MK Robinson et al. (1999) International C. elegans Meeting "Towards the cloning of two genes involved in sex myoblast migration"

MK Robinson, MJ Stern

[International C. elegans Meeting, 1999]

In the Caenorhabditis elegans hermaphrodite a pair of M derived cells, known as the sex myoblasts (SMs), migrate anteriorly during the second larval stage to take up positions that precisely flank the center of the developing gonad. The SMs subsequently divide and differentiate to give rise to the egg-laying muscles. The migrations of these cells are guided by multiple, genetically separable mechanisms. These include a gonad-independent mechanism which is required for the coarse positioning of the SMs and a gonad-dependent mechanism necessary for the precise positioning of the SMs. 1 To better understand the mechanisms underlying SM migration guidance we are working to determine the molecular identities of two genes, unc-71 and sem-3 , that both affect the migrations of the SMs when mutated. Mutations in unc-71 affect both the gonad-independent mechanism of SM migration and axon outgrowth and fasciculation. unc-71 maps to the right arm of linkage group III and was rescued by germline transformation with either of two overlapping YACs, Y37D8 and Y52B8. 2 This region of overlap is not spanned by cosmids. We have determined the extent of overlap between these two YACs and are using fragments derived from this region to perform transformation rescue experiments. We are also performing a non-complementation screen in an attempt to identify deletions in unc-71 that will facilitate the identification of this gene by Southern blot or PCR-based methods. sem-3(n1655) was identified as a spontaneous mutation that caused subtle defects in sex myoblast migration and vulval muscle attachment. This mutation maps to linkage group IV and is allelic to let-654 . We are interested in cloning sem-3 to characterize its roles in SM migration and vulval muscle attachment as well as to understand its essential function. sem-3 maps between the endpoints of the deficiencies sDf62 and sDf8 . 3 We have performed transformation rescue experiments with cosmids from this region and have preliminary results suggesting that we have rescued the Egl defect of sem-3(n1655) . Work to confirm these results and to determine the identity of the open reading frame corresponding to the sem-3 gene will be discussed. 1. Chen & Stern, TIGS 14:322. 2. L. DeLong, personal communication. 3. Clark & Baillie, Mol Gen Genet 232:97

Robinson NT et al. (1993) International C. elegans Meeting "mig-13, a gene that affects anteriorward migration of neuroblasts."

Kenyon CJ, Robinson NT

[International C. elegans Meeting, 1993]

Matthew K Robinson et al. (2001) International C. elegans Meeting "sem-3 encodes a cis-prenyltransferase homolog, an enzyme required for production of an essential lipid."

Matthew K Robinson, Michael J Stern

[International C. elegans Meeting, 2001]

In C. elegans , the egg-laying muscles are descended from a pair of M-derived cells known as the sex myoblasts (SMs). The SMs are born in the posterior of the animal during the L2 stage and migrate anteriorly, in response to multiple cues, to flank the center of the developing gonad.1 The development of functional egg-laying muscles requires their proper differentiation, attachment, and innervation in addition to the proper migration of the SMs. sem-3(n1655) was identified as a spontaneous Egl mutation with defects in SM migration and vulval muscle attachment. The final positions of the SMs in sem-3(n1655) hermaphrodites lack the precision found in wild type, but are very close to normal. The sex muscles derived from these SMs are normal both in number and in their expression of appropriate reporter constructs, but often fail to attach properly. The subtle nature of the SM migration defect makes it likely that the vulval muscle attachment defects are the cause of the Egl phenotype of sem-3(n1655) mutants. sem-3 was mapped to linkage group IV and found to be allelic to let-654. Standard germline transformation rescue of the Let and Egl phenotypes was used to identify the sem-3 open reading frame from among cosmids in the region; the presence of lesions in multiple sem-3 alleles confirmed the identification of the sem-3 gene. By sequence analysis, sem-3 encodes a member of the cis-prenyltransferase class of enzymes. This class of enzymes is responsible for the production of dolichol, a lipid molecule with essential functions in N-linked glycosylation, formation of GPI anchors, and regulation of membrane fluidity.2 BLAST analysis shows that SEM-3 is the only member of this class of enzymes in C. elegans and suggests that the lethality associated with sem-3 mutations may be due to a lack of dolichol. Consistent with an essential role for SEM-3 in a ubiquitous process such as N-linked glycosylation a sem-3 ::GFP reporter construct is expressed in a wide variety of tissues throughout development. Models for SEM-3 function in vulval muscle attachment will be discussed. 1. Chen, E.B. and Stern, M.J. (1998) TIGS 14, 322-327 2. Chojnacki, T. and Dallner, G. (1988) Biochem. J. 251, 1-9

Sym M et al. (2000) West Coast Worm Meeting "Two new genes regulating neuroblast migration in C. elegans"

Kenyon CJ, Yang L, Sym M, Ch'ng Q-L

[West Coast Worm Meeting, 2000]

In C. elegans, the Q cells are bilaterally symmetric neuroblasts present in the posterior body region of the worm at hatching. During the first larval stage, the Q cells divide and migrate. QR and its descendents migrate anteriorly whereas QL and its descendents migrate posteriorly. Several genes that regulate the anterior migrations of QR and its descendents have been identified. These include: 1) lin-39, a homeobox gene required in QR and its descendents for migration (Wang B. et al, 1993; Clark S. et al, 1993); 2) mig-13, a novel transmembrane protein required outside of QR and its descendents for migration (Sym M et al, 1999); and 3) egl-20, a Wnt homolog expressed in cells in the tail region (Whangbo J.. and Kenyon C, 1999). Two mutant screens (Mary Sym and Queelim Ch'ng) were conducted to identify additional genes that regulate the migration of QR and its descendents. From these screens, mutations in two new genes were identified that cause certain cells in the QR lineage to stop migrating prematurely. These two genes seem likely to be involved in guidance rather than in providing cells with the ability to migrate because, in these mutants, other cells sometimes migrate in the wrong direction. Current efforts are directed toward cloning these two genes and determining how these genes regulate the migration of QR and its descendents together with genes previously known to regulate these migrations. References: Clark SG, Chisholm AD, and Horvitz HR. 1993. Control of Cell Fates in the Central Body Region of C. elegans by the Homeobox Gene lin-39. Cell 74: 43-55. Sym M, Robinson N, Kenyon C. 1999. mig-13 Positions Migrating Cells Along Anteroposterior Body Axis of C. elegans. Cell 98: 26-36. Wang BB, Muller-Immergluck MM, Austin, J, Robinson, NT, Chisholm, A, Kenyon, C. A Homeotic Gene Cluster Patterns the Anteroposterior Body Axis of C.. elegans. Cell 74: 29-42. Whangbo J, Kenyon, C. A Wnt Signaling System that Specifies Two Patterns of Cell Migration in C. elegans. Molecular Cell 4: 851-858.

Angeles-Albores, D. et al. (2017) International Worm Meeting "Epistasis analysis using RNA-seq."

Sternberg, P.W., Williams, B.J., Angeles-Albores, D., Puckett Robinson, C., Wold, B.A.

[International Worm Meeting, 2017]

RNA-seq analysis of transcriptomes is commonly used to identify genetic modules that respond to perturbations. In single cells, transcriptomes have been used as phenotypes, but this concept has not been applied to whole-organism RNA-seq. Linear models can quantify expression effects of individual mutants and identify epistatic effects in double mutants in individual genes, resulting in thousands of epistasis measurements. Interpreting these high-dimensional measurements is not intuitive. We thus developed a single coefficient to quantify epistasis from vectorial phenotypes that accurately reflects the underlying genetic interactions. To demonstrate the power of our approach, we sequenced four single and two double mutants of C. elegans. From these mutants, we successfully reconstructed the known hypoxia pathway that was derived from classic double mutant analyses in which EGL-9 and VHL-1 inhibit the activity of HIF-1. In addition, we uncovered a class of 31 genes that have opposing changes in expression in egl-9(lf) and vhl-1(lf) but for which the egl-9(lf);vhl-1(lf) mutant has the same expression phenotype as does egl-9(lf). These changes violate the classical model of HIF-1 regulation, but can be explained by postulating a role of hydroxylated HIF-1 in transcriptional control, opposing the function of non-hydroxylated HIF-1 at selected targets. As a second example, we used transcription profiling to explore the role of sperm in regulation of C. elegans life cycle, and identified a substrate-dependent pathway linking fog-2 and aging through sperm depletion. Our ongoing work demonstrates the usefulness of transcriptomic phenotypes for interpreting epistatic interactions and resolving molecular pathways in detail . We believe our method holds promise for quantitative assays that involve multiple molecular species resulting in measurements of complex phenotypes.

WB Derry et al. (1999) International C. elegans Meeting "Analysis of the C. elegans homolog of the FHIT tumor suppressor gene"

WB Derry, S van Kreeveld, JH Rothman

[International C. elegans Meeting, 1999]

Alterations in the FHIT gene occur frequently in the development of several human cancers (1). The Fhit protein is a diadenosine P 1 , P 3 -triphosphate hydrolase and is a member of the histidine triad superfamily of nucleotide binding proteins (2). The cellular mechanism of Fhit activity and the relationship between Fhit signaling and tumorigenesis are presently unknown. The C. elegans and Drosophila FHIT genes encode a fusion protein in which the Fhit domain is fused with a novel domain showing homology to bacterial and plant nitrilases, and are referred to as NitFhit (3). We are interested in understanding the role of NitFhit in development and programmed cell death. RNAi of C. elegans NitFhit causes an embryonic arrest phenotype, suggesting an essential role for this gene in development. We are currently analyzing the loss-of-function phenotype and the effect of ectopic NitFhit expression on viability and programmed cell death in the worm. (1) Huebner, K., Garrison, P.N., Barnes, L.D. & Croce, C.M. (1998). Ann. Rev. Genet ., 32 : 7-31. (2) Barnes, L.D., Garrison, P.N., Siprashvili, Z., Guranowski, A, Robinson, A.K., Ingram, S.W., Croce, C.M., Ohta, M. & Huebner, K. (1996). Biochemistry , 35 : 11529-11535. (3) Pekarsky, Y., Campiglio, M., Siprashvili, Z, Druck, T., Sedkov, Y, Tillib, S., Draganescu, A., Wermuth, P., Rothman, J.H., Huebner, K., Buchberg, A.M., Mazo, A., Brenner, C. & Croce, C.M. (1998). Proc. Natl. Acad. Sci. USA , 95 : 8744-8749.

Bhagwati P Gupta et al. (2002) West Coast Worm Meeting "Genetic analysis of the vulval mutants in C. briggsae"

Shahla Gharib, Bhagwati P Gupta, Paul W Sternberg, Jennifer X Li

[West Coast Worm Meeting, 2002]

To understand the evolution of developmental mechanisms, we are doing a comparative analysis of vulval patterning in C. elegans and C. briggsae. C. briggsae is closely related to C. elegans and has identical looking vulval morphology. However, recent studies have indicated subtle differences in the underlying mechanisms of development. The recent completion of C. briggsae genome sequence by the C. elegans Sequencing Consortium is extremely valuable in identifying the conserved genes between C. elegans and C. briggsae.

Oomura, S. et al. (2019) International Worm Meeting "Early embryogenesis of C. inopinata, a sibling species of C.elegans."

Matsumura, Y., Haruta, N., Hoshi, Y., Sugimoto, A., Oomura, S.

[International Worm Meeting, 2019]

C. inopinata is a newly discovered sibling species of C. elegans. Despite their phylogenetic closeness, they have many differences in morphology and ecology. For example, while C. elegans is hermaphroditic, C. inopinata is gonochoristic; C. inopinata is nearly twice as long as C. elegans. A comparative analysis of C. elegans and C. inopinata enables us to study how genomic changes cause these phenotypic differences. In this study, we focused on early embryogenesis of C. inopinata. First, by the microparticle bombardment method we made a C. inopinata line that express GFP::histone in whole body, and compared the early embryogenesis with C. elegans by DIC and fluorescent live imaging. We found that the position of pronuclei and polar bodies were different between these two species. In C. elegans, the female and male pronuclei first become visible in anterior and posterior sides, respectively, then they meet at the center of embryo. On the other hand, the initial position of pronuclei were more closely located in C. inopinata. Also, the polar bodies usually appear in the anterior side of embryo in C. elegans, but they appeared at random positions in C. inopinata. Therefore, we infer that C. inopinata may have a different polarity formation mechanism from that in C. elegans. We are also analyzing temperature dependency of embryogenesis in C. inopinata, whose optimal temperature is ~7 degree higher than that in C. elegans.

Karin Kiontke et al. (2008) Development & Evolution Meeting "New Phylogeny Of Caenorhabditis Including Recently Discovered Species"

David Fitch, Karin Kiontke, Marie-Anne FELIX

[Development & Evolution Meeting, 2008]

Recently, seven new Caenorhabditis have been discovered, bringing the number of Caenorhabditis species in culture to 17, 10 of which are undescribed. To elucidate the relationships of the new species to the five species with sequenced genomes, we have used sequence data from two rRNA genes and several protein-coding genes for reconstructing the phylogenetic tree of Caenorhabditis. Four new species (spp. 5, 9, 10, 11) group within the so-called Elegans group of Caenorhabditis, with C. elegans being the first branch. Whereas none of them is likely to be the sister species of C. elegans, we now know of two close relatives of C. briggsae-C. sp. 5 and C. sp. 9. C. sp. 9 can hybridize with C. briggsae in the laboratory [see abstract by Woodruff et al.]. Of the remaining new species, C. sp. 7 branches off between C. elegans and C. japonica. This species is easier to cultivate than C. japonica and may be a better candidate for comparative experimental work. Two of the new species branch off before C. japonica as sister species of C. sp. 3 and C. drosophilae+C. sp. 2, respectively. Only one of the new species, C. sp. 11, is hermaphroditic. The position of C. sp. 11 in the phylogeny suggests that hermaphroditism evolved three times within the Elegans group. Two of the new species were isolated from rotting leaves and flowers, and five from rotting fruit. Rotting fruit is also the habitat in which C. elegans has been found to proliferate (Barriere and Felix, Genetics 2007) and from which C. briggsae, C. brenneri and C. remanei were repeatedly isolated. This suggests that the habitat of the stem species of Caenorhabditis after the divergence of the earliest branches (C. plicata, C. sonorae and C. sp. 1) was rotting fruit. The rate of discovery of new Caenorhabditis species has steadily increased since the description of C. elegans in 1899, with a leap in the last two years. There is no indication that we are even close to knowing all species in this genus.

Schartner, Caitlin M. et al. (2015) International Worm Meeting "X-chromosome evolution: divergence of X-sequence motifs that drive dosage compensation across Caenorhabditis species."

Meyer, Barbara J., Lo, Te-Wen, Schartner, Caitlin M.

[International Worm Meeting, 2015]

Dosage compensation (DC) across Caenorhabditis species exemplifies an essential process that has undergone rapid co-evolution of protein-DNA interactions central to its mechanism. In C. elegans, recruitment elements on X (rex sites) recruit a condensin-like DC complex (DCC) to hermaphrodite X chromosomes to balance gene expression between the sexes. Recruitment assays in vivo showed that C. elegans rex sites do not recruit the DCC of C. briggsae, and vice versa. To understand how DC complexes and X chromosomes evolved to use different X targeting sequences, we compared DCC subunits and binding sites in C. elegans to those in three species of the C. briggsae clade (15-30 MYR diverged): C. briggsae, its close relative C. nigoni (C. sp. 9), and C. tropicalis (C. sp. 11). By raising antibodies and introducing endogenous tags with TALENs or CRISPR/Cas9, we showed that homologs of both SDC-2, the pivotal X targeting factor, and DPY-27, a DCC-specific condensin subunit, bind X chromosomes of XX animals. Although the DCC shares key components across these four species, the binding sites differ. First, ChIP-seq studies in C. briggsae and C. nigoni identified DCC binding sites that are homologous across these close relatives but differ from C. elegans sites in sequence and location. Second, C. elegans sites use motifs enriched on X (MEX and MEXII) to drive DCC binding, but these motifs are not in C. briggsae or C. nigoni DCC sites and are not X-enriched. Third, we found an X-enriched motif at DCC binding sites of C. briggsae and C. nigoni that is not X-enriched in C. elegans. An oligo with the C. briggsae motif recruits the DCC in C. briggsae, but a similar oligo lacking the motif fails to recruit, establishing the importance of the motif. Fourth, another motif was found in C. briggsae and C. nigoni that shares a few nucleotides with MEX, but its functional divergence was shown by C. elegans recruitment assays. Fifth, two endogenous C. briggsae X-chromosome regions with strong C. elegans MEX motifs fail to recruit the C. briggsae DCC, as assayed by ChIP-seq and recruitment assays. None of these DCC motifs is enriched on the C. tropicalis draft X sequence, supporting further binding site divergence within the C. briggsae clade. Ongoing ChIP-seq studies in C. tropicalis will help determine how C. elegans and C. briggsae clade motifs are evolutionarily related. Comparison of DCC targeting mechanisms across these four species allows us to characterize a rarely captured event: the recent co-evolution of a protein complex and its rapidly diverged target sequences across an entire X chromosome.