Shu P et al. (1995) International C. elegans Meeting "MORE ABOUT EGO GENES."

Maine EM, Smardon AM, Stacey SC, Spoerke JM, Lissemore JL, Qiao L, Shu P

[International C. elegans Meeting, 1995]

The glp-1 gene encodes a transmembrane receptor that serves as part of an inductive pathway during embryonic and larval development. Zygotic glp-1 is essential for induction of mitotic proliferation in the germ line beginning in mid-L2 and continuing throughout adulthood, while maternal glp-1 product is essential for several inductive events in embryogenesis. In the absence of glp-1 function, cells that normally undergo mitosis enter meiosis. To further understand the process of glp-1 mediated cell signaling, we have isolated extragenic enhancers of a weak glp-1 phenotype. Two of these enhancer genes, ego-1 and ego-2(enhancer of glp-1) are described here. Five other genes are described in the abstract by Qiao et al. Hermaphrodites with ego-1 mutations display a delay in the onset of meiosis as well as abnormal oogenesis. Therefore, gametogenesis is also delayed. ego-1(om18) males appear wild type. Some ego-1 mutants produce small, irregular oocytes while others lack oocytes altogether. Any embryos produced are always inviable. ego-1 appears to be a loss of function mutation due to the fact that ego-1/Df and ego-1 mutants have similar phenotypes. However, ego-1(om18)/ozDf5 worms have a more severe oogenesis defect than do ego-1(om18)/nDf25 worms and therefore ego-1(om18) may be only a partial loss of function allele. ego-1 appears to act downstream of glp-1 because ego-1(-) suppresses germline overproliferation in glp-1(gf/lf) animals. The ego-2(om33) chromosome contains ts spermatogenesis (Spe) and maternal effect lethal (Mel) phenotypes. The Ego and Mel phenotypes are uncovered by hDf9, while the Spe phenotype is not. Current work is attempting to determine if the ego and mel mutations can be separated. Progeny of ego-2(om33) mothers may die at anytime from embryogenesis through early adulthood. Since ego-2(-) does not suppress glp-1(gf) in the germline, we believe ego-2 acts upstream of or regulates glp-1.

Maine EM et al. (1994) Worm Breeder's Gazette "Mutations That Enhance glp-1 Identify Genes Required For Various Aspects of Germline Development."

Gelber MB, Smardon AM, Shu P, Qiao L, Maine EM, Lissemore JL

[Worm Breeder's Gazette, 1994]

Mutations that enhance glp-1 identify genes required for various aspects of germline development. Eleanor Maine, Li Qiao, Jim Lissemore-, Pei Shu, Anne Smardon, and Melanie Gelber. Biology Dept., Syracuse University, Syracuse, NY 13244 and Biology Dept., John Carroll University, Cleveland, OH 44118.

McClendon TB et al. (2016) Genetics "Promotion of Homologous Recombination by SWS-1 in Complex with RAD-51 Paralogs in Caenorhabditis elegans."

Bernstein K, McClendon TB, Yanowitz J, Sullivan M

[Genetics, 2016]

Homologous recombination (HR) repairs cytotoxic DNA double-strand breaks (DSBs) with high fidelity. Deficiencies in HR result in genome instability. A key early step in HR is the search for and invasion of a homologous DNA template by a single-stranded RAD-51 nucleoprotein filament. The Shu complex, comprised of a SWIM domain-containing protein and its interacting RAD51 paralogs, promotes HR by regulating RAD51 filament dynamics. Despite Shu complex orthologs throughout eukaryotes, our understanding of its function has been most extensively characterized in budding yeast. Evolutionary analysis of the SWIM domain identified Caenorhabditis elegans sws-1 as a putative homolog of yeast Shu complex member, Shu2. Using a CRISPR-induced nonsense allele of sws-1, we show that sws-1 promotes HR in mitotic and meiotic nuclei. sws-1 mutants exhibit sensitivity to DSB-inducing agents and fail to form mitotic RAD-51 foci following treatment with camptothecin. Phenotypic similarities between sws-1 and the two RAD-51 paralogs, rfs-1 and rip-1, suggest they function together. Indeed, we detect direct interaction between SWS-1 and RIP-1 by yeast-two-hybrid that is mediated by the SWIM domain in SWS-1 and the Walker B motif in RIP-1. Furthermore, RIP-1 bridges an interaction between SWS-1 and RFS-1, suggesting RIP-1 facilitates complex formation with SWS-1 and RFS-1. We propose that SWS-1, RIP-1, and RFS-1 comprise a C. elegans Shu complex. Our work provides a new model for studying Shu complex disruption in the context of a multicellular organism that has important implications as to why mutations in the human RAD51 paralogs are associated with genome instability.

Schneider K et al. (2000) East Coast Worm Meeting "EGO-1: A link between development and RNAi"

Vought VE, Schneider K, Maine EM

[East Coast Worm Meeting, 2000]

ego-1 gene function is required for normal germline development and, consequently, for fertility (1, 2). In addition to its pleiotropic germline phenotype, ego-1 is also required for a robust response to RNAi by many germline-expressed genes (2). EGO-1 protein is a member of the RNA-directed RNA polymerase (RdRP) family, whose other members include N. crassa QDE-1, A. thaliana SGS-2, and tomato RdRP. These proteins have been implicated in RNA silencing phenomena, as well. Consistent with these results, ego-1 appears to be expressed mainly, if not entirely, in the germ line (2). We are studying ego-1 to understand better its role in development and if/how its developmental role is related to its role in RNAi. We are carrying out a variety of studies aimed at describing the EGO-1 expression pattern in the gonad and identifying potential targets of EGO-1 regulation. For comparison with other RNAi-defective mutants (Rde and Mut genes), we are examining whether transgenes are desilenced in the ego-1 mutant germ line, and whether Rde mutations enhance the Ego-1 developmental phenotype. The results of these ongoing studies will be presented. (1) Qiao, L., Lissemore, J.L., Shu, P., Smardon, A., Gelber, M.B., and Maine, E.M. (1995) Genetics 141:551-569. (2) Smardon, A., Spoerke, J.M., Stacey, S.C., Klein, M.E., Mackin, N., and Maine, E.M. (2000) Current Biology 10:169-178.

Nadeem Moghal et al. (2001) International C. elegans Meeting "Modulation of inductive signaling during C. elegans vulval development"

Nadeem Moghal, Paul W Sternberg, L Rene Garcia, Chieh Chang

[International C. elegans Meeting, 2001]

In C. elegans hermaphrodites, the vulval precursor cells (VPCs) P3.p-P8.p can adopt vulval or hypodermal fates. In wild-type animals, the anchor cell secretes LIN-3, which activates its receptor, LET-23, and causes P5.p-P7.p to adopt vulval fates. Although P3.p, P4.p, and P8.p also express LET-23, it is thought that their distance from the anchor cell prevents levels of activated LET-23 from accumulating necessary to overcome default hypodermal fates. We have used two approaches to study modulation of responses to this inductive signal. In one approach, we tested candidate loci for interaction with this pathway. We find that a loss of function mutation in the egl-15 negative regulator, clr-1 , a receptor tyrosine phosphatase, increases responsiveness of all 6 VPCs to the inductive signal. In contrast, an activated allele of egl-30 Gq alpha facilitates responsiveness of P5.p-P7.p, but not P3.p, P4.p, and P8.p. In a second approach, we identified EMS-induced mutations that enhance the choice of vulval fates by P3.p, P4.p, and P8.p in the presence of a gain-of-function allele of let-23 . Some of these mutations, such as sy622 , strongly enhance the responsiveness of P3.p, P4.p, and P8.p, while only weakly affecting P5.p-P7.p. These results indicate that there are both pan-Pn.p, as well as Pn.p-specific mechanisms operating to modulate vulval induction. Pan-Pn.p mechanisms might function as buffers to lower the overall noise inappropriately contributing to vulval induction. In conjunction with pathways defined by egl-30(gf) and sy622 which increase responsiveness of P5.p-P7.p and decrease responsiveness of P3.p, P4.p, and P8.p, respectively, a robust response by only a subset of cells might be guaranteed during inductive signaling.

Qiao L et al. (1995) International C. elegans Meeting "ENHANCERS OF glp-1 DEFINE A SET OF GENES REQUIRED FOR GERMLINE DEVELOPMENT"

Lissemore JL, Shu P, Smardon AM, Qiao L, Gelber MB, Maine EM

[International C. elegans Meeting, 1995]

The distal tip cell (DTC) regulates the choice between proliferation and differentiation in the C. elegans germ line by an inductive mechanism. Cell signaling requires a receptor in the germ line that is encoded by the glp-1 gene and a signal from the DTC that is encoded by the lag-2 gene. These molecules are also essential for other inductive events during embryonic and larval development. To identify other genes involved in the induction of germline proliferation, we have screened for extragenic mutations that enhance a weak glp-1 mutation, bn18(ts). We find that mutations in seven genes, including lag-1, glp-4 and five new ego (for enhancer of glp-1) genes, strongly enhance a weak glp-1 loss of function phenotype in the germline. Genetic experiments suggest that ego-1 and ego-3 act downstream while ego-2 acts upstream of (or regulates) the GLP-1 protein. In addition to enhancement of glp-1, mutations in each ego gene have visible phenotypes in a glp-1(+) background. We will describe the phenotypes of ego-3, lag-1(Ego) and glp-4(Ego) mutants. Mutant phenotypes of other ego geneswill be described in the abstract by Shu et al. Our results suggest that ego genes function in multiple aspects of development. Currently, we are testing the interactions between different ego genes. Molecular analysis of ego-1 and ego-3 gene is underway to further define their functions in development.

Min Zheng et al. (2001) International C. elegans Meeting "Cell fate specification during vulva formation in Pristionchus pacificus"

Ralf J Sommer, Min Zheng

[International C. elegans Meeting, 2001]

We compare vulva development between Caenorhabditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C.elegans , P8.p is part of the vulva equivalence group and adopts a 3deg fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P.pacificus represents a new cell type. First, P8.p is unable to respond to an inductive signal from the gonad and fuses with the epidermis, but can respond to lateral signal from P6.p and form vulval tissue. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p to adopt a 1deg fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification we screened for mutants with P8.p specification defects and identified three candidate mutants in which P8.p behaves like a normal VPC. In one of the mutants, named tu151 , the nucleus of P8.p has an abnormal size. Cell ablation experiments in tu151 revealed that P8.p is able to respond to the inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal so that after P6.p ablation, P5.p and P7.p can adopt the 1deg fate. In addtion, P8.p loses the function of providing negative signal in tu151 mutant animals since gonad-independent vulva formation occurs. Positional cloning of tu151 is currently in progress.

Min Zheng et al. (2002) European Worm Meeting "Genetic and molecular analysis of the vulval patterning mutants ped-7 and ped-17 in Pristionchus pacificus"

Min Zheng, Ralf J Sommer

[European Worm Meeting, 2002]

We compare vulva development between Caenorhaditis elegans and Pristionchus pacificus to study the evolution of developmental processes. Cell fate specification during vulva development differs in these two nematodes. In C. elegans, P8.p is a member of the vulva equivalence group and adopts a 3 fate under wild-type conditions. In contrast, several experiments indicate that P8.p in P. pacificus represents a novel cell type. First, P8.p is unable to respond to an inductive signal to form vulval tissue, but it can respond to lateral signal from the neighboring 1 cell P6.p. Second, P8.p provides a lateral inhibitory signal that prevents P5.p and P7.p, but not P6.p, from adopting a 1 fate. Third, P8.p provides a negative signal that inhibits gonad-independent vulva formation of P(5-7).p. To study P8.p specification, we screened for mutants with P8.p specification defects and identified three mutants in which P8.p behaves like a normal VPC. In one of these mutants, namely ped-17, the nucleus of P8.p has an abnormal size. Cell ablation experiments in ped-17 revealed that P8.p is able to respond to inductive signal from the gonad to form vulval tissue. Also, P8.p in this mutant cannot provide the lateral inhibitory signal. After P6.p ablation in these animals, either P5.p or P7.p can adopt a 1 fate. In addition, P8.p loses the function of providing negative signal in ped-17 mutant animals since gonad- independent vulva formation occurs. In another mutant, ped-7, P7.p migrates towards P8.p and forms an ectopic invagination. Genetic analysis revealed that ped-7 represents a multivulva mutation. We have mapped both mutants to the genetic linkage map of P. pacificus. ped-7 maps to chromosome V and is located between the markers S130 and S13. ped-17 is on chromosome II and is located between S111 and S24. Positional cloning of ped-7 and ped-17 is currently ongoing.

Sternberg PW et al. (1982) Worm Breeder's Gazette "cell-cell interactions in P redivivus ventral hypodermis development."

Sternberg PW, Horvitz HR

[Worm Breeder's Gazette, 1982]

In C. elegans the gonad, specifically the anchor cell, induces the ventral hypodermal precursors P(5-7).p to generate the vulva (Kimble, Develop. Biol. 87, 286, 1981). Ablation of any of these vulval precursor cells leads to regulation (Sulston and White, Develop. Biol. 78, 577, 1980). Similar regulation is seen in C. elegans males. We have performed a series of gonadal, P, and Pn.p cell laser ablation experiments in P. redivivus females and males. In P. redivivus, P(5- 8).p generate the vulva; the other Pn.p cells join the hypodermal syncytium. [In C. elegans, P(3,4,8).p divide and their progeny join the syncytium; P(1,2,9-11).p join the syncytium directly.] Our results can be summarized as follows. (1) The P. redivivus gonad is required for female Pn.p divisions and vulval development. The anchor cell (Z4. aaa) is required for the third round of Pn.p divisions. Z4 is required for vulval induction, although a few Pn.p cells can divide in its absence. In males of both species the gonad is not required for Pn.p divisions. (2) Regulation occurs as in C. elegans: the ablation of certain Pn.p cells can result in the replacement of the ablated cell by another Pn.p cell. (3) As in C. elegans, there is a hierarchy of replacements. A higher ranked cell can be replaced by a lower ranked cell but will not replace a lower ranked cell. In C. elegans hermaphrodites, the fate of P6.p is primary (ranks highest in the hierarchy), the fates of P(5,7).p are secondary, and the fates of P(3,4,8).p are tertiary. In P. redivivus females, the fates of P(6,7) .p are primary, the fates of P(5,8).p are secondary, and the fates of P(4,9).p are tertiary. (4) In C. elegans hermaphrodites, P(3-8).p can participate in vulval development and thus define the vulval equivalence group. In P. redivivus females, at least P(4-9).p can participate in vulval development. Thus, the vulval equivalence group differs between the species. (5) In males of both species, P10.p and P11.p generate cells of a pre-anal sensillum (called the hook sensillum in C. elegans); P9.p joins the ventral hypodermal syncytium or, in some C. elegans males generates two daughters that join the syncytium. As in C. elegans, P9.p can replace P10.p. Thus, in P. redivivus, P9.p belongs to both the male and female equivalence groups, indicating that equivalence groups in the two sexes need not be distinct. The differences in vulval regulation between the two species suggest that there are at least four independent levels of control specifying Pn.p fate in vulval development. Different sets of instructions seem to specify: (a) which Pn.p cells can participate in vulval development; (b) which Pn.p cells do participate in vulval development; (c) which Pn.p cells follow primary or secondary fates; and (d) what lineage is generated by cells with the primary, secondary and tertiary fates.

Alper SD et al. (1997) International C. elegans Meeting "Regulation of Pn.p Cell fusion in C. elegans"

Kenyon CJ, Alper SD

[International C. elegans Meeting, 1997]

Cell fusion is a ubiquitous process in C. elegans, with approximately one third of all somatic nuclei in adults found in variably sized multinucleate syncytia. In hermaphrodites, the hypodermal cells P1.p, P2.p, and P(9-11).p all fuse with hyp7 during late L1 while P(3-8).p remain unfused and become members of the vulval equivalence group. In males, P(3-6).p and P(9-11).p remain unfused; P(9-11).p subsequently become the male equivalence group. The Pn.p fusion decision is regulated by the Hox genes lin-39 and mab-5. lin-39 is expressed in P(3-8).p and in hermaphrodites prevents fusion of these cells. mab-5 is expressed in P(7-11).p. In males, lin-39 and mab-5 each individually prevent Pn.p cell fusion in P(3-6).P and P(9-11).p, respectively. However, in P7.p and P8.p, where both Hox genes are expressed in the same cell, they somehow neutralize one another's effects, so that P7.p and P8.p fuse with hyp7. We are carrying out a screen to identify additional genes that affect Pn.p cell fusion. In a lin-12(d) background, those Pn.p cells that remain unfused all generate ectopic pseudovulvae or hooks that protrude from the ventral surface of the worm. We are screening for mutations that change the pattern of ventral protrusion in lin-12(n137); him-5(e1467); thus far, we have identified five mutants with extra ventral protrusions in the hermaphrodite and one mutant with extra protrusions in the male. MH27, a monoclonal antibody that stains adherins junctions, has been used to verify that extra Pn.p cells fail to fuse with hyp7 in some of these mutants. Preliminary experiments suggest that some of these mutations may affect upstream regulators of the Hox genes. We also hope to find genes that integrate the Hox signals as well as genes required for the actual process of cell fusion.