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Caenorhabditis briggsae as a companion species for Caenorhabditis elegans has played an increasingly important role in study of evolution of development and genome and gene regulation. Aided by the isolation of its sister spices, it has recently been established as a model for speciation study. To take full advantage of the species for comparative study, an effective transgenesis method especially those with single-copy insertion is important for functional comparison. Here, we improved a transposon-based transgenesis methodology that had been originally developed in C. elegans but worked marginally in C. briggsae. By incorporation of a heat shock step, the transgenesis efficiency in C. briggsae with a single-copy insertion is comparable to that in C. elegans. We used the method to generate 54 independent insertions mostly consisting of a mCherry tag over the C. briggsae genome. We demonstrated the use of the tags in identifying interacting loci responsible for hybrid male sterility between C. briggsae and Caenorhabditis nigoni when combined with the GFP tags we generated previously. Finally, we demonstrated that C. briggsae tolerates the C. elegans toxin, PEEL-1, but not SUP-35, making the latter a potential negative selection marker against extrachromosomal array., (© The Author(s) 2022. Published by Oxford University Press on behalf of Genetics Society of America.)

Early embryonic cell cycles usually alternate between S and M phases without any gap phase. When the gap phases are developmentally introduced in various cell types remains poorly defined especially during embryogenesis. To establish the cell-specific introduction of gap phases in embryo, we generate multiple fluorescence ubiquitin cell cycle indicators (FUCCI) in C. elegans . Time-lapse 3D imaging followed by lineal expression profiling reveals sharp and differential accumulation of the FUCCI reporters, allowing the systematic demarcation of cell cycle phases throughout embryogenesis. Accumulation of the reporters reliably identifies both G1 and G2 phases only in two embryonic cells with an extended cell cycle length, suggesting that the remaining cells divide either without a G1 phase, or with a brief G1 phase that is too short to be picked up by our reporters. In summary, we provide an initial picture of gap phase introduction in a metazoan embryo. The newly developed FUCCI reporters pave the way for further characterization of developmental control of cell cycle progression., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Wong, Ho, Huang, Chan, Xie, Li, Ren, Guan, Ma, Hu, Yan and Zhao.)

Background: Ribosomal DNAs (rDNAs) are arranged in purely tandem repeats, preventing them from being reliably assembled onto chromosomes during generation of genome assembly. The uncertainty of rDNA genomic structure presents a significant barrier for studying their function and evolution., Results: Here we generate ultra-long Oxford Nanopore Technologies (ONT) and short NGS reads to delineate the architecture and variation of the 5S rDNA cluster in the different strains of C. elegans and C. briggsae. We classify the individual rDNA's repeating units into 25 types based on the unique sequence variations in each unit of C. elegans (N2). We next perform assembly of the cluster by taking advantage of the long reads that carry these units, which led to an assembly of 5S rDNA cluster consisting of up to 167 consecutive 5S rDNA units in the N2 strain. The ordering and copy number of various rDNA units are consistent with the separation time between strains. Surprisingly, we observed a drastically reduced level of variation in the unit composition in the 5S rDNA cluster in the C. elegans CB4856 and C. briggsae AF16 strains than in the C. elegans N2 strain, suggesting that N2, a widely used reference strain, is likely to be defective in maintaining the 5S rDNA cluster stability compared with other wild isolates of C. elegans or C. briggsae., Conclusions: The results demonstrate that Nanopore DNA sequencing reads are capable of generating assembly of highly repetitive sequences, and rDNA units are highly dynamic both within and between population(s) of the same species in terms of sequence and copy number. The detailed structure and variation of the 5S rDNA units within the rDNA cluster pave the way for functional and evolutionary studies., (© 2022. The Author(s).)

The invariant development and transparent body of the nematode Caenorhabditis elegans enables complete delineation of cell lineages throughout development. Despite extensive studies of cell division, cell migration and cell fate differentiation, cell morphology during development has not yet been systematically characterized in any metazoan, including C. elegans. This knowledge gap substantially hampers many studies in both developmental and cell biology. Here we report an automatic pipeline, CShaper, which combines automated segmentation of fluorescently labeled membranes with automated cell lineage tracing. We apply this pipeline to quantify morphological parameters of densely packed cells in 17 developing C. elegans embryos. Consequently, we generate a time-lapse 3D atlas of cell morphology for the C. elegans embryo from the 4- to 350-cell stages, including cell shape, volume, surface area, migration, nucleus position and cell-cell contact with resolved cell identities. We anticipate that CShaper and the morphological atlas will stimulate and enhance further studies in the fields of developmental biology, cell biology and biomechanics.

hlh-1 is a myogenic transcription factor required for body-wall muscle specification during embryogenesis in Caenorhabditis elegans. Despite its well-known role in muscle specification, comprehensive regulatory control upstream of hlh-1 remains poorly defined. Here, we first established a statistical reference for the spatiotemporal expression of hlh-1 at single-cell resolution up to the second last round of divisions for most of the cell lineages (from 4- to 350-cell stage) using 13 wild-type embryos. We next generated lineal expression of hlh-1 after RNA interference (RNAi) perturbation of 65 genes, which were selected based on their degree of conservation, mutant phenotypes, and known roles in development. We then compared the expression profiles between wild-type and RNAi embryos by clustering according to their lineal expression patterns using mean-shift and density-based clustering algorithms, which not only confirmed the roles of existing genes but also uncovered the potential functions of novel genes in muscle specification at multiple levels, including cellular, lineal, and embryonic levels. By combining the public data on protein-protein interactions, protein-DNA interactions, and genetic interactions with our RNAi data, we inferred regulatory pathways upstream of hlh-1 that function globally or locally. This work not only revealed diverse and multilevel regulatory mechanisms coordinating muscle differentiation during C. elegans embryogenesis but also laid a foundation for further characterizing the regulatory pathways controlling muscle specification at the cellular, lineal (local), or embryonic (global) level.

Mitochondrial genome (mtDNA) carries not only well-conserved protein coding, tRNA and rRNA genes, but also highly variable non-coding regions (NCRs). However, the NCRs show poor conservation across species, making their function and evolution elusive. Identification and functional characterization of NCRs across species would be critical for addressing these questions. To this end, we devised a computational pipeline and performed de novo assembly and annotation of mtDNA from 19 Caenorhabditis species using next-generation sequencing (NGS) data. The mtDNAs for 14 out of the 19 species are reported for the first time. Comparison of the 19 genomes reveals species-specific sampling of partial displacement-loop (D-loop) sequence as a novel NCR inserted into a unique tRNA cluster, suggesting an important role of the D-loop and the tRNA cluster in shaping NCR evolution. Intriguingly, RNA-Seq analysis suggests that a novel NCR resulting from a recent duplication of NADH dehydrogenase subunit 5 (ND5) could be utilized as a 3' UTR for up-regulation of its upstream gene. The expression analysis shows a species- and sex-specific expression of mitochondrial genes encoded by mtDNA and nucleus, respectively. Our analyses provide important insights into the function and evolution of mitochondrial NCRs and pave the way for further studying the function and evolution of mitochondrial genome.

Intercellular signaling interactions play a key role in breaking fate symmetry during animal development. Identification of signaling interactions at cellular resolution is technically challenging, especially in a developing embryo. Here, we develop a platform that allows automated inference and validation of signaling interactions for every cell cycle of Caenorhabditis elegans embryogenesis. This is achieved by the generation of a systems-level cell contact map, which consists of 1114 highly confident intercellular contacts, by modeling analysis and is validated through cell membrane labeling coupled with cell lineage analysis. We apply the map to identify cell pairs between which a Notch signaling interaction takes place. By generating expression patterns for two ligands and two receptors of the Notch signaling pathway with cellular resolution using the automated expression profiling technique, we are able to refine existing and identify novel Notch interactions during C. elegans embryogenesis. Targeted cell ablation followed by cell lineage analysis demonstrates the roles of signaling interactions during cell division in breaking fate symmetry. Finally, we describe the development of a website that allows online access to the cell-cell contact map for mapping of other signaling interactions by the community. The platform can be adapted to establish cellular interactions from any other signaling pathway., (Copyright © 2018 by the Genetics Society of America.)

DNA recombination is required for effective segregation and diversification of genomes and for the successful completion of meiosis. Recent studies in various species hybrids have demonstrated a genetic link between DNA recombination and speciation. Consistent with this, we observed a striking suppression of recombination in the hybrids between two nematodes, the hermaphroditic Caenorhabditis briggsae and the gonochoristic C. nigoni. To unravel the molecular basis underlying the recombination suppression in their hybrids, we generated a C. nigoni genome with chromosome-level contiguity and produced an improved C. briggsae genome with resolved gaps up to 2.8 Mb. The genome alignment reveals not only high sequence divergences but also pervasive intra- and inter-chromosomal sequence re-arrangements between the two species, which are plausible culprits for the observed suppression. Comparison of recombination boundary sequences suggests that recombination in the hybrid requires extensive sequence homology, which is rarely seen between the two genomes. The new genomes and genomic libraries form invaluable resources for studying genome evolution, hybrid incompatibilities and sex evolution for this pair of model species.

Caenorhabditis briggsae has emerged as a model for comparative biology against model organism C. elegans. Most of its cell fate specifications are completed during embryogenesis whereas its cell growth is achieved mainly in larval stages. The molecular mechanism underlying the drastic developmental changes is poorly understood. To gain insights into the molecular changes between the two stages, we compared the proteomes between the two stages using iTRAQ. We identified a total of 2,791 proteins in the C. briggsae embryos and larvae, 247 of which undergo up- or down-regulation between the two stages. The proteins that are upregulated in the larval stages are enriched in the Gene Ontology categories of energy production, protein translation, and cytoskeleton; whereas those upregulated in the embryonic stage are enriched in the categories of chromatin dynamics and posttranslational modification, suggesting a more active chromatin modification in the embryos than in the larva. Perturbation of a subset of chromatin modifiers followed by cell lineage analysis suggests their roles in controlling cell division pace. Taken together, we demonstrate a general molecular switch from chromatin modification to metabolism during the transition from C. briggsae embryonic to its larval stages using iTRAQ approach. The switch might be conserved across metazoans.

This study examined the benefits of pharmaceutical pictograms for improving comprehension of medication information for older people. Fifty Hong Kong Chinese older people completed a medical information comprehension task for five drugs. Participants in the control group were presented with text labels while those in the experimental group were given the text labels plus supplementary pharmaceutical pictograms, and then all reported their understanding of the medication information conveyed. Lower educated older people had poorer understanding of medication information. The addition of pharmaceutical pictograms significantly improved the comprehension of medication information for older people. The majority of older people tested with pictograms favored adding pictograms to text and thought the pictograms were useful for conveying medical information rather than using written text alone. The findings suggested that pharmaceutical and health care professionals should include pharmaceutical pictograms on labels to better convey instructions on medication to older people., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Metazoan development demands not only precise cell fate differentiation but also accurate timing of cell division to ensure proper development. How cell divisions are temporally coordinated during development is poorly understood. Caenorhabditis elegans embryogenesis provides an excellent opportunity to study this coordination due to its invariant development and widespread division asynchronies. One of the most pronounced asynchronies is a significant delay of cell division in two endoderm progenitor cells, Ea and Ep, hereafter referred to as E2, relative to its cousins that mainly develop into mesoderm organs and tissues. To unravel the genetic control over the endoderm-specific E2 division timing, a total of 822 essential and conserved genes were knocked down using RNAi followed by quantification of cell cycle lengths using in toto imaging of C. elegans embryogenesis and automated lineage. Intriguingly, knockdown of numerous genes encoding the components of general transcription pathway or its regulatory factors leads to a significant reduction in the E2 cell cycle length but an increase in cell cycle length of the remaining cells, indicating a differential requirement of transcription for division timing between the two. Analysis of lineage-specific RNA-seq data demonstrates an earlier onset of transcription in endoderm than in other germ layers, the timing of which coincides with the birth of E2, supporting the notion that the endoderm-specific delay in E2 division timing demands robust zygotic transcription. The reduction in E2 cell cycle length is frequently associated with cell migration defect and gastrulation failure. The results suggest that a tissue-specific transcriptional activation is required to coordinate fate differentiation, division timing, and cell migration to ensure proper development., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)