similar to depletion of progenes. We conclude that the C. elegans distal sheath is particularly sensitive to alterations in ribosome biogenesis and that ribosome biogenesis defects in one tissue can non-autonomously influence proliferation in an adjacent tissue.

9. Fisher, Jasmin, Piterman, Nir, Hubbard, E Jane Albert, Stern, Michael J, Harel, David. "Computational insights into Caenorhabditis elegans vulval development," Proceedings of the National Academy of Sciences of the United States of America 2005 Feb 8;102(6):1951-6.   (MEDL:15684055 PMID: 15684055 #J0126778)    

   Studies of Caenorhabditis elegans vulval development provide a paradigm for pattern formation during animal development. The fates of the six vulval precursor cells are specified by the combined action of an inductive signal that activates the EGF receptor mitogen-activated PK signaling pathway (specifying a primary fate) and a lateral signal mediated by LIN-12/Notch (specifying a secondary fate). Here we use methods devised for the engineering of complex reactive systems to model a biological system. We have chosen the visual formalism of statecharts and use it to formalize Sternberg and Horvitz's 1989 model [Sternberg, P. W. & Horvitz, H. R. (1989) Cell 58, 679-693], which forms the basis for our current understanding of the interaction between these two signaling pathways. The construction and execution of our model suggest that different levels of the inductive signal induce a temporally graded response of the EGF receptor mitogen-activated PK pathway and make explicit the importance of this temporal response. Our model also suggests the existence of an additional mechanism operating during lateral specification that prohibits neighboring vulval precursor cells from assuming the primary fate.

10. Hubbard, E Jane Albert, Greenstein, David. "Introduction to the germ line," WormBook : the online review of C. elegans biology 2005 Sep 1:1-4.   (MEDL:18050415 PMID: 18050415 #J0155953)    

11. Killian, Darrell J, Hubbard, E Jane Albert. "Caenorhabditis elegans germline patterning requires coordinated development of the somatic gonadal sheath and the germ line," Developmental biology (Orlando) 2005 Mar 15;279(2):322-35.   (MEDL:15733661 PMID: 15733661 #J0126776)    

    Interactions between the somatic gonad and the germ line influence the amplification, maintenance, and differentiation of germ cells. In Caenorhabditis elegans, the distal tip cell/germline interaction promotes a mitotic fate and/or inhibits meiosis through GLP-1/Notch signaling. However, GLP-1-mediated signaling alone is not sufficient for a wild-type level of germline proliferation. Here, we provide evidence that specific cells of the somatic gonadal sheath lineage influence amplification, differentiation, and the potential for tumorigenesis of the germ line. First, an interaction between the distal-most pair of sheath cells and the proliferation zone of the germ line is required for larval germline amplification. Second, we show that insufficient larval germline amplification retards gonad elongation and thus delays meiotic entry. Third, a more severe delay in meiotic entry, as is exhibited in certain mutant backgrounds, inappropriately juxtaposes undifferentiated germ cells with cells of the proximal sheath lineage, leading to the formation of a proximal germline tumor derived from undifferentiated germ cells. Tumors derived from dedifferentiated germ cells, however, respond to the proximal interaction differently depending on the mutant background. Our study underscores the importance of strict developmental coordination between neighboring tissues. We discuss these results in the context of mechanisms that may underlie tumorigenesis.

12. Maciejowski, John, Ahn, James Hyungsoo, Cipriani, Patricia Giselle, Killian, Darrell J, Chaudhary, Aisha L, Lee, Ji Inn, Voutev, Roumen, Johnsen, Robert C, Baillie, David L, Gunsalus, Kristin C, Fitch, David H A, Hubbard, E Jane Albert. "Autosomal genes of autosomal/X-linked duplicated gene pairs and germ-line proliferation in Caenorhabditis elegans," Genetics 2005 Apr;169(4):1997-2011.   (MEDL:15687263 PMID: 15687263 #J0126777)    

    We report molecular genetic studies of three genes involved in early germ-line proliferation in Caenorhabditis elegans that lend unexpected insight into a germ-line/soma functional separation of autosomal/X-linked duplicated gene pairs. In a genetic screen for germ-line proliferation-defective mutants, we identified mutations in rpl-11.1 (L11 protein of the large ribosomal subunit), pab-1 [a poly(A)-binding protein], and glp-3/eft-3 (an elongation factor 1-alpha homolog). All three are members of autosome/X gene pairs. Consistent with a germ-line-restricted function of rpl-11.1 and pab-1, mutations in these genes extend life span and cause gigantism. We further examined the RNAi phenotypes of the three sets of rpl genes (rpl-11, rpl-24, and rpl-25) and found that for the two rpl genes with autosomal/X-linked pairs (rpl-11 and rpl-25), zygotic germ-line function is carried by the autosomal copy. Available RNAi results for highly conserved autosomal/X-linked gene pairs suggest that other duplicated genes may follow a similar trend. The three rpl and the pab-1/2 duplications predate the divergence between C. elegans and C. briggsae, while the eft-3/4 duplication appears to have occurred in the lineage to C. elegans after it diverged from C. briggsae. The duplicated C. briggsae orthologs of the three C. elegans autosomal/X-linked gene pairs also display functional differences between paralogs. We present hypotheses for evolutionary mechanisms that may underlie germ-line/soma subfunctionalization of duplicated genes, taking into account the role of X chromosome silencing in the germ line and analogous mammalian phenomena.

13. Hansen, Dave, Hubbard, E Jane Albert, Schedl, Tim. "Multi-pathway control of the proliferation versus meiotic development decision in the Caenorhabditis elegans germline," Developmental biology (Orlando) 2004 Apr 15;268(2):342-57.   (MEDL:15063172 PMID: 15063172 #J0126779)    

    An important event in the development of the germline is the initiation of meiotic development. In Caenorhabditis elegans, the conserved GLP-1/Notch signaling pathway regulates the proliferative versus meiotic entry decision, at least in part, by spatially inhibiting genes in the gld-1 and gld-2 parallel pathways, which are proposed to either inhibit proliferation and/or promote meiotic development. Mutations that cause constitutive activation of the GLP-1 pathway, or inactivation of both the gld-1 and gld-2 parallel pathways, result in a tumorous germline in which all cells are thought to be proliferative. Here, to analyze proliferation and meiotic entry in wild-type and mutant tumorous germlines, we use anti-REC-8 and anti-HIM-3 specific antibodies as markers, which under our fixation conditions, stain proliferative and meiotic cells, respectively. Using these makers in wild-type animals, we find that the border of the switch from proliferation to meiotic entry is staggered in late-larval and adult germlines. In wild-type adults, the switch occurs between 19 and 26 cell diameters from the distal end, on average. Our analysis of mutants reveals that tumorous germlines that form when GLP-1 is constitutively active are completely proliferative, while tumors due to inactivation of the gld-1 and gld-2 pathways show evidence of meiotic entry. Genetic and time course studies suggest that a third pathway may exist, parallel to the GLD-1 and GLD-2 pathways, that promotes meiotic development.

14. Killian, Darrell J, Hubbard, E Jane Albert. "C. elegans pro-1 activity is required for soma/germline interactions that influence proliferation and differentiation in the germ line," Development 2004 Mar;131(6):1267-78.   (MEDL:14973273 PMID: 14973273 #J0126780)    

    Strict spatial and temporal regulation of proliferation and differentiation is essential for proper germline development and often involves soma/germline interactions. In C. elegans, a particularly striking outcome of defective regulation of the proliferation/differentiation pattern is the Pro phenotype in which an ectopic mass of proliferating germ cells occupies the proximal adult germ line, a region normally occupied by gametes. We describe a reduction-of-function mutation in the gene pro-1 that causes a highly penetrant Pro phenotype. The pro-1 mutant Pro phenotype stems from defects in the time and position of the first meiotic entry during early germline development. pro-1(RNAi) produces a loss of somatic gonad structures and concomitant reduction in germline proliferation and gametogenesis. pro-1 encodes a member of a highly conserved subfamily of WD-repeat proteins. pro-1(+) is required in the sheath/spermatheca lineage of the somatic gonad in its role in the proper establishment of the proliferation/differentiation pattern in the germline. Our results provide a handle for further analysis of this soma-to-germline interaction.

15. Hubbard, E Jane Albert, Pera, Renee A Reijo. "A germ-cell odyssey: fate, survival, migration, stem cells and differentiation. Meeting on germ cells," EMBO reports 2003 Apr;4(4):352-7.   (MEDL:12671678 PMID: 12671678 #J0126782)    

16. Mishra, Bud, Daruwala, Raoul-Sam, Zhou, Yi, Ugel, Nadia, Policriti, Alberto, Antoniotti, Marco, Paxia, Salvatore, Rejali, Marc, Rudra, Archisman, Cherepinsky, Vera, Silver, Naomi, Casey, William, Piazza, Carla, Simeoni, Marta, Barbano, Paolo, Spivak, Marina, Feng, Jiawu, Gill, Ofer, Venkatesh, Mysore, Cheng, Fang, Sun, Bing, Ioniata, Iuliana, Anantharaman, Thomas, Hubbard, E Jane Albert, Pnueli, Amir, Harel, David, Chandru, Vijay, Hariharan, Ramesh, Wigler, Michael, Park, Frank, Lin, Shih-Chieh, Lazebnik, Yuri, Winkler, Franz, Cantor, Charles R, Carbone, Alessandra, Gromov, Mikhael. "A sense of life: computational and experimental investigations with models of biochemical and evolutionary processes," Omics : a journal of integrative biology 2003 Fall;7(3):253-68.   (MEDL:14583115 PMID: 14583115 #J0125778)    

    We collaborate in a research program aimed at creating a rigorous framework, experimental infrastructure, and computational environment for understanding, experimenting with, manipulating, and modifying a diverse set of fundamental biological processes at multiple scales and spatio-temporal modes. The novelty of our research is based on an approach that (i) requires coevolution of experimental science and theoretical techniques and (ii) exploits a certain universality in biology guided by a parsimonious model of evolutionary mechanisms operating at the genomic level and manifesting at the proteomic, transcriptomic, phylogenic, and other higher levels. Our current program in 'systems biology' endeavors to marry large-scale biological experiments with the tools to ponder and reason about large, complex, and subtle natural systems. To achieve this ambitious goal, ideas and concepts are combined from many different fields: biological experimentation, applied mathematical modeling, computational reasoning schemes, and large-scale numerical and symbolic simulations. From a biological viewpoint, the basic issues are many: (i) understanding common and shared structural motifs among biological processes; (ii) modeling biological noise due to interactions among a small number of key molecules or loss of synchrony; (iii) explaining the robustness of these systems in spite of such noise; and (iv) cataloging multistatic behavior and adaptation exhibited by many biological processes.

17. Pepper, Anita S-R, Lo, Te Wen, Killian, Darrell J, Hall, David H, Hubbard, E Jane Albert. "The establishment of Caenorhabditis elegans germline pattern is controlled by overlapping proximal and distal somatic gonad signals," Developmental biology (Orlando) 2003 Jul 15;259(2):336-50.   (MEDL:12871705 PMID: 12871705 #J0126781)    

    We investigated the control of proliferation and differentiation in the larval Caenorhabditis elegans hermaphrodite germ line through analysis of glp-1 and lag-2 mutants, cell ablations, and ultrastructural data. After the first several rounds of germ cell division, GLP-1, a receptor of the LIN-12/Notch family, governs germline proliferation. We analyzed the proximal proliferation (Pro) phenotype in glp-1(ar202) and found that initial meiosis was delayed and spatially mispositioned. This is due, at least in part, to a heightened response of the mutant GLP-1 receptor to multiple sources of the somatic ligand LAG-2, including the proximal somatic gonad. We investigated whether proximal LAG-2 affects germline proliferation in the wild type. Our results indicate that (1) LAG-2 is necessary for GLP-1-mediated germline proliferation and prevention of early meiosis, and (2) several distinct anatomical sources of LAG-2 in the larval somatic gonad functionally overlap to promote proliferation and prevent early meiosis. Ultrastructural studies suggest that mitosis is not restricted to areas of direct DTC-germ line contact and that the germ line shares a common cytoplasm in larval stages. We propose that downregulation of the GLP-1 signaling pathway in the proximal germ line at the time of meiotic onset is under tight temporal and spatial control.

18. Pepper, Anita S-R, Killian, Darrell J, Hubbard, E Jane Albert. "Genetic analysis of Caenorhabditis elegans glp-1 mutants suggests receptor interaction or competition," Genetics 2003 Jan;163(1):115-32.   (MEDL:12586701 PMID: 12586701 #J0126783)    

    glp-1 encodes a member of the highly conserved LIN-12/Notch family of receptors that mediates the mitosis/meiosis decision in the C. elegans germline. We have characterized three mutations that represent a new genetic and phenotypic class of glp-1 mutants, glp-1(Pro). The glp-1(Pro) mutants display gain-of-function germline pattern defects, most notably a proximal proliferation (Pro) phenotype. Each of three glp-1(Pro) alleles encodes a single amino acid change in the extracellular part of the receptor: two in the LIN-12/Notch repeats (LNRs) and one between the LNRs and the transmembrane domain. Unlike other previously described gain-of-function mutations that affect this region of LIN-12/Notch family receptors, the genetic behavior of glp-1(Pro) alleles is not consistent with simple hypermorphic activity. Instead, the mutant phenotype is suppressed by wild-type doses of glp-1. Moreover, a trans-heterozygous combination of two highly penetrant glp-1(Pro) mutations is mutually suppressing. These results lend support to a model for a higher-order receptor complex and/or competition among receptor proteins for limiting factors that are required for proper regulation of receptor activity. Double-mutant analysis with suppressors and enhancers of lin-12 and glp-1 further suggests that the functional defect in glp-1(Pro) mutants occurs prior to or at the level of ligand interaction.

19. Hubbard, E J, Greenstein, D. "The Caenorhabditis elegans gonad: a test tube for cell and developmental biology," Developmental dynamics 2000 May;218(1):2-22.   (MEDL:10822256 PMID: 10822256 #J0126813)    

    Sexual reproduction of multicellular organisms depends critically on the coordinate development of the germ line and somatic gonad, a process known as gonadogenesis. Together these tissues ensure the formation of functional gametes and, in the female of many species, create a context for production and further development of the zygote. Since the future of the species hangs in the balance, it is not surprising that gonadogenesis is a complex process involving conserved and multi-faceted developmental mechanisms. Genetic, anatomical, cell biological, and molecular experiments have established the nematode Caenorhabditis elegans as a paradigm for studying gonadogenesis. Furthermore, these studies demonstrate the utility of C. elegans gonadogenesis for exploring broad issues in cell and developmental biology, such as cell fate specification, morphogenesis, cell signaling, cell cycle control, and programmed cell death. The synergy of molecular genetics and cell biology conducted at single-cell resolution in real time permits an extraordinary depth of analysis in this organism. In this review, we first describe the embryonic and post-embryonic development and morphology of the C. elegans gonad. Next we recount seminal experiments that established the field, highlight recent results that provide insight into conserved developmental mechanisms, and present future prospects for the field.

20. Wu, G, Hubbard, E J, Kitajewski, J K, Greenwald, I. "Evidence for functional and physical association between Caenorhabditis elegans SEL-10, a Cdc4p-related protein, and SEL-12 presenilin," Proceedings of the National Academy of Sciences of the United States of America 1998 Dec 22;95(26):15787-91.   (MEDL:9861048 PMID: 9861048 #J0155955)    

    Mutations in either of two human presenilin genes (PS1 and PS2) cause Alzheimer's disease. Here we describe genetic and physical interactions between Caenorhabditis elegans SEL-10, a member of the Cdc4p family of proteins, and SEL-12, a C. elegans presenilin. We show that loss of sel-10 activity can suppress the egg-laying defective phenotype associated with reducing sel-12 activity, and that SEL-10 can physically complex with SEL-12. Proteins of the Cdc4p family have been shown to target proteins for ubiquitin-mediated turnover. The functional and physical interaction between sel-10 and sel-12 therefore offers an approach to understanding how presenilin levels are normally regulated.

21. Hubbard, E J, Wu, G, Kitajewski, J, Greenwald, I. "sel-10, a negative regulator of lin-12 activity in Caenorhabditis elegans, encodes a member of the CDC4 family of proteins," Genes & development 1997 Dec 1;11(23):3182-93.   (MEDL:9389650 PMID: 9389650 #J0126814)    

    Mutations that influence lin-12 activity in Caenorhabditis elegans may identify conserved factors that regulate the activity of lin-12/Notch proteins. We describe genetic evidence indicating that sel-10 is a negative regulator of lin-12/Notch-mediated signaling in C. elegans. Sequence analysis shows that SEL-10 is a member of the CDC4 family of proteins and has a potential human ortholog. Coimmunoprecipitation data indicate that C. elegans SEL-10 complexes with LIN-12 and with murine Notch4. We propose that SEL-10 promotes the ubiquitin-mediated turnover of LIN-12/Notch proteins, and discuss potential roles for the regulation of lin-12/Notch activity by sel-10 in cell fate decisions and tumorigenesis.

22. Hubbard, E J, Dong, Q, Greenwald, I. "Evidence for physical and functional association between EMB-5 and LIN-12 in Caenorhabditis elegans," Science 1996 Jul 5;273(5271):112-5.   (MEDL:8658178 PMID: 8658178 #J0126815)    

    The Caenorhabditis elegans LIN-12 and GLP-1 proteins are members of the LIN-12/Notch family of receptors for intercellular signals that specify cell fate. Evidence presented here suggests that the intracellular domains of LIN-12 and GLP-1 interact with the C. elegans EMB-5 protein and that the emb-5 gene functions in the same pathway as the lin-12 and glp-1 genes. EMB-5 is similar in sequence to a yeast protein that controls chromatin structure. Hence, a direct consequence of LIN-12 or GLP-1 activation may be an alteration of chromatin structure that produces changes in transcriptional activity.

23. Hubbard, E J, Jiang, R, Carlson, M. "Dosage-dependent modulation of glucose repression by MSN3 (STD1) in Saccharomyces cerevisiae," Molecular & cellular biology 1994 Mar;14(3):1972-8.   (MEDL:8114728 PMID: 8114728 #J0155954)    

    The SNF1 protein kinase of Saccharomyces cerevisiae is required to relieve glucose repression of transcription. To identify components of the SNF1 pathway, we isolated multicopy suppressors of defects caused by loss of SNF4, an activator of the SNF1 kinase. Increased dosage of the MSN3 gene restored invertase expression in snf4 mutants and also relieved glucose repression in the wild type. Deletion of MSN3 caused no substantial phenotype, and we identified a homolog, MTH1, encoding a protein 61% identical to MSN3. Both are also homologous to chicken fimbrin, human plastin, and yeast SAC6 over a 43-residue region. Deletion of MSN3 and MTH1 together impaired derepression of invertase in response to glucose limitation. Finally, MSN3 physically interacts with the SNF1 protein kinase, as assayed by a two-hybrid system and by in vitro binding studies. MSN3 is the same gene as STD1, a multicopy suppressor of defects caused by overexpression of the C terminus of TATA-binding protein (R. W. Ganster, W. Shen, and M. C. Schmidt, Mol. Cell. Biol. 13:3650-3659, 1993). Taken together, these data suggest that MSN3 modulates the regulatory response to glucose and may couple the SNF1 pathway to transcription.

24. Hubbard, E J, Yang, X L, Carlson, M. "Relationship of the cAMP-dependent protein kinase pathway to the SNF1 protein kinase and invertase expression in Saccharomyces cerevisiae," Genetics 1992 Jan;130(1):71-80.   (MEDL:1310088 PMID: 1310088 #J0126818)    

    The SNF1 protein kinase and the associated SNF4 protein are required for release of glucose repression in Saccharomyces cerevisiae. To identify functionally related proteins, we selected genes that in multicopy suppress the raffinose growth defect of snf4 mutants. Among the nine genes recovered were two genes from the cAMP-dependent protein kinase (cAPK) pathway, MSI1 and PDE2. Increased dosage of these genes partially compensates for defects in nutrient utilization and sporulation in snf1 and snf4 null mutants, but does not restore invertase expression. These results suggest that SNF1 and cAPK affect some of the same cellular responses to nutrients. To examine the role of the cAPK pathway in regulation of invertase, we assayed mutants in which the cAPK is not modulated by cAMP. Expression of invertase was regulated in response to glucose and was dependent on SNF1 function. Thus, a cAMP-responsive cAPK is dispensable for regulation of invertase.

25. Robinson, L C, Hubbard, E J, Graves, P R, DePaoli-Roach, A A, Roach, P J, Kung, C, Haas, D W, Hagedorn, C H, Goebl, M, Culbertson, M R. "Yeast casein kinase I homologues: an essential gene pair," Proceedings of the National Academy of Sciences of the United States of America 1992 Jan 1;89(1):28-32.   (MEDL:1729698 PMID: 1729698 #J0126817)    

    We report the isolation of an essential pair of Saccharomyces cerevisiae genes that encode protein kinase homologues. The two genes were independently isolated as dosage-dependent suppressors. Increased dosage of YCK1 suppressed defects caused by reduced SNF1 protein kinase activity, and increased dosage of YCK2 relieved sensitivity of wild-type cells to salt stress. The two genes function identically in the two growth assays, and loss of function of either gene alone has no discernible effect on growth. However, loss of function of both genes results in inviability. The two predicted protein products share 77% overall amino acid identity and contain sequence elements conserved among protein kinases. Partial sequence obtained for rabbit casein kinase I shares 64% identity with the two yeast gene products. Moreover, an increase in casein kinase I activity is observed in extracts from cells overexpressing YCK2. Thus YCK1 and YCK2 appear to encode casein kinase I homologues.

26. Yang, X, Hubbard, E J, Carlson, M. "A protein kinase substrate identified by the two-hybrid system," Science 1992 Jul 31;257(5070):680-2.   (MEDL:1496382 PMID: 1496382 #J0126816)    

    A genetic method, the two-hybrid system, was used to identify four genes encoding proteins that interact with the SNF1 protein kinase from yeast. One of the genes, SIP1, was independently isolated as a multicopy suppressor of defects caused by reduced SNF1 kinase activity, and genetic evidence supports its function in the SNF1 pathway. The SIP1 protein co-immunoprecipitated with SNF1 and was phosphorylated in vitro. Thus, the two-hybrid system, which is applicable to any cloned gene, can be used to detect physical interactions between protein kinases and functionally related substrate proteins.