Green, Rebecca et al. (2019) International Worm Meeting "4D High Content Imaging and Automated Phenotypic Profiling of C. elegans Embryogenesis."

Ochoa, Stacy, Wang, Shaohe, Green, Rebecca, Desai, Arshad, Hendel, Jeff, Chow, Tiffany, Oegema, Karen, Khaliullin, Renat, Zhao, Zhiling

[International Worm Meeting, 2019]

An important challenge is to functionally classify the ~2000 genes (>1400 conserved) that control cell-fate specification and morphogenesis during embryogenesis. Here, we perform a 4D high-content screen by filming embryogenesis using two custom-engineered C. elegans reporter strains, following individual RNAi-based knockdown (>20,000 individual movies). We monitor (1) changes in cell fate specification, by dynamically tracking fluorescently labeled endoderm, mesoderm and ectoderm nuclei, and (2) morphogenic changes during epithelial and neuronal development by monitoring tissue position and tissue shape. Consistent and timely analysis of 20,000 movies requires automation, however, the range and complexity of 4D developmental phenotypes are not easily captured by existing automated methods. To address this challenge, we manually curated a pilot set of 500 genes (>7000 movies) and used this reference to guide the development of custom automated analysis algorithms; this effort ensured that our final automated analysis method captured observed phenotypes across a spectrum of developmental defects. For each RNAi condition, our automated analysis yields phenotypic signatures consisting of >100 continuous parameters. To evaluate the phenotypic similarity between RNAi conditions, we measure the distance between phenotypes in continuous space. To correct for the fact that a strict measure of Euclidean distance penalizes genes with more severe phenotypes, we measure the angle between the average phenotypes for the two conditions (phenotypic angle of deviation; PAD). Finally, we optimized the set of parameters used for automated comparison by assessing performance of the algorithm on a manually-annotated set of phenotypic groups. Our resulting automated method effectively identifies genes whose knockdown leads to similar phenotypes; this allows partitioning of genes into functional groups that are predicted to reflect developmental pathways and will yield a systems-level view of embryonic development. This work represents the first fully automated high-content screen of an intact developing organism and is the most complex morphological profiling effort to date.

Green, Rebecca et al. (2015) International Worm Meeting "­Decoding Embryonic Developmental Pathways Using 4D-High Content Imaging of C. elegans embryos."

Zhao, Zhiling, Desai, Arshad, Wang, Shaohe, Ochoa, Stacy, Biggs, Ronald, Gerson-Gurwitz, Adina, Khaliullin, Renat, Green, Rebecca, Oegema, Karen

[International Worm Meeting, 2015]

­Embryogenesis is a complex process requiring coordination of cell division, signaling, migration, differentiation, and death. Systematically defining the genetic pathways that drive these morphogenetic events during embryogenesis is an important current challenge. Our goal is to construct a comprehensive functional network map of essential developmental genes for the model metazoan, C. elegans. To this end, we have developed a 4D-high-content screening based approach to functionally classify ~2600 developmental genes, using two-specifically engineered marker strains that readout defects in (1) germ layer specification and positioning and (2) cell shape changes and cell migration during morphogenesis. Following RNAi of targets, we image C. elegans embryos throughout the developmental time course (~10hrs) using a CV1000 spinning disk confocal high-content imaging system, which enables collection of developmental data for 50-100 embryos in a single experiment. To date, we have completed a pilot set of >500 genes. Among these, we have recovered expected phenotypes for well described developmental genes as well as severe developmental phenotypes for many uncharacterized genes, validating our overall experimental approach. This pilot data set is being used to develop custom data management algorithms (cropping, orienting, and indexing embryos) and data analysis protocols, including: manual and automated scoring of phenotypic features (Imaris and custom). Using this approach, each individual embryogenesis movie is scored and genes are clustered according to phenotypic profiles. When complete, this will be the first systems-level view of embryonic development in a complex multicellular organism. We anticipate such an effort will translate to higher organisms and help reveal the genetic basis for congenital defects, such as neural tube, craniofacial, and ventral body wall closure abnormalities.

Memar, Nadin et al. (2009) International Worm Meeting "A view on the embryogenesis of the Caenorhabditis Family."

Schnabel, Ralf, Memar, Nadin, Martin, Katharina

[International Worm Meeting, 2009]

The molecular differences of the four Caenorhabditis species C. elegans, C. briggsae, C. remanei and C. brenneri are currently of great interest, however little is known about development. Zhao et al. (2008) reported an automatic lineage of C. briggsae and came - based mostly on the cleavage pattern and cell positions - to the conclusion that the embryogenesis of the two species is very similar. We now present detailed 4D analyses of the species including the terminal differentiation patterns. All analyses including bioinformatical quantifications of cell behaviour show a huge similarity between those species. Immunochemical analyses of the tissue distributions only reveal a difference in the intestinal differentiation of C. brenneri. Interestingly hybrid embryos always appear to fail in different ways in embryogenesis.

Weinreb, A. et al. (2017) International Worm Meeting "Analysis of activity-dependent synaptogenesis in the nematode Caenorhabditis elegans."

Bessereau, JL., Weinreb, A., Jospin, M.

[International Worm Meeting, 2017]

In various systems, activity of neurons or muscle leads to various forms of plasticity, thus shaping network connectivity and regulating synaptic strength. In C. elegans, activity was demonstrated in a few systems to play a role in synapse formation and function. We chose to analyze the development of the 3 cholinergic SAB motor neurons that are innervating the head muscles. In this system, electrical silencing of the muscle cells during development was demonstrated to regulate SAB morphology (Zhao and Nonet, 2000). Using fluorescently-tagged acetylcholine receptors (AChR), we observed SAB overgrowth and ectopic synapse formation in unc-13 and unc-18 mutant worms in which neuromuscular transmission was disrupted. We could confirm that this effect is not due to the loss of movement because there is no SAB overgrowth in the unc-54 myosin mutants that are paralyzed. To silence the electrical activity of muscle cells, we specifically expressed in muscles the Drosophila HisCl1 histamine-gated chloride channel and the TWK-18 temperature-dependent potassium channel. In both conditions, inhibition of muscle cell activity causes SAB overgrowth, suggesting that a retrograde factor(s) controls SAB development. We could further pinpoint a critical developmental window at the L1 stage during which SAB development is plastic. In addition, we demonstrated that chronic - but not acute - increase of synaptic transmission through acetylcholinesterase inhibition leads to a decrease in the number of synaptic AChRs, suggesting an activity-dependent regulation of AChR number during development. Through a transcriptomic approach, we expect to find genes involved in the overgrowth of the SAB and the regulation of AChR number. We are using RNA-Seq to detect genes differentially expressed upon electrical manipulation of the muscle cells. In parallel, we are using the tools that we developed to better define the conditions leading to SAB overgrowth and AChR downregulation, as well as testing a number of candidate genes. References: Zhao, H., and Nonet, M.L. (2000). A retrograde signal is involved in activity-dependent remodeling at a C. elegans neuromuscular junction. Development 127, 1253-1266.

Shuo Luo et al. (2005) International Worm Meeting "Dissection of the retrograde signal at C. elegans neuromuscular junctions"

Michael L Nonet, Shuo Luo

[International Worm Meeting, 2005]

Retrograde signals play important roles in regulating synapse plasticity during neural development. In the fruit fly D. melanogaster, the retrograde signal TGF modulates presynaptic morphology as well as synaptic transmission at both neuromuscular junctions (NMJs) and central synapses. In the nematode C. elegans, retrograde signal(s) also regulate the morphology of developing SAB head motor neurons (Zhao & Nonet, Development 127: 1253, 2000). Here we tested whether TGF-pathway could contribute to the retrograde signaling operating at SAB synapses. We examined a variety of TGF- pathway mutants for defects in SAB development but none exhibited SAB morphology defects. In addition, when synaptic transmission was disrupted in these mutants, all of them developed over-sprouted SAB axons, a phenotype similar to what has been observed in TGF normal animals. We conclude the TGF pathway does not play major roles in activity-dependent axon sprouting at SAB NMJs in worms. My current effort is to screen for the molecular components of retrograde signal at C. elegans NMJs. Various techniques including RNAi and chemical mutagenesis are being employed.

Herrera, R. Antonio et al. (2009) International Worm Meeting "Laser-Capture Microdissection and Microarray Profiling of C. elegans Male Tail Tip Morphogenesis."

Fitch, David H. A., S, Sindhoora, Herrera, R. Antonio

[International Worm Meeting, 2009]

Regulation of C. elegans male tail tip morphogenesis involves the input of several distinct molecular pathways. The heterochronic [1], Wnt signaling [2], Hox patterning [4] and sex determination [3] pathways each contribute to the proper development of the male tail tip syncytium. Mutations in certain genes result in unretracted adult male tail tips. Our lab and others have confirmed the intersection between these molecular pathways by examining the expression of transgenic reporters in various mutant backgrounds [3]. Our current understanding of the genetic network is limited, as many of these interactions are indirect. To identify candidate genes that are involved in tail tip retraction, we are performing a microarray analysis of gene expression in tail tips isolated from synchronized males and hermaphrodites prior to male L4 tail tip morphogenesis. We are using laser-capture microdissection to obtain tail tips, from which total RNA is isolated for hybridization onto Affymetrix C. elegans GeneChips. The tail tip lends itself particularly well to analysis of post-embryonic gene expression in specific somatic cells because the four tail tip cells can be removed by a single slice perpendicular to the anterior-posterior axis. Work is in progress to identify genes that are differentially expressed and/or change in expression profile from late L3 to middle L4. To confirm their role in morphogenesis, we will examine tail tip phenotypes in RNAi knockdowns or mutants of these genes. [1] Del Rio-Albrechtsen et al. 2006, Dev. Biol. 297:74. [2] Zhao et al. 2002, Development 129:1497. [3] Mason et al. 2008, Development 135:2373. [4] see MD Nelson Abstract.

Ajitha Pillai et al. (2005) International Worm Meeting "Evolution of nematode ASABF and cecropin-type antimicrobial peptides"

Yusuke Kato, Ajitha Pillai, Hong Zhang

[International Worm Meeting, 2005]

Many organisms including nematodes produce antimicrobial peptides, which are selectively toxic to microbes, for defense against microbial infection. The antimicrobial peptides are categorized based on chemical structure (alpha-helical, CS alpha beta, etc). Although the antimicrobial peptides whose chemical structure is similar can be found in evolutionarily distant organisms, their phylogenetic relationship is often ambiguous due to higher sequence divergence caused by competitive evolution against pathogens. Previously, we reported the alpha-helical antimicrobial peptide, nematode cecropins, as positively induced factors by bacterial injection in the pig round worm, Ascaris suum. Peptides similar to nematode cecropins have been reported in insects (insect cecropins) and tunicates (styelins). Although insect cecropins and styelins are similar near their secretory signal-mature peptide junction, the C-terminal acidic pro-region is found only in styelins but not in insect cecropins [1]. We determined 9 precursors of nematode cecropins. All nematode cecropin precursors contained the C-terminal acidic pro-regions observed in styelins. In addition, the length of each region (secretory signal, mature peptide, and acidic pro-region) was almost identical between nematode cecropins and styelins. The criteria of sequence similarity, precursor organization, and regional length suggest that nematode, insect, and tunicate cecropin-type antimicrobial peptides could have diversified from a common ancestor, opposite to what we previously expected (Pillai et al., 2004 East Asia Meeting). Moreover, another nematode antimicrobial peptide, ASABF, is specifically similar to mollusk antimicrobial peptides, MGDs and myticins. These results suggest that immunity by these antimicrobial peptides observed in nematodes cannot be adaptive convergence but generated in the early stage of animal evolution and still function in some organisms. [1] Zhao et al. (1997) FEBS Lett. 412, 144.

Noriko Sasakawa et al. (2007) International Worm Meeting "Identification and analyses of mutants defective in the asymmetric T cell division."

Hitoshi Sawa, Noriko SASAKAWA, Hisako Takeshita

[International Worm Meeting, 2007]

Asymmetric cell division is an important mechanism to produce cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, the polarity of the T cell division is controlled by lin-17/frizzled and lin-44/wnt. In wild type, the anterior daughter (T.a) of the T cell produces hypodermal cells and the posterior one (T.p) makes neural cells including phasmid socket cells. In lin-17 mutants, both daughters produce hypodermal cells. To identify genes involved in asymmetric cell divisions, we screened for mutants that lack phasmid socket cells (the phenotype is called Psa for phasmid socket cell absent). We have identified 110 mutants of 53 different genes. To characterize these mutants, we are examining expression and localization of two genes that are regulated by Wnt signaling. In wild type, expression of tlp-1::GFP is stronger in the posterior T cell daughter (T.p) than the anterior one (T.a) after the asymmetric division (Zhao et al. 2002). We analyzed tlp-1::GFP expression in psa mutants of 21 different genes so far that have not cloned or characterized in other studies. 13 mutants showed abnormal expression pattern. 6 mutants showed symmetric expression while 9 showed weak or not expression. These mutants are probably defective in either polarity of the T cell or transcriptional regulation of the tlp-1 gene. To identify mutants defective in polarity of the T cell, we are analyzing localization of WRM-1::GFP in mutants that showed abnormal expression of tlp-1::GFP. In wild type, WRM-1::GFP is localized to the anterior cortex before and during division and to the posterior (T.p) nucleus after the division (Takeshita and Sawa 2005). So far, we observed abnormal localization of WRM-1 in 9 mutants, suggesting that these mutants are defective in the polarity of the T cell. We will continue the analyses to identify more genes involved in the T cell polarity.

Noriko Sasakawa et al. (2006) East Asia C. elegans Meeting "Identification and analyses of mutants defective in the asymmetric T cell division"

Hisako Takeshita, Noriko SASAKAWA, Hitoshi Sawa

[East Asia C. elegans Meeting, 2006]

Asymmetric cell division is an important mechanism to produce cellular diversity during development. In C. elegans, asymmetric divisions of many cells are regulated by Wnt signaling. In particular, the polarity of the T cell division is controlled by lin-17/frizzled and lin-44/wnt. In wild type, the anterior daughter (T.a) of the T cell produces hypodermal cells and the posterior one (T.p) makes neural cells including phasmid socket cells. In lin-17 mutants, both daughters produce hypodermal cells. To identify genes involved in asymmetric cell divisions, we screened for mutants that lack phasmid socket cells (the phenotype is called Psa for phasmid socket cell absent). We have identified 110 mutants of 55 different genes. To characterize these mutants, we are examining expression and localization of two genes that are regulated by Wnt signaling. In wild type, Expression of tlp-1::GFP is stronger in the posterior T cell daughter (T.p) than the anterior one (T.a) after the asymmetric division (Zhao et al. 2002). We analyzed tlp-1::GFP expression in psa mutants of 22 different genes so far that have not cloned or characterized in other studies. 15 mutants showed abnormal expression pattern. One mutants showed reversed expression (higher in T.a than in T.p), 7 showed symmetric expression, while 7 showed weak or not expression. These mutants are probably defective in either polarity of the T cell or transcriptional regulation of the tlp-1 gene. To identify mutants defective in polarity of the T cell, we are analyzing localization of WRM-1::GFP in mutants that showed abnormal expression of tlp-1::GFP. In wild type, WRM-1::GFP is localized to the anterior cortex before and during division and to the posterior (T.p) nucleus after the division (Takeshita and Sawa 2005). So far, we observed abnormal localization of WRM-1 in three mutants, suggesting that these mutants are defective in the polarity of the T cell. We will continue the analyses to identify more genes involved in the T cell polarity.

Blackwell, Alexander et al. (2021) International Worm Meeting "Studying chromatin regulation at single cell resolution during C. elegans postembryonic development"

Van den Heuvel, Sander, Gaarenstroom, Tessa, Blackwell, Alexander

[International Worm Meeting, 2021]

Despite containing identical genomes, developing cells differentiate into a plethora of diverse cell types. Distinct patterns of gene expression now form the basis for classifying different cell fates. How the combinatorial activity of transcription factors, chromatin regulators and histone modifications achieve the proper spatiotemporal patterns of gene expression is a major question in developmental biology. Biologists increasingly appreciate the need to investigate gene expression regulation at the single-cell level because much heterogeneity and complexity is lost when averaging across populations of cells. However, profiling chromatin at the single cell level is challenging due to limited input material. Chromatin immunocleavage with sequencing (ChIC-seq) is an efficient method to study chromatin modifications from low input samples. ChIC-seq utilises antibody targeted micrococcal nucleases, leading to controlled, binding-dependent enzymatic digestion of DNA. This releases short fragments which become preferentially incorporated during library preparation and enables high resolution mapping of genomic positions. Crucially, the absence of crosslinking and immunoprecipitation steps, required in less sensitive techniques such as ChIP-seq, leads to minimal material loss. Recently, ChIC-seq was used to profile histone modifications in single human cells [1]. Adapting ChIC-seq to profile histone modifications in C. elegans will provide a powerful tool for studying the epigenetic regulation of development. Here, we present progress in optimising ChIC-seq for profiling chromatin modifications at single-cell level across a developmental time-course in C. elegans. Specifically, we combine Cre/Lox lineage tracing with cell isolation and FACS procedures in order to isolate postembryonic mesoderm cells. Following prolonged quiescence, the mesoblast precursor resumes proliferation and produces fourteen muscle cells, two scavenger cells, and two migratory bipotent myoblasts over 24-hours. By profiling chromatin modifications at high temporal resolution, we aim to reveal regulatory processes controlling cellular proliferation and differentiation. This work will shed light on how epigenetic modifications contribute to cellular decision making in a living animal. [1] Ku WL, Nakamura K, Gao W, Cui K, Hu G, Tang Q, Ni B, Zhao K. (2019) Single-cell chromatin immunocleavage sequencing (scChIC-seq) to profile histone modification. Nature Methods, vol. 16, pages 323-325.