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.

Aoki, Scott Takeo et al. (2013) International Worm Meeting "Structural characterization of the P-granule protein scaffold."

Aoki, Scott Takeo, Kimble, Judith

[International Worm Meeting, 2013]

C. elegans P-granules are essential for both the development and maintenance of the germline tissue. Disruption of P-granule formation interferes with proper germ cell proliferation and differentiation, resulting in sterility (1, 2). P-granule assembly requires structural scaffold proteins PGL-1 and PGL-3 (3, 4). These nematode-specific paralogs are sufficient to form granules in cells and multimerize through self-association (5, 6). We aim to understand the structural organization of the P-granule scaffold to better understand how the organelle regulates mRNA trafficking and turnover. We are able to express and purify recombinant PGL-1 and PGL-3. Full-length recombinant protein self-assembles into large soluble aggregates. Protease digestion analyses identify a single domain that dimerizes in solution. We are currently trying to obtain a high-resolution crystal structure of the protease-protected fragment, as well as determine the role of the N- and C- terminal regions in scaffold oligomerization. Several different types of RNA granules are required in eukaryotes for cell homeostasis, differentiation, and response to stress. The fundamental mechanisms involved in P-granule organization will undoubtedly shed light on other granules involved in RNA regulation. References: 1. Updike, D., Strome, S. (2010) J Androl 31: 53-60. 2. Voronina, E., Paix, A., Seydoux, G. (2012) Development 139: 3732-3740. 3. Kawasaki, I., et al. (1998) Cell 94: 635-645. 4. Kawasaki, I., et al. (2004) Genetics 167: 645-661. 5. Updike, D.L., Hachey, S.J., Kreher, J., Strome, S. (2011) J Cell Biol 192: 939-948. 6. Hanazawa, M., Yonetani, M., Sugimoto, A. (2011) J Cell Biol 192: 929-937..

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.

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.

Pires da Silva A et al. (2000) European Worm Meeting "Novel mutants defective in vulva induction in Pristionchus pacificus"

Sommer RJ, Pires da Silva A

[European Worm Meeting, 2000]

More than 50 genes involved in vulva development have been characterized in the rhabditid C. elegans. The extensive characterization of this model system provides a framework for studying the genetic basis of evolutionary modifications. Cell fate specification during vulva formation differs significantly between C. elegansand the diplogasterid nematode Pristionchus pacificus. Cell ablation experiments in P. pacificus demonstrate the existence of several novel cell-cell interactions (Sigrist and Sommer, 1999; Sommer and Sternberg, 1996). To understand the molecular mechanisms underlying vulval pattern formation in P. pacificus, we performed several TMP/UV and EMS mutagenesis screens. The mutants isolated in these screens fall into two categories: I) "generation-vulvaless" mutants do not generate Pn.p cells; II) "induction-vulvaless" mutants generate Pn.p cells, but these fail to differentiate. We have isolated and characterized a new class of induction vulvaless mutants, in which P6.p differentiates as a normal 1 cell, but P5.p and P7.p adopt a non-vulval fate (3). In wild type P. pacificus, as in C. elegans, P6.p adopts the 1 fate, whereas P5.p and P7.p generate the 2 lineage. Four mutants, tu114(TMP/UV), tu132(EMS), tu48(EMS) and tu51 (EMS), display this defect to different extents. Complementation test among these mutants are in progress. We are using a PCR-based genomic substraction strategy (RDA) to identify the deletion associated with the tu114 mutant. The identification of the genes involved in the induction of the vulva in P. pacificus may help us understand how molecular mechanisms change during evolution. Sigrist, C. B., and Sommer, R. J. (1999). Vulva formation in Pristionchus pacificus relies on continuous gonadal induction. Dev Genes Evol 209, 451-9. Sommer, R. J., and Sternberg, P. W. (1996). Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 6, 52-9.

Sommer RJ et al. (2000) European Worm Meeting "Novel mutants defective in vulva induction in Pristionchus pacificus"

Pires da Silva A, Sommer RJ

[European Worm Meeting, 2000]

More than 50 genes involved in vulva development have been characterized in the rhabditid C. elegans. The extensive characterization of this model system provides a framework for studying the genetic basis of evolutionary modifications. Cell fate specification during vulva formation differs significantly between C. elegansand the diplogasterid nematode Pristionchus pacificus. Cell ablation experiments in P. pacificus demonstrate the existence of several novel cell-cell interactions (Sigrist and Sommer, 1999; Sommer and Sternberg, 1996). To understand the molecular mechanisms underlying vulval pattern formation in P. pacificus, we performed several TMP/UV and EMS mutagenesis screens. The mutants isolated in these screens fall into two categories: I) "generation-vulvaless" mutants do not generate Pn.p cells; II) "induction-vulvaless" mutants generate Pn.p cells, but these fail to differentiate. We have isolated and characterized a new class of induction vulvaless mutants, in which P6.p differentiates as a normal 1 cell, but P5.p and P7.p adopt a non-vulval fate (3). In wild type P. pacificus, as in C. elegans, P6.p adopts the 1 fate, whereas P5.p and P7.p generate the 2 lineage. Four mutants, tu114 (TMP/UV), tu132 (EMS), tu48 (EMS) and tu51 (EMS), display this defect to different extents. Complementation test among these mutants are in progress. We are using a PCR-based genomic substraction strategy (RDA) to identify the deletion associated with the tu114 mutant. The identification of the genes involved in the induction of the vulva in P. pacificus may help us understand how molecular mechanisms change during evolution. Sigrist, C. B., and Sommer, R. J. (1999). Vulva formation in Pristionchus pacificus relies on continuous gonadal induction. Dev Genes Evol 209, 451-9. Sommer, R. J., and Sternberg, P. W. (1996). Apoptosis and change of competence limit the size of the vulva equivalence group in Pristionchus pacificus: a genetic analysis. Curr Biol 6, 52-9.

Simske JS et al. (1995) International C. elegans Meeting "SEQUENTIAL SIGNALING during vulval development."

Kim SK, Simske JS

[International C. elegans Meeting, 1995]

During the induction of the C. elegans vulva, cell signaling causes initially equipotent cells to express a reproducible pattern of cell fates. The position of the anchor cell determines the pattern of vulval precursor cell fates, such that the closest precursor cell (P6.p) expresses the primary cell fate, the next closest cells (P5.p and P7.p) both express the secondary cell fate, and each of the precursor cells located at a distance (P3.p, P4.p, and P8.p) express the uninduced, tertiary cell fate. We present data indicating that this stereotypical pattern of cell fates can be generated by sequential signals. We identified genetic mosaic animals in which P5.p and P7.p were defective in the anchor cell signal transduction pathway and observed that these cells adopted the secondary cell fate, indicating that anchor cell signal transduction in P5.p and P7.p is not required for the expression of the secondary cell fate. These results suggest that the anchor cell induces P6.p to express the primary cell fate, and that P6.p subsequently induces P5.p and P7.p to express the secondary cell fate.

Jungblut B et al. (1998) European Worm Meeting "The Pristionchus pacificus mab-5 gene is involved in the regulation of ventral epidermal cell fates."

Sommer RJ, Jungblut B

[European Worm Meeting, 1998]

The comparison of cell fate specification in the vulva equivalence group between C. elegans and Pristionchus pacificus reveals several evolutionary differences. In P. pacificus, P8.p loses its competence to adopt vulval cell fate early in the L1 stage and can only adopt an epidermal fate. In mutagenesis screens of a total of appr. 100 000 gametes many egg-laying defective mutations were isolated. In 11 of these mutations the specification of P8.p was affected. In most mutant animals analyzed, a posterior vulva-like structure was formed by P8.p. The specification of P7.p was also affected: cell lineage analysis in 5 strains revealed that P7.p adopted the 1! fate in approximately 5% of animals. In such animals, the main invagination was generated by the progeny of P(6,7).p and P6.p had the 2! fate. The AC in these cases was displaced posteriorly so that it was now located above P7.p. Complementation tests revealed that all 11 mutations are allelic to one another and to the previously isolated mutation Ppa-ped-4. Further genetic analysis indicated that Ppa-ped-4 is linked to the visible mutation Ppa-dpy-1 which has been shown to be closely linked to the homeotic gene Ppa-lin-39. This result suggests, that Ppa-ped-4 might be associated with, or might be a member of the Hox genes of P. pacificus. One candidate gene would be mab-5. To test if Ppa-ped-4 corresponds to P. pacificus mab-5, we cloned the Ppa-mab-5 homeodomain by PCR and isolated genomic and cDNA clones. We analyzed the complete Ppa-mab-5 coding region in 5 of the 12 ped-4 mutant strains and found mutations in all of them indicating that Ppa-ped-4 is identical to the homeotic gene mab-5. To simplify nomenclature we renamed Ppa-ped-4 as Ppa-mab-5. When we ablated Z(1,4) in Ppa-mab-5 mutant animals, P7.p and P8.p could differentiate into vulval cells, whereas P(5,6).p remained epidermal. These results suggest that P(7,8).p can differentiate in a gonad-independent way in Ppa-mab-5 mutant animals, indicating that P(7,8).p are distinct from P(5,6).p. The gonad-independent differentiation properties of P(7,8).p might occur in response to an unknown signal or because of an intrinsic property of these cells to undergo vulva-like differentiation. In wild-type animals, Ppa-mab-5 functions as part of a negative signaling system that antagonizes the differentiation properties of P(7,8).p. The Ppa-mab-5-mediated process prevents the inappropriate differentiation of P8.p by making this cell incompetent to adopt vulval fate. This effect of Ppa-mab-5 is eventually restricted to P8.p, as the activity in P7.p is overruled by gonadal signaling that induces vulva formation. Comparing the mab-5 lof phenotypes, the strongest difference between species is that in Cel-mab-5 no ectopic vulva differentiation occurs. Furthermore, Cel-mab-5 mutant animals have VPCs that are identical in their developmental potential. In contrast, in Ppa-mab-5 mutations P(7,8).p still differ from P(5,6).p resulting in gonad independent differentiation. This observation indicates that factors other than mab-5 distinguish P(7,8).p from P(5,6).p or vice versa. One major function of Ppa-MAB-5 is to antagonize gonad-independent differentiation of P(7,8).p. Mechanistically, MAB-5 might act in different genetic circuits with different target genes in both species. For example, Ppa-mab-5 might be part of a locally restricted negative signaling system that is only present in P. pacificus P(7,8).p. However, we cannot rule out that only quantitative differences of MAB-5 activity in the regulation of identical downstream target genes led to the observed competence patterns in both species.

Jagan Srinivasan et al. (2002) West Coast Worm Meeting "Mapping and cloning of generation vulvaless mutants of Pristionchus pacificus"

Jagan Srinivasan, Ralf J Sommer

[West Coast Worm Meeting, 2002]

We are studying the evolution of cell fate specification, using vulva development as a model system and compare C. elegans with Pristionchus pacificus. In P. pacificus, seven ventral epidermal cells P(1-4,9-11).p undergo programmed cell death during late embryogenesis.P(5-7.p) form the vulva and P8.p remains epidermal but influences the fate of P(5-7).p. We performed various genetic screens to isolate a number of mutants defective in vulva formation, many of which result in phenotypes unknown in C.elegans . It has previously been shown that the lin-39 homologue of P. pacificus prevents apoptosis of P(5-8).p and that mutations in Ppa-lin-39 result in a generation vulvaless phenotype. Double mutants of lin-39 with the cell death gene ced-3 indicated that Ppa lin-39 has an early role in vulva development in P. pacificus. It prevents cell death but is dispensable for vulva induction, which is in contrast to Cel lin-39.