Moya, Nicolas D. et al. (2021) International Worm Meeting "Improved reference genomes for Caenorhabditis briggsae"

Chitrakar, Rojin, Stevens, Lewis, Baugh, L. Ryan, Moya, Nicolas D., Walhout, Marian, Tanny, Robyn E., Dekker, Job, Na, Huimin, Andersen, Erik C.

[International Worm Meeting, 2021]

Decades of research have led to the development of comprehensive genome resources that have been essential to study the Caenorhabditis elegans species. In parallel, the emergence of Caenorhabditis briggsae as a model system has been useful to make interspecies comparisons. Despite the importance of C. briggsae as a model, its genome resources have not been developed to the same extent as C. elegans. The current genome of C. briggsae reference strain AF16 contains thousands of unresolved gaps and numerous mis-assemblies. Because of these issues, C. briggsae gene models remain incomplete and have numerous structural errors in protein-coding genes. We sought to exploit the latest sequencing technologies and computational tools to provide the highest quality C. briggsae genome resources to date. First, we generated high-quality genome assemblies for two strains of C. briggsae: QX1410 (a "tropical" strain isolated in Saint Lucia that is closely related to AF16) and VX34 (a divergent strain isolated in China). These genome assemblies incorporate high coverage Oxford Nanopore PromethION long reads and chromosome conformation capture (Hi-C) data. Second, we genotyped 99 recombinant inbred lines generated from reciprocal crosses between QX1410 and VX34. Using these data, we produced a high-quality recombination map that validated the placement of scaffolds after genome assembly. Third, we sequenced the transcriptomes of each strain to high coverage using Pacific Biosciences SMRT and Illumina platforms. We developed a computational pipeline that leverages long and short RNA reads to generate a genome annotation for each strain. These new genome annotations have improved accuracy and completeness relative to the AF16 genome. Fourth, our research group currently maintains over 1,600 C. briggsae wild strains, comprising the largest collection worldwide. We sequenced the genomes of this entire collection to high coverage using the Illumina platform. We mapped the sequences of all wild strains to the QX1410 genome to call single nucleotide variants across the entire population. These high-quality genome resources will facilitate new avenues of research, including quantitative and population genetic studies of C. briggsae, and enable informative comparisons with C. elegans.

Sullivan-Brown, Jessica et al. (2021) International Worm Meeting "Characterization of RNAi phenotypes in C. elegans from understudied genes in a Cell and Molecular Biology course"

Sowa, Jessica, Sullivan-Brown, Jessica

[International Worm Meeting, 2021]

The characterization of relatively understudied genes in C. elegans using RNA interference is an excellent approach for designing undergraduate research-based labs. Here we describe a successful lab course in which Cell and Molecular Biology students at West Chester University of Pennsylvania (WCU) make novel discoveries associated with knock down of dcaf-13, a likely oncogene in humans. Each class represents the flow of a typical study of gene function, beginning with a journal club reading the primary literature. Then students use bioinformatics to identify the human protein sequence of DCAF13, perform a BLAST search to find C. elegans homologs, and use WormBase to identify the C. elegans gene sequence. An RNAi knock down experiment is then performed with dcaf-13. Throughout the lab, students characterize the resulting phenotypes, using IMAGEJ/FIJI for quantification of worm length and fluorescence microscopy for GFP analysis of an intestinal cell fate marker. Students then isolate RNAs using Trizol RNA extraction and perform cDNA synthesis. Students then design primers to test if dcaf-13 knock down affects the expression of different cell cycle genes by performing RT-PCR. At the end of the course, students present their work orally in a poster presentation and in written communication in a primary research style paper. We have recently added a component in which students design a future experiment in the format of a small grant proposal and after peer review, two projects are chosen to perform in lab. The lab serves two primary functions: to provide students opportunities to advance their experimental techniques so they are competitive in the job market and create an experience that mimics a typical research project by making real contributions to science. Our goals are to publish the work the students generated. Importantly, this framework can be applied to other understudied genes in C. elegans in which the RNAi phenotypes are not well documented, benefiting both students and the C. elegans community.

Mary C Abraham et al. (2003) International Worm Meeting "Was it the butler? The curious case of linker cell murder."

Mary C Abraham, Shai Shaham

[International Worm Meeting, 2003]

Apoptosis sculpts tissues during development, is required to remove self-reactive immune cells and can act as a defense mechanism against cancer. Apoptosis occurs in these very diverse contexts across many different organisms, yet at its core lies conservation of molecules and morphology of cell death. The C. elegans linker cell death is an example of a developmentally regulated cell death. The linker cell migrates as a leader cell guiding the male gonad as it grows and develops. It dies at the late L4 stage when its migratory job is complete and the gonad is about to reach and fuse with the proctodeum, allowing the cloaca to act as a sperm exit channel. There are two particularly striking observations about linker cell death compared with other cell deaths in C. elegans. First, the linker cell is the only known example of a C. elegans cell that consistently dies in animals harboring a strong ced-3/caspase loss-of-function mutation (1). This observation suggests the linker cell may have a caspase-independent cell death pathway. Second, the signal initiating linker cell death may come from outside the cell. Specifically, ablation of the U cell prevents linker cell death (2). It is thought, therefore, that a signal from the U cell descendants, which neighbor the linker cell at the moment of its demise, commits cellular murder. No intercellular pathways initiating apoptosis have been identified in C. elegans. Work reported will include further analysis of linker cell death in a variety of mutants, including the cell death pathway mutants, engulfment mutants and linker cell migration mutants. Engulfment of the linker cell was studied using GFP markers such as EGL-5::GFP expressed in the engulfing U cell descendant. Current progress will be reported on an EMS mutagenesis screen, using a strain with LAG-2::GFP to mark the linker cell, aimed at finding mutants defective in linker cell death. References: (1) Ellis H.M. and Horvitz H.R. (1986) Genetic Control of programmed cell death in the nematode C. elegans. Cell, 44:817-29; (2) Sulston J.E., Albertson D.G and Thompson J. N. (1980) The Caenorhabditis elegans male: postembryonic development of nongonadal structures. Dev Biol, 78:542-76.

O Hobert et al. (1999) International C. elegans Meeting "ceh-1 regulates ttx-3: An initiation-maintenance switch paradigm"

G Ruvkun, O Hobert, Z Altun-Gultekin

[International C. elegans Meeting, 1999]

It seems intuitive that cell fate specification should be subdivided into an initiation step and a subsequent maintenance step which ensures the preservation of specific differentiation features. This simple model has indeed been proven true for many developmental processes. The differentiation of the AIY interneuron appears to follow this rule as well. We define a genetic cascade of two transcription factors that are required in consecutive steps to initiate and then maintain AIY cell fate. ceh-10 is a homeobox gene that is expressed during early proliferative embryonic stages in a few head neurons, including AIY (Svedsen and McGhee, 1995). ttx-3 encodes a LIM homeobox gene that is expressed in AIY and also in a few other neurons (Hobert et al., 1997; this poster). We define a 243 bp intronic fragment of the ttx-3 gene that is sufficient to drive GFP expression in AIY; its expression is then maintained throughout adulthood. Given that ttx-3 is potentially involved in synpatic maturation of AIY (see abstract by Hall et al.), the ttx-3::GFP reporter gene fragment can be considered to be a marker for the terminal differentiation of AIY. We find that ceh-10 is both required and sufficient for ttx-3::GFP expression in AIY. In ceh-10 null mutants, a neuron that is in the correct position for AIY is formed (Forrester et al., 1998), yet ttx-3::GFP expression is not initiated. On the other hand, ectopic expression of ceh-10 using the heat shock promoter induces ectopic expression of ttx-3::GFP reporter gene constructs. Notably, ceh-10 expression disappears after hatching , yet ttx-3 expression is maintained. This process appears to rely on an autoregulatory mechanism since in ttx-3 null mutant animals, ttx-3::GFP expression is initiated but not as strongly maintained past the embryonic stage. Taken together these data suggest that ceh-10 is required to initiate the AIY differentiation program by initiating the expression of certain AIY differentiation marker(s). After ceh-10 has performed this initial task, the protein disappears, yet one of the proteins that is induced by CEH-10, namely TTX-3, now take over the job of maintaining its own expression and presumably that of other AIY cell fate markers as well.