Pilgrim, Dave et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "THE SEX DETERMINING PATHWAY IN CAENORHABDITIS; TRA SUPPRESSORS AND FEM GENES"

Reidy, Keith, Pilgrim, Dave, Egydio de Carvalho, Carlos, Dewar, Jill

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

Sex determination in Caenorhabditis is a rapidly evolving process that can provide insights into how biological pathways can be modified from a common set of ancestral genes. C. elegans and C. briggsae are two of three androdiocious species within the Elegans group. These two species are both morphologically and developmentally similar but their genetic control of hermaphroditism is different. XX animals in these species are somatically female but are capable of producing and storing sperm before switching to oocyte production. Previous work from our lab and Eric Haag's showed that the function of the Tra genes was similar between the two species, while mutations in C. briggsae fem-2 or fem-3 genes resulted in somatic but not germline feminization, suggesting that the sperm/oocyte switch must be controlled downstream of the Fem genes in C. briggsae XX animals. We have now extended that work, by isolating and characterizing suppressors of Cb-tra-2 and Cb-tra-3 ts mutations, in hope of identifying missense mutations which will help with the analysis of Fem protein function.Over 70 tra-2 suppressors were identified in the Pilgrim and Haag labs and several tra-3 suppressors in our lab by forward genetics (looking for the restoration of self-fertility to tra(ts) animals at the restrictive temperature). The results of these screens and the ongoing characterization will be presented. The majority of the tra-2 suppressors are alleles of the three fem genes, as expected, including a large set of mutations in Cb-fem-1, which had not been previously isolated by reverse genetics. A small set of interesting mutations complemented the fem genes and are phenotypically distinct from any other mutants described in C. elegans or C. briggsae. WGS has identified candidates for these suppressors and we are currently in the process of mapping the mutations to confirm that the lesion is responsible for their unique phenotypes, and the alleles are being separated from the tra-2 background to allow characterization of their phenotype.

Dave Pilgrim et al. (2001) International C. elegans Meeting "UNC-45 is an evolutionarily conserved myosin-interacting protein"

Dave Pilgrim, Tamara Checkland, Wanyuan Ao, Serene Wohlgemuth

[International C. elegans Meeting, 2001]

unc-45 is essential for normal thick filament development in body wall muscles. The protein product (UNC-45) contains TPR repeats and has similarity to the yeast She4p protein which is involved in asymmetric segregation of specific mRNAs, but its biochemical function is unknown. We have previously shown that UNC-45 differentially co-localizes in body wall muscle thick filaments with myosin heavy chain B (MHC B) but not MHC A. In contrast to other body wall muscle thick filament components, unc-45 is also maternally contributed and the protein and mRNA are present in all cells of the early embryo. However, zygotic unc-45 expression is only detected in the developing muscle cells. Yeast two-hybrid screens show that UNC-45 interacts with at least two non-muscle myosins, including NMY-2, a type II myosin necessary for asymmetric cell division. UNC-45 and NMY-2 also co-localize in vivo, at the cortex of the cell in early embryos, and localization of UNC-45 is dependent on the presence of NMY-2. Rather than being a muscle-specific protein, this would argue that the UNC-45 protein may have a more general role as a myosin chaperone, stabilizer, or assemblase. We have recently found UNC-45 sequence homologues in Drosophila and humans, and a lethal mutation in the Drosophila gene, leading us to propose that this family of proteins may be an evolutionarily conserved class of myosin interacting proteins.

Pelletier, Katharine et al. (2015) International Worm Meeting "Evolution of hermaphroditism in Caenorhabiditis species."

Pelletier, Katharine, Pilgrim, Dave, Reidy, Keith

[International Worm Meeting, 2015]

Hermaphroditism (sperm production in the XX ovotestis) is one of the traits that has made C. elegans such a powerful genetic model, but most species in the genus have a male/female mating system. The genetic pathway used to determine somatic and germline sex in C. elegans is well understood; however the genes in the pathway are poorly conserved within Caenorhabditis. We have been interested in how sex determination, a rapidly evolving process in Caenorhabditis, can provide insights into how biological pathways can be modified from a common set of ancestral genes to provide novel traits, such as the XX hermaphrodite. Previous work from our lab and the Haag lab has shown that the control of sperm production in a female gonad is different in C. briggsae as compared to C. elegans. fem mutants in C. elegans result in XX and XO females, while in C. briggsae both XX and XO fem mutants are hermaphrodites.We have now extended that work, by isolating and characterizing suppressors of Cb-tra-2 ts mutations, in hope of identifying missense mutations which will help with the analysis of Fem protein function. Over 70 tra-2 suppressors were identified in the Pilgrim and Haag labs by looking for the restoration of self-fertility to tra(ts) animals at the restrictive temperature. As expected, most of these lie in the Cb-fem genes, however some represent novel phenotypes/alleles not previously observed in either system. I will present the current understanding of the C. briggsae sex determination pathway and how this differs from C. elegans. Additionally, I will discuss how comparative genomics within the Caenorhabditis species may provide us with clues to the relationship between molecular changes and the evolution of novel traits. .

Torah M. Kachur et al. (2006) European Worm Meeting "Creation of Germ Cells: Characterization of Germline Cytokinesis"

Dave Pilgrim, Torah M. Kachur

[European Worm Meeting, 2006]

Torah M. Kachur and Dave Pilgrim. The creation of germ cells as discrete cells is essential to the fecundity of metazoan species. The process of separating developing germ cells into distinct compartments requires an actomyosin based contractile apparatus. We have identified components of the germline cellularization mechanism that includes: UNC-45, a myosin chaperone, and its associated myosin, NMY-2. Lack of either myosin or its chaperone results in complete sterility with a failure to create oocytes in the proximal gonad. Partial defects in germline cellularization are apparent with different sized embryos that develop from unc-45 or nmy-2 partially depleted adults resulting from a defect in neighboring oocytes to complete cytokinesis. In the absence of UNC-45, there is a failure to organize the actomyosin cytoskeleton into a characteristic hexagonal pattern in the distal syncytial gonad and a subsequent failure in the proximal gonad to separate nucleii into oocytes. Both males and hermaphrodites require intact myosin function for cellularization in the germline where sperm formation fails in both hermaphrodites and sperm does not accumulate in males. We present a detailed analysis of germline cytokinesis in C. elegans indicating that cellularization requires an intact actomyosin cytoskeleton in order to properly cellularize both types of primordial germ cells.

Eric S Haag et al. (2003) International Worm Meeting "Sex determination mutants in C. briggsae"

Eric S Haag, Marnie R Layton, Danielle F Kelleher

[International Worm Meeting, 2003]

Sex determination is accomplished via highly disparate mechanisms in different phyla, and the homologous sex determination genes of closely related species often undergo rapid molecular evolution. To systematically explore how sex determination evolves over short, tractable time scales, we are developing Caenorhabditis briggsae as a true genetic system. Until now, the function of C. briggsae sex determination genes has been assessed using RNAi. However, RNAi rarely eliminates all target expression and it is not suitable for suppressor screens, epistasis and mosaic analyses, etc. Such genetic approaches are essential for accurate reconstruction of an entire pathway. Further, RNAi targeting of the C. briggsae and C. remanei fem genes suggests there may be large differences in germline fem function between these species1-3, a result that, if verified, would have major implications for the evolution of hermaphroditism in the genus. These results may in fact be due to divergence in the tissue-specificity of fem function, but could also merely reflect limited efficacy of RNAi. This can be resolved by the isolation of true mutations, done most readily in the hermaphrodite C. briggsae.Here, we describe the isolation of loss-of-funtion Cb-tra mutant alleles, work being carried out in conjunction with the Pilgrim Lab at the Univ. of Alberta (see poster by de Carvalho and Pilgrim). Using standard C. elegans methods, we have obtained six such alleles. 4 are completely penetrant at all temperatures, whereas 2 are partially rescued at 15 degrees . The alleles appear to fall into two complementation groups, one resembling Ce-tra-1 and the other Ce-tra-2 in phenotype. Mapping with visible and SNP markers is consistent with the tra-2-like complementation group (nm1 and nm10 alleles) being actual Cb-tra-2 mutations. Sequencing of these alleles is underway. If they are indeed Cb-tra-2 mutants, then the penetrance of Cb-tra-2(RNAi)), about 10%1,4, is much lower than that of true mutants. This suggests that the qualitative species differences seen in fem gene RNAi phenotypes may also be the result of incomplete target inactivation.1Haag, ES, Wang, S, and Kimble, J (2002). Current Biol. 12: 2035-41; 2Stodhard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282; 3Stodhard, P, and Pilgrim, D (2003). BioEssays 25: 221-31; 4Kuwabara, PE (1996). Genetics 144: 597-607.

Carlos Carvalho de Egyido et al. (2004) West Coast Worm Meeting "More on C. briggsae sex determination mutants"

Carlos Carvalho de Egyido, David B Pilgrim, Danielle F Kelleher, Eric S Haag

[West Coast Worm Meeting, 2004]

To explore how sex determination evolves over short, tractable time scales, we are developing C. briggsae as a true genetic system. C. briggsae sex determination genes have been functionally assessed by RNA interference (RNAi). However, RNAi rarely eliminates all target expression and it is not suitable for the genetic approaches essential for pathway reconstruction. RNAi targeting of the C. briggsae and C. remanei fem genes suggests differences in germline fem function between species (1-3). Recent work using a Cb-fem-2 deletion mutant indicates that fem function has changed, and has major implications for the evolution of hermaphroditism in the genus. Here, we describe the isolation of Cb-tra mutant alleles. The best-characterized alleles fall into two linkage groups, one group resembling Ce-tra-1 and linked to Cb-tra-1 and the other group resembling Ce-tra-2 and linked to Cb-tra-2. Suppression of Cb-tra-2 further suggests the role of fem genes may have changed between species. Epistasis analysis of Cb-tra-1, Cb-tra-2, and Cb-fem-2 mutants indicates that gene interactions and order may be similar between species. Lastly, Cb-tra-2 mutants show that the penetrance of Cb-tra-2(RNAi), about 10% (1,4), is much lower than true mutants, which supports the idea that real mutants will be crucial for accurate reconstruction of the C. briggsae sex determination pathway. 1 Haag, ES, Wang, S, and Kimble, J (2002). Curr. Biol. 12: 2035-41. 2 Stothard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282. 3 Stothard, P, and Pilgrim, D (2002). BioEssays 25: 221-31. 4 Kuwabara, PE (1996). Genetics 144: 597-607

Danielle F Kelleher et al. (2005) International Worm Meeting "C. briggsae tra mutants"

Eric S Haag, David B Pilgrim, John Salogiannis, Carlos Carvalho, Marnie R Layton, Danielle F Kelleher

[International Worm Meeting, 2005]

To explore how sex determination evolves over short, tractable time scales, we are developing Caenorhabditis briggsae, a close relative of the model nematode C. elegans, as a true genetic system. The function of C. briggsae sex determination genes has previously been assessed by RNA interference (RNAi). RNAi against the C. briggsae and C. remanei fem genes suggests differences in their germline function between species (1-3). However, RNAi rarely eliminates all target expression, and it is not suitable for the genetic approaches essential for pathway reconstruction (1,4). Here, we describe the isolation and characterization of Cb-tra mutant alleles. The best-characterized tra alleles fall into three linkage groups tightly linked to Cb-tra-1, Cb-tra-2, and Cb-tra-3. Sequencing of reference alleles from the Cb-tra-1 and Cb-tra-2 classes revealed missense and nonsense mutations, suggesting these are Cb-tra-1 and Cb-tra-2 mutants. Sequencing of a likely Cb-tra-3 reference allele is in progress. Intra-species suppression of the Cb-tra(ed24ts) Tra phenotype by genomic variants in the wild-type strain HK104 suggests that molecular interactions underlying sex determination are changing between two C. briggsae populations. Epistasis analysis of Cb-tra-2 with Cb-fem-2 and other fem-like Cb-tra-2 suppressors (see posters by Carvalho et al. and Hill et al.) indicates that the order of sex determination gene interactions in the soma is similar between the two species.1 Haag, ES, Wang, S, and Kimble, J (2002). Curr. Biol. 12: 2035-41. 2 Stothard, P, Hansen, D, and Pilgrim, D (2002). J. Mol. Evol. 54: 267-282. 3 Stothard, P, and Pilgrim, D (2003). BioEssays 25: 221-31. 4 Kuwabara, PE (1996). Genetics 144: 597-607.

G Dalpe et al. (1999) International C. elegans Meeting "Toward the cloning of vab-6, a gene involved in axon guidance and morphogenesis"

G Dalpe, PJ Roy, J Culotti

[International C. elegans Meeting, 1999]

C.elegans morphogenesis is a complex process requiring numerous short-range migrations, rearrangements, and concerted cell movements. Several existing C.elegans mutants disrupt essential morphogenic events since they result in severe body deformities, classically described as a vab (variable-abnormal) phenotype. Several studies have demonstrated that molecules shown to play cell and axon guidance roles in other model systems have homologues in the worm that play broad roles in hypodermal morphogenesis (George et al., 1998, Cell 92, 633-643; Wang et al., 1999, submitted). For example, mab-20 mutants that have disruptions in the C.elegans homologue of Drosophila Semaphorin II, have severe morphological deformities, axon guidance defects and fusion of sensory rays in the male tail. Ectopic hypodermal cell contacts and other observations in mab-20 mutants suggest that Semaphorin II is required to repel cell extensions (Roy et al., 1999, in preparation). Another previously isolated mutant called vab-6 has several phenotypic traits that are strikingly similar to those observed in mab-20 mutants, including ectopic hypodermal seam cell contacts, male tail sensory ray fusions and misguided axons. Although mab-20 and vab-6 mutants may affect the same developmental events, a double mutant between a putative mab-20 null mutation (ev574) and vab-6(e697 ) could not be made on several attempts. This suggests that mab-20 and vab-6 may be in parallel pathways. Consistent with this hypothesis, double mutants containing a temperature sensitive allele of mab-20(bx61) (Baird et al., 1991, Dev. 113, 515-526) and vab-6(e697) display embryonic lethality and severe morphological defects at restrictive temperatures. vab-6 genetically maps to position -27.17 on LGIII near unc-45 (-27.13) (Dave Pilgrim, personal communication). To better understand the function of VAB-6, we are presently trying to clone the gene. We show that the morphological abnormalities of vab-6 can be rescued with the cosmid F10C5 that resides to the left of unc-45 . F10C5.2 is a predicted transmembrane protein and F10C5.1 has a region highly homologous to the TPR domains found in yeast CDC16, CDC23 and CDC27. We are now trying to identify a smaller rescuing DNA fragment within that cosmid.

Yiqing Guo et al. (2006) Development & Evolution Meeting "glf-1 Controls Hermaphrodite Development in C. briggsae"

Yiqing Guo, Ronald Ellis

[Development & Evolution Meeting, 2006]

In C. elegans, XX animals are self-fertile hermaphrodites, and XO animals are males. The only other male/hermaphrodite species in this genus is C. briggsae. However, our phylogeny shows that these species are not sisters. Furthermore, work by Eric Haag and Dave Pilgrim indicates that the fem genes do not regulate germ cell fates in C. briggsae, although they are essential in C. elegans. Thus, it seems likely that hermaphroditism evolved separately in these species. How did this happen? And why did it happen twice within this genus? To answer these questions, we are taking two approaches. Here, we describe a genetic analysis of C. briggsae, to identify the genes that control hermaphrodite development. In a separate abstract, C. Baldi and R. Ellis describe what changes would be needed in the male/female species C. remanei to create hermaphrodites. By screening for mutations that transform XX animals into females, we identified these genes: glf-1 IV. GLF-1 is required for XX animals to develop as hermaphrodites, but plays no role in males. Thus, it acts like fog-2 does in C. elegans. However, Tim Schedl could not identify a homolog of fog-2 in C. briggsae, so GLF-1 probably acts by a novel mechanism. The original allele, v35, is temperature-sensitive, and we have isolated several suppressors. We also found two more alleles in our screen for XX females, and four alleles in a non-complementation screen. All are ts, and all affect only XX animals. Three alleles on LGI that cause both sexes to produce oocytes. We suspect that two are alleles of fog-1, and that one is an allele of fog-3, and are testing this hypothesis by sequencing DNA from each strain. At least five alleles on LGII that cause both sexes to produce oocytes. These mutations fail to complement each other, and a new gene, glf-2. At least one allele of tra-1 on LGIII that causes oogenesis in both sexes, perhaps like rare tra-1(gf) alleles do in C. elegans. Taken together, these results suggest that some parts of the sex-determination pathway are similar in both C. elegans and C. briggsae, like the roles of fog-1, fog-3 and tra-1. Other parts, in particular the functions of glf-1 and glf-2, are likely to be novel. Since glf-1 appears to be the key to hermaphroditic development in C. briggsae, we have been using SNP mapping to clone it. We have narrowed its location down to one of two finger-printed supercontigs on LGIV, and are using fine-scale mapping to identify the locus.

Pilgrim DB et al. (1991) International C. elegans Meeting "MOLECULAR ANALYSIS OF THE DAUER CONSTITUTIVE daf-7 GENE."

Johnsen RC, Pilgrim DB, Riddle DL

[International C. elegans Meeting, 1991]

Mutations in daf-7 result in constitutive formation of dauer larvae, even in abundant food. This gene has been localized by a combination of genetic and RFLP mapping, DNA transformation, and Tcl tagging. On a chromosomal walk toward fem-2, one of us (D. Pilgrim) identified a cosmid (DE9) that transformed a daf-7 mutant to wild-type when injected into the mutant germline. Cosmid DE9, which contains an insert of <30kb, was used to probe both a northern blot and a CDNA library constructed by Stuart Kim. The northern blot revealed a single hybridizing RNA, and ten cDNA clones of various sizes up to 2.4kb were isolated from the library. Hybridization results indicate that all these clones share common sequences. Probing restriction digested genomic DNA isolated from the Tcl-insertion mutant DR1039, daf-7 (m434) , revealed differences consistent with a 1.6kb Tcl insertion in DR1039 DNA when compared with nonmutant DNA. For example, a 2.2kb EcoRl fragment found only in mutant DNA hybridized to both DE9 and to Tcl. We intend to determine the sequence of a daf-7 cDNA in order to obtain information regarding its possible function, and possible relationship to the daf-l and daf-4 receptor protein kinases.