Hubbard EJA et al. (1994) Worm Breeder's Gazette "Genetic Analysis of sel-10."

Greenwald IS, Hubbard EJA

[Worm Breeder's Gazette, 1994]

Genetic analysis of sel-10 E. Jane Albert Hubbard and Iva Greenwald Department of Biochemistry and Molecular Biophysics and Howard Hughes Medical Institute, Columbia University, New York, New York, 10032

David Michaelson et al. (2006) Development & Evolution Meeting "Soma-germline Interactions Required for Robust Larval Germline Proliferation"

Chris Ryan, John Maciejowski, E. Jane Hubbard, Dina Esposito, Rachael Lader, Jessica Dy-Johnson, David Michaelson

[Development & Evolution Meeting, 2006]

Proper control of cell proliferation is critical for normal development. While the molecular basis for the proliferation/differentiation decision is relatively well-characterized in certain developmental contexts, less is known about cell-cell interactions that control of the extent of proliferation within a developing organ. The C. elegans germ line offers a tractable model system for exploring this problem. Previous work (McCarter et al., 1997; Killian and Hubbard, 2005), indicates that the distal pair of somatic gonadal sheath cells (Sh1) promote robust larval germline proliferation. We are taking several approaches to identify the molecular basis for this interaction. Forward genetic screens, molecular analysis and directed RNAi studies identified a critical role for optimal ribosome biogenesis in the sheath (Killian and Hubbard, 2004; Voutev et al., submitted) for its germline proliferation-promoting activity. To identify additional players, a genome-wide RNAi screen and candidate screens are underway. One screening strategy is based on the observation that reduced larval germline proliferation inhibits gonad arm extension, delays meiotic entry, and thereby uncouples the coordination between somatic gonad and germline development. As a result, under certain conditions, reducing larval germline proliferation can enhance proximal germline tumor formation (Pro phenotype; Killian and Hubbard, 2005; see also abstract by Maciejowski and Hubbard). Thus we are screening for soma-autonomous RNAi-induced enhancers of Pro. In addition, candidate screens suggest that the insulin pathway contributes to robust larval germline proliferation.

Cynthia Wagner et al. (2006) Development & Evolution Meeting "The Role of gak-1 in Meiosis"

Cynthia Wagner, Judith Yanowitz, Rachel Webster, Gabrielle Howard, Dana Hubbard

[Development & Evolution Meeting, 2006]

We performed an RNAi feeding screen to identify chromatin associated gene products, that when mutant, result in an increase in recombination. We screened greater than 300 genes and identified 20 genes that give between a two-fold and four-fold enhancement of recombination. We are in the process of characterizing one of these genes, gak-1. gak-1 has previously been reported to function in meiotic chromosome segregation, including an increase in X-chromosome nondisjunction, which results in a him phenotype. We will present our cytological analysis on gak-1 mutants as well as detailed information on recombination effects in gak-1 mutants.

Diana Dalfo et al. (2008) Development & Evolution Meeting "RNAi screening to identify enhancers of the germline tumor phenotype in C. elegans"

Diana Dalfo, E. Jane Hubbard

[Development & Evolution Meeting, 2008]

There is significant interplay between the control of cell fate decisions and cell proliferation during development, yet the mechanisms underlying this interplay are not well understood. We are using the C. elegans germ line to investigate these mechanisms. C. elegans, germline polarity is established when a subset of previously undifferentiated germ cells differentiate and enter meiosis at a distinct developmental time and position. We have isolated mutants that disrupt the pattern of germline development such that the initial meiotic onset is delayed. The ultimate consequence of this delay is a large mass of proliferating germ cells in the proximal germ line of the adult. This phenotype, proximal proliferation (Pro), is observed with certain alleles of glp-1 (Pepper et al., 2003(a,b)). glp-1 is a receptor of the LIN-12/Notch family, and its activity promotes the proliferative or undifferentiated state and/or inhibits differentiation (meiosis) in the germ line (Austin and Kimble, 1987; Yochem and Greenwald, 1989). We are using a glp-1(Pro) allele as a starting point to identify additional factors that affect early germline proliferation and initial meiotic entry. Our previous data indicate that the distal pair of somatic gonadal sheath cells influence germline proliferation and act as an additional, non-DTC-mediated, mechanism to promote robust larval germline proliferation (Killian and Hubbard, 2005). We also observed that ablation of these cells enhances the glp-1(Pro) phenotype (Killian and Hubbard, 2005). Therefore, to identify the sheath-autonomous proliferation-promoting activity, we are performing candidate and genome-wide RNAi screens for enhancers of the Pro phenotype.

Marie McGovern et al. (2008) Development & Evolution Meeting "A Mechanism for Tumor Initiation in the C. elegans germ line"

John Maciejowski, Marie McGovern, E. Jane Hubbard

[Development & Evolution Meeting, 2008]

We are studying the molecular and cellular basis of tumor formation in the germ line of C. elegans as a paradigmatic model for tumor seeding and its relationship to stem cell niches. Germ cells in the distal gonad remain undifferentiated in response to a signal produced by the Distal Tip Cell (DTC) (Kimble and White 1981). The DTC produces the DSL ligand LAG-2 that interacts with the GLP-1/Notch receptor in the germ line to maintain a self-renewing population of neighboring germ cells (Austin and Kimble, 1987; Yochem and Greenwald, 1989; Lambie and Kimble, 1991; Henderson et al., 1994). During larval stages, the DTC moves away from the midline, and this movement is critical for proper germline patterning. Two independent mechanisms underlie early DTC migration: an intrinsic migration program and the extent of germline proliferation. As a result of these two mechanisms, proximal-most germ cells eventually lie outside the influence of the DTC and differentiate, setting up the distal-proximal germline pattern. Proximal germline tumors (Pro phenotype) can form when cells in the developing germ line fail to differentiate at the appropriate time (Pepper et al., 2003(a,b); Killian and Hubbard, 2004, Voutev et al., 2006). We have found three different genetic/cellular defects that cause proximal tumor formation, each of which results in a delay in germline differentiation. This delay inappropriately juxtaposes undifferentiated germ cells in the proximal oviduct with certain somatic gonad cells of the proximal sheath lineage. Based on cell ablation studies (Killian and Hubbard, 2005), we showed that the Pro phenotype occurs in response to a latent proliferation-promoting signal produced by the proximal sheath lineage. To determine the molecular basis for this latent proliferation-promoting signal, we examined the other 9 putative DSL ligands in the genome. We found that apx-1 is expressed in the correct cells of the somatic gonad, and that reduction of apx-1 activity can suppress the Pro phenotype in three different mutant conditions, each of which have a different anatomical focus of activity but share the same delay in germline differentiation. We further found that apx-1 and glp-1 are required continuously to maintain the proximal germline tumors. These results suggest a novel general mechanism for tumor formation.

Dalfo, Diana et al. (2009) International Worm Meeting "RNAi-based identification of genes involved in germline proliferation."

Dalfo, Diana, Albert Hubbard, E. Jane

[International Worm Meeting, 2009]

Precise control of cell proliferation is necessary to create organs of the correct size and shape. We are interested in understanding the molecular basis for the control cell proliferation in vivo, and we are using the C. elegans germ line as a model. In C. elegans, as in other organisms, germline proliferation is a key step in the development of a reproductively competent individual. Previously, we isolated mutants that disrupt the pattern of germline development and ultimately produce a large ectopic mass of proliferating germ cells (a germline tumor) in the proximal part of the gonad arm (Pepper et al., 2003a,b; Killian and Hubbard, 2004; Voutev et al., 2006). This phenotype, proximal proliferation (Pro), is observed with certain mutant alleles of glp-1 (Pepper et al., 2003). glp-1 encodes a receptor of the LIN-12/Notch family, and its activity promotes the proliferative or undifferentiated state and/or inhibits differentiation (meiosis) in the germ line (Austin and Kimble, 1987; Yochem and Greenwald, 1989). We are performing a genome-wide RNAi screen for enhancers of the Pro phenotype to look for factors that affect early germline proliferation and initial meiotic entry.

Coux O (2003) Biochem Soc Trans "An interaction map of proteasome subunits."

Coux O

[Biochem Soc Trans, 2003]

Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits (Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821-828). A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three alpha-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming 'interaction clusters' of

Roumen Voutev et al. (2005) International Worm Meeting "Pro mutants, germline tumors, sheath cells, and ribosome biogenesis"

Darrell J Killian, James Hyungsoo Ahn, Roumen Voutev, E Jane Albert Hubbard

[International Worm Meeting, 2005]

Increasing evidence suggests that general cellular processes influence development in specific ways, likely reflecting tissue-specific differences in cellular demands. Gonadogenesis is proving an excellent model system to explore the intersection of ribosome biogenesis and development. Our data suggest that certain somatic gonad functions that influence germline proliferation are very sensitive to perturbations in ribosome biogenesis.Our analysis of pro (PROximal PROliferation) mutants led us to consider the intersection of development and ribosome biogenesis. The Pro phenotype is characterized by a proximal germline tumor. Our results support a model whereby the pro-1(na48) Pro phenotype stems from a cascade of events starting with an early reduction in germline proliferation due to functional defects in the distal sheath. This germline proliferation defect retards gonad arm extension, which in turn, causes a delay in meiotic entry. The delay in meiotic entry inappropriately juxtaposes mitotic germ cells with cells of the proximal sheath lineage and thereby allows these germ cells to respond to a latent proliferation-promoting activity of the proximal sheath lineage. Thus, due to developmental asynchrony between the somatic gonad and germ line, a proximal germline tumor eventually develops in the initially under-proliferated germ line (Killian and Hubbard, 2005).Several lines of evidence suggest that ribosome biogenesis is a limiting process for the germline proliferation-promoting activity of the distal sheath. PRO-1 is a conserved WD-repeat protein that acts in the sheath lineage of the somatic gonad (Killian and Hubbard, 2004). The budding yeast ortholog of PRO-1, IPI3, has been implicated in several processes including pre-rRNA processing (Peng et al., 2003; Krogan et al., 2004). Several C. elegans orthologs of direct IPI3 interactors that are also involved in ribosome biogenesis cause a Pro phenotype when depleted by RNAi. Reduction of ncl-1 increases rDNA transcription (Frank and Roth, 1998; Frank et al., 2002) and suppresses the pro-1 Pro phenotype. Reduction of lin-35/Rb in the soma also suppresses the Pro phenotype. This result is particularly intriguing since Rb negatively regulates rDNA transcription in mammals. In addition, the relative levels of rRNA processing intermediates are altered in pro-1(na48) mutants. Finally, pro-2, as defined by pro-2(na27), likely encodes an ortholog of yeast NOC2 which is implicated in nucleolar/nucleoplasmic transport of the large ribosomal subunit.

Soderlund CA et al. (1990) Worm Breeder's Gazette "gm Sequence Analysis System Update"

Shanmugam P, Fields CA, Soderlund CA

[Worm Breeder's Gazette, 1990]

A number of improvements have been made in the gm automated DNA sequence analysis system: (1) The ratio of AT-containing to CG-containing dinucleotides has been added as a test for introns. This works better than AT frequency alone in C. elegans.(2) A branch site consensus sequence or an enhanced dinucleotide ratio can be required as an additional test on introns. (3) Predicted amino-acid sequence files are generated in a format appropriate for input to the Dana-Farber motif-identification program plsearch. (4) A graphic interface based on X-windows, version 11 is available as an option. (5) The complete analysis algorithm is significantly faster than the previous version. (6) A greedy model evaluation algorithm is available as an option. This algorithm generates the longest, non-overlapping models that cover a sequence and is much faster than the complete analysis algorithm. The program has been tested on Sun3, Sun4 and VAX machines running Unix (Ultrix on the VAX). Results for a series of tests run on a Sun 4/60 are shown in the table. [See Figure 1] gm can be run remotely on our machine, using the Internet. To do this, telnet to haywire.nmsu.edu, and logon as gm_guest with password gmuser. Read the README file for information on running gm. We are also distributing gm as C source code to nonprofit laboratories, either via anonymous ftp to haywire.nmsu.edu, or on tape. If you would like to receive gm on tape, send a 1/4' cartridge or 1/2' reel tape to: Chris Fields, Box 30001/3CRL, New Mexico State University, Las Cruces, New Mexico 88003-0001, USA; Telephone (505) 646-2848.

Tiffany Pica et al. (2006) Development & Evolution Meeting "Identification Of Genes Necessary For Gonadal Sheath Development Using RNAi"

Laura Vallier, Tiffany Pica

[Development & Evolution Meeting, 2006]

The gonadal sheath of Caenorhabditis elegans is essential to the correct development of male and female gametes. The gonadal sheath and the distal tip cell (DTC) play an essential role in germ line proliferation, and the gonadal sheath additionally is vital for germ cell fate specification, meiotic progression from pachytene and ovulation of oocytes into the spermatheca. The absence of a gonadal sheath results in reproductive disorders such as sterility. Double stranded RNA interference (dsRNAi), the process by which homologous double-stranded RNA is introduced to a cell and interferes with the expression of a gene, was employed to determine the genes essential to the gonadal sheath. Strains DG1575 tnIs6[lim-7::GFP, Rol-6(su1006)] (kind gift of D. Greenstein) and GC678 tnIs5[lim-7::GFP; Rol-6(su1006)]; qIs19[lag-2::GFP, Rol-6(su1006)] (kind gift of E. J. A. Hubbard), each carrying a GFP reporter system that auto-fluoresces in the gonadal sheath (and the DTC in GC678) were utilized to assess genes essential for gonadal sheath production in a dsRNAi screen. Hermaphrodites from either of these strains were fed bacteria containing individual dsRNAi clones to examine the requirement of genes on Chromosomes I and II for development of the gonadal sheath. Hermaphrodites exposed to individual dsRNAi clones were examined for loss of gonadal sheath tissue as assessed by loss of GFP fluorescence and consequent sterility. To date, we have screened all the genes on Chromosome I and the majority of the genes on Chromosome II. Progress and subsequent findings will be reported.