Schwarz JZ et al. (2000) Midwest Worm Meeting "What can a muscle cell do with a neuronal integrin?"

Schwarz JZ, McDonald KA, Williams BD

[Midwest Worm Meeting, 2000]

Integrins, transmembrane proteins that bind the extracellular matrix (ECM), are heterodimers composed of distinct a and b subunits. Vertebrates make a variety of a and bsubunits, with each b subunit capable of pairing with several different asubtypes to produce heterodimers with distinct ligand binding and cell signaling functions. The complexity of the vertebrate integrin repertoire complicates the analysis of in vivo integrin function. C. elegans expresses a limited set of integrins, allowing for analysis of the functional significance of different integrin isoforms in vivo. To date, only two C. elegans a integrin subunits have been identified: PAT-2 and INA-1. These subunits are expressed in different tissues, both pairing with a single b subunit, PAT-3. aINA-1 bPAT-3 heterodimers are expressed in a variety of cells, including neurons, where they function in migration and axon fasciculation. In contrast, the aPAT-2 bPAT-3 heterodimer is expressed in muscle and myoepithelial cells, localizing to adhesion structures attaching body wall muscle cells to the hypodermis. Both pat-2 and pat-3 loss-of-function mutations block the assembly of anchorage structures and the associated contractile apparatus. To ask if the PAT-2 subunit of muscle integrins performs unique, cell-specific functions required for muscle development, we are assessing the ability of INA-1 to replace PAT-2 during muscle development. By transforming wild-type worms with a functional ina-1::gfp fusion (provided by Baum and Garriga) driven by the endogenous pat-2 promoter, we have shown muscle cells are capable of assembling an aINA-1 bPAT-3 heterodimer. This heterodimer is expressed on the surface of body wall muscles and localizes to both dense bodies and M lines, the sites of normal aPAT-2 bPAT-3 localization. Although localized properly, aINA-1 bPAT-3 has dominant effects on adhesion structure assembly, indicated by occasional spontaneous detachment of the myofilament lattice from the body wall. Localization of aINA-1 bPAT-3 heterodimers to dense bodies and M lines suggests this integrin heterodimer is, at least, partially functional in body wall muscle and is can bind to ECM and/or cytoplasmic proteins that direct it to nascent attachment structures. With evidence aINA-1 bPAT-3 functions at least partially in muscle cells, we looked to see if the heterodimer could rescue muscle development in pat-2 loss-of-function mutants. pat-2 mutants are characterized by the failure to assemble muscle cytoskeleton, which causes complete embryonic paralysis. INA-1 partially rescued muscle activity, but did not support complete myofilament lattice organization. These results provide further evidence INA-1 is partially functional in the muscle cell, but also suggest the PAT-2 subunit performs specialized functions necessary for the normal muscle cell developmental program. Currently, we are constructing chimeras between INA-1 and PAT-2 to determine whether these specializations map to the extracellular and/or cytoplasmic domains of PAT-2.

KA McDonald et al. (1999) International C. elegans Meeting "Constitutively activated integrin during muscle development"

JZ Schwarz, KA McDonald, BD Williams

[International C. elegans Meeting, 1999]

Integrins are heterodimeric transmembrane proteins that bind components of the extracellular matrix (ECM). Recent advances have shown that integrin-mediated signaling can control fundamental cell properties such as motility, cytoskeletal remodeling, differentiation, and gene expression. We are investigating integrin function during muscle cell development in C. elegans. Body wall muscle cells express the integrin a subunit PAT-2 and b subunit PAT-3, both of which are located in regularly patterned cytoskeletal anchorage structures analogous to vertebrate muscle Z and M lines. pat-2 and pat-3 mutations disrupt cytoskeletal assembly, resulting in the Pat developmental arrest phenotype. The integrin heterodimer can modulate its affinity for ECM ligands and the ability of its cytoplasmic domain to interact with the cytoskeleton and associated signaling molecules. The "non-activated" heterodimer has a low affinity for extracellular ligands and does not show cytoskeletal association and related signaling. Conversely, the "activated" heterodimer has high affinity for ligand and does associate with the cytoskeleton. In our working model of body wall muscle development, integrin is activated by binding to an ECM ligand, and in response, initiates cytoskeletal assembly. To test this model, we have introduced a mutation into a functional pat-3::gfp construct that is predicted to constitutively "activate" the integrin heterodimer (Hughes et al., 1996, JBC 271: 6571). This mutation disrupts a highly conserved potential salt bridge between the a and b subunits. When expressed transgenically in a wild-type background the "activated" PAT-3::GFP protein localizes normally to muscle cell attachment structures. We have observed mispositioned sex muscle insertions in these animals, indicating dominant effects on cell migration and/or insertion. Surprisingly, the "activated" pat-3::gfp transgene rescues pat-3 loss-of-function homozygotes, but only at very low frequency. Most animals expressing the transgene arrest as Pats. We are currently characterizing the defects in muscle cell development that occur in these animals. The relatively few rescued adults often show examples of mispositioned body wall and sex muscles, again consistent with migration/insertion defects.

Ni, J. et al. (2017) International Worm Meeting "The triggering mechanism of C. elegans germline nuclear RNAi at the native targets."

Ni, J., Gu, S., Kalinava, N.

[International Worm Meeting, 2017]

We wish to understand the molecular mechanisms that distinguish "self" and "non-self" DNA in the germline genome of C. elegans. Nuclear RNAi refers to a set of small RNA-guided chromatin-based gene-silencing pathways (e.g., heterochromatin formation and transcriptional silencing), and plays an essential role in genome surveillance in yeast, plants, and animals. We previously identified a large set of genomic loci that are targeted by germline nuclear RNAi in C. elegans [1,2]. The triggering mechanisms at these native targets appear to be highly complex and are poorly understood. We are using two different approaches to resolve this gap. (1) We are identifying aberrant features associated with germline nuclear RNAi-targeted transcripts. To this end, we are using an unbiased RNA-seq approach to characterize both polyA and non-polyA transcripts in both wild type and mutant animals. In addition, subcellular localization of the native target transcripts are being examined using single-molecule FISH and RNA-seq combined with biochemical fractionation. (2) We are using CRISPR-mediated genome editing to test whether the silencing responses at native targets are indeed triggered by the aberrant features that are identified in (1). To date, we have found that "self" and "non-self" transcripts differ in RNA-pocessing, as well as subcellular localization. By using CRISPR, we have identified cis-regulatory elements that are required for the silencing response. Reference: 1. Ni JZ, Chen E, Gu SG. BMC Genomics. 2014. PMID: 25534009 2. Ni JZ, Kalinava N, Chen E, Huang A, Trinh T, Gu SG. Epigenetics Chromatin. 2016. PMID: 26779286

Moon, K. et al. (2019) International Worm Meeting "Gait selection of Caenorhabditis elegans is regulated by mechanosensitive DEG/ENaC channels."

Moon, K., Kim, K., Kim, J., Yeon, J.

[International Worm Meeting, 2019]

Animals change their locomotion or gaits in response to environmental condition. In vertebrates, gait transition has been shown to be mediated by monoamines, which is conserved across many species including the nematode Caenorhabditis elegans (Vidal-Gadea et al., 2011). However, molecular mechanisms of gait transition are still unclear. C. elegans exhibits two gaits, swimming in liquids and crawling on dense gels. C. elegans genome contains evolutionarily conserved 28 DEG/ENaC channels, of which functions may be involved in mechanosensory transduction and locomotion (Goodman and Schwarz, 2003). We first hypothesized that mechanosensitive channels could act as a gait transition initiator and examined crawl-to-swim transition phenotype in DEG/ENaC mutants. We found that while acd-5 mutants show normal crawling, transition from crawling to swimming upon liquid exposure is defective, suggesting roles of ACD-5 in gait transition. We are currently generating acd-5 rescue lines and examining expression pattern of acd-5.

Chiang, Y. et al. (2017) International Worm Meeting "Wild-isolates of C. elegans exhibit variable attraction to the sex-pheromone mimicry compound produced by the nematode-trapping fungus A. oligospora."

Chiang, Y., Hsueh, Y.

[International Worm Meeting, 2017]

The nematode-trapping fungus A. oligospora produces odors that mimic sex and food cues to attract many nematode species (Hsueh et al. 2017). One of the compounds (MMB) that likely mimics male pheromone in Canerhbiditis nematodes is highly attractive to two lab strains of C. elegans (N2 and Hawaiian). To investigate whether this attraction is highly conserved among the wild-isolates, we analyzed the MMB chemotaxis behavior among the C. elegans wild isolates from the CeNDR collection. We found that MMB-attraction is highly polymorphic trait. Six out of forty wild-isolates tested exhibited weak attraction to MMB. Genetic analysis showed that this trait is controlled by QTL in some strains but a single locus in others. The high incidence of MMB-insensitive strains among the wild-isolates suggests that lost of MMB attraction might offer benefit to C. elegans in the natural environments. Hsueh YP, Gronquist MR, Schwarz EM, Nath RD, Lee CH, Gharib S, Schroeder FC, Sternberg PW. 2017. Nematophagous fungus Arthrobotrys oligospora mimics olfactory cues of sex and food to lure its nematode prey. eLife 6.

Ted Ririe et al. (2007) International Worm Meeting "cis- and trans-regulation of zmp-1 expression in the C. elegans vulva."

Ted Ririe, Jolene Fernandes, Paul Sternberg

[International Worm Meeting, 2007]

GFP reporters for approximately 32 genes are expressed in the differentiated C. elegans vulval cell types; each with its own spatial and temporal pattern of expression. The C. elegans vulva comprises seven different cell types (vulA, vulB1, vulB2, vulC, vulD, vulE and vulF). zmp-1 (zinc metalloproteinase) is expressed in vulD and vulE starting at the late-L4 stage of development and in vulA starting in young adulthood. We are using several approaches to identify the regulatory network that controls differentiation of the vulval cell types. Vulval enhancer elements have previously been identified by analysis of the cis-regulatory regions of the zmp-1 promoter. Using Cistematic (see abstract by Schwarz/Mortazavi) four conserved motifs were found in the vulval enhancer element of zmp-1. Disruption of two of these sites revealed demonstrable function in driving gene expression, and disruption of a third revealed potential repressor activity. Concurrently we have conducted RNAi screens of over 500 transcription factors to identify genes involved in the regulation of zmp-1 expression. We have identified factors that promote zmp-1 expression as well as factors that inhibit ectopic zmp-1 expression in unexpected cell types. Here we report our progress in the characterization of these regulatory factors.

Kalinava, N. et al. (2017) International Worm Meeting "H3K9me3 plays a complex role in RNAi-mediated transcriptional silencing and transgenerational epigenetic inheritance in C. elegans."

Chen, E., Kalinava, N., Ni, J., Gu, S., Peterman, K.

[International Worm Meeting, 2017]

In many organisms, nuclear siRNA can guide transcriptional silencing and/or heterochromatin formation at the homologous genomic regions. Such heterochromatin response can be maintained over several generations in C. elegans. However, the function of the heterochromatin response remains elusive. We use biochemical, genetic and whole-genome approaches to investigate the requirements of H3K9me3 heterochromatin for initiation and maintenance of transcriptional silencing in germline nuclear RNAi pathway in C. elegans. First we identified three histone methyltransferase (HMT) - MET-2, SET-25 and SET-32 that, in combination, are required for the full H3K9me3 response in the germline nuclear RNAi pathway. Our results showed that H3K9me3 is not required for the heritable RNAi. In addition, complete depletion of H3K9me3 in met-2 set-25;set-32 mutants does not result with transcriptional de-silencing of nuclear RNAi targets. Therefore, the maintenance of transcriptional silencing by nuclear RNAi pathway can occur in H3K9me3-independent manner [1]. We recently developed a CRISPR-based system to examine the establishment of transcriptional silencing by nuclear RNAi at the native targets. This work revealed additional complexity for the silencing mechanism at the native targets in that H3K9me3 and RNAi play distinct roles at the establishment and maintenance stages of transgenerational silencing. We will report these novel findings at the meeting. Reference: 1. Kalinava N, Ni JZ, Peterman K, Chen E, Gu SG. Epigenetics Chromatin. 2017. PMID: 28228846

Haskell, D. et al. (2017) International Worm Meeting "Forward genetic screen to identify regulators of microRNA strand choice."

Haskell, D., Zinovyeva, A., Li, L.

[International Worm Meeting, 2017]

Precisely controlled gene regulation is essential for proper organism development and homeostasis. A species of small non-coding RNAs called microRNAs (miRNAs) play a critical role in post-transcriptional regulation of gene expression. miRNAs regulate gene expression by binding to partially complementary sites in mRNA targets, repressing their translation, or targeting the mRNA for degradation through recruitment of miRNA induced silencing complex (miRISC) components. One of the key steps in miRNA biogenesis is Dicer-mediated processing of the hairpin precursor miRNA to generate a double stranded miR::miR* duplex. As the miRNA duplex loads into Argonaute, the orientation of the duplex loading dictates which miRNA strand will become the more abundant, mature miRNA. Most of the time, this process of miRNA strand selection is highly asymmetric, with one miRNA strand dominantly selected. It is known that 5' nucleotide (nt) identity and thermodynamic stabilities of the miRNA duplex ends are important determinants of small RNA strand selection in vitro1,2. Interestingly, evidence recently emerged that miRNA strand choice may be regulated in vivo, with miR and miR* levels changing in response to physiological or environmental cues, suggesting the miRNA duplex extrinsic factors may play a role in miRNA strand selection. We have generated a sensor that is designed to differentially express fluorescent proteins based on the relative abundance of miR and miR* strands of a given miRNA. We will use this sensor to conduct a forward genetic screen to identify factors that may be important for miRNA strand selection in vivo. 1 Ghildiyal, M., Xu, J., Seitz, H., Weng, Z. & Zamore, P. D. Sorting of Drosophila small silencing RNAs partitions microRNA* strands into the RNA interference pathway. RNA 16, 43-56 (2010). 2 Schwarz, D. S. et al. Asymmetry in the assembly of the RNAi enzyme complex. Cell 115, 199-208 (2003).

Stevens, Lewis et al. (2019) International Worm Meeting "Nothing in Caenorhabditis biology makes sense except in the light of evolution: The Caenorhabditis Genomes Project."

Blaxter, Mark, Stevens, Lewis

[International Worm Meeting, 2019]

Caenorhabditis elegans is one of the preeminent model organisms in modern biology, but only recently have we started to understand the details of its natural ecology and evolutionary history. Studying C. elegans alongside its closest relatives provides an important evolutionary context for the origins and constraints that have resulted in the particular instance analyzed in the laboratory. A focussed search for new Caenorhabditis species over the last decade has led to the discovery of over 50 species of Caenorhabditis. As part of an international collaboration, the Caenorhabditis Genomes Project (CGP), we have sequenced the genomes of all Caenorhabditis species currently in culture. We exploit these new genome sequences to perform the most comprehensive reconstruction of the Caenorhabditis phylogeny to date, providing an essential evolutionary framework for downstream analyses. We reveal extensive variation in genome size in the genus, ranging from the 48 Mb genome of C. drosophilae to the 160 Mb genome of C. vivipara. Investigating the origins of this variation, we find that protein-coding gene number is highly correlated with genome size, with C. drosophilae possessing just 13,000 genes due to extensive gene loss. Other genomic features, such as repeat (mobile element) proliferation also contribute to genome size changes. Among the non-coding portions of these genomes, we identified rapid and extensive intron loss across the genus, including in the group that includes C. elegans. We demonstrate the utility of these genome sequences for C. elegans research by analyzing the evolutionary history of key signaling pathways, revealing unexpected complexity. We have made these genomic resources, including genus-wide orthology sets, publicly available via the CGP website (http://www.caenorhabditis.org) which includes a dedicated genome browser and a BLAST server. These new species and their associated genomic resources add to the arsenal of tools available to the C. elegans researcher to interrogate biology of this important nematode. The CGP is a collaboration between the following labs: Blaxter (Edinburgh), Felix (Paris), Ailion (Washington), Andersen (Northwestern), Braendle (Nice), Cutter (Toronto), Fitch (NYU), Fierst (Alabama), Kikuchi (Miyazaki), Rockman (NYU), Schwarz (Cornell), Wang (Academia Sinica).

Carmi I et al. (2000) European Worm Meeting "Characterization of the egg-laying system of two diplogasterids, Pristionchus pacificus and Diplogastrellus gracilis"

Sommer RJ, Carmi I

[European Worm Meeting, 2000]

The nematode egg-laying system is a complex structure consisting of several different cell types: epidermal vulval cells, vulval and uterine muscles, neurons and gonadal cells. In C. elegans, a variety of signaling processes are required among these cells both to specify cell fates and to guide various cells to their correct final positions. We would like to know how these interactions and cell migrations change during evolution, and particularly how they adapt to changes in the position of the vulva. For this reason, we are focusing our studies on two related nematodes from the family Diplogasteridae: P. pacificus, a central-vulva nematode, and D. gracilis, a posterior-vulva nematode. We are currently examining the development of various components of the egg-laying system of these nematodes. Our analysis of vulva development in D. gracilis and P. pacificus has shown that, although the same set of ventral epidermal precursor cells forms the vulva in both species, cellular fates are specified differently. In both species seven of 12 ventral epidermal precursor cells die by programmed cell death, while three of the remaining cells, P(5-7).p, form the vulva. In D. gracilis, these cells then migrate posteriorly, followed by posterior extension of the gonad. Ablation studies suggest that cell interactions among vulval precursor cells have evolved differently in the two species, leading to differences in cell competence for individual precursor cells. Furthermore, in contrast to P. pacificus, vulval differentiation in D. gracilis is gonad independent. Gonad-independent differentiation has been previously observed in conjunction with posterior vulva development in two other nematode families, suggesting convergent evolution. In addition to vulval development, we have also begun cell lineage studies of muscle development in P. pacificus, and, in collaboration with Heinz Schwarz (Max Planck electron microscopy lab, Tuebingen), we have initiated an electron microscope study to compare the gonad morphologies of diplogasterids with that of C. elegans. Finally, we are trying to develop a transformation system for diplogasterids. We will report our progress on all these projects at the meeting.