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[
East Asia Worm Meeting,
2010]
The study of protein interactions in the physiological context is a valuable tool for the investigation of biological regulatory mechanisms. Here, we are interested in the interactions between molecular motors and small adaptor proteins that might have a regulatory function. For example, our previous study shows a regulatory function of an active zone protein liprin-alpha (SYD-2) on UNC-104 (KIF1A). However, factors that control UNC-104/SYD-2 interactions are not known. Similarly, it is known that UNC-16 (JIP3) acts as a scaffold for kinesin-1 (KLC-2) regulating the transport of synaptic vesicles; while it is unknown which factors regulate the UNC-16/KLC-2 interaction. To investigate factors that activate or suppress the interactions between these kinesins and their adaptor proteins, we use a novel method BiFC (Bimolecular fluorescence complementation) in combination with forward and reverse mutagenesis. Here, we fuse fluorescent protein complementary fragments (hybrids of fluorophores) to each protein in the complex. In detail, we express the N-terminal half of a YFP fused to one protein and at the same time the C-terminal half fused to another protein in the complex. As it is known that YFP-N can complement with YFP-C to make a functional YFP, this method enables us to investigate physical interactions between two proteins in the living worm. Specifically, we use a native, pan-neuronal promoter pUnc104 to drive the expression of the following proteins in the nervous system of C. elegans: YN::SYD-2/UNC-104::YC, UNC-16::YN/KLC-2::YC as well as UNC-104::YN/UNC-104::YC. As a positive control we use bJUN::YN and bFOS::YC that are known to express and strongly interact in the nucleus. The investigation of UNC-104/UNC-104 interaction is of special interest as in the literature it is highly discussed whether this kinesin-3 exists as a monomer or dimer when activated or deactivated. The next important step in this research will be to use forward (EMS) and reverse (genome-wide RNAi screen) mutagenesis to identify genes that might disrupt the interaction between the BiFC pairs. Therefore we would be able to investigate novel regulators in the kinesin/adaptor protein complexes.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
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.
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[
International Worm Meeting,
2005]
Fluorescent Timer, a proprietary fluorescent reporter from Clontech, has the unique property of shifting its emission fluorescence from green (480nm) to red (583nm) in a time/intramolecular reaction dependent manner. Red fluorescence was documented to occur two to four hours after initial green fluorescence is detected (Terskikh et al. 2000). I have attempted to use this reporter to assay for message turnover under the control of various upstream regulatory regions. Regulatory regions were selected on the basis of showing limited temporal expression during early development and/or in various tissues to test the efficacy of the reporter as a general assay of transcriptional regulation. Regulatory regions were linked to the reporter via PCR fusion (Hobert, O. 2002). All transgenic lines containing upstream regulatory regions fused to the raw reporter failed to produce fluorescence. Upon the addition of a 3UTR and the insertion of two adenosine residues directly upstream of the translational start site, 10/14 transgenic lines displayed fluorescence. Embryonic expression, which was observed using the same upstream regulatory regions fused to GFP, was not observed in any of the Fluorescent Timer reporter lines constructed. To possibly counter to effects of cytoplasmic toxicity of the Fluorescent Timer protein, and to concentrate the signal to the nucleus, I fused a 5NLS to the construct. Unexpectedly, only 3/14 lines displayed fluorescence leading to the assumption that Fluorescent Timer may be more toxic when concentrated to the nucleus. This result seems to agree with the results that no Fluorescent Timer expression was observed during early development possibly due to toxicity of the construct. Fluorescent Timer may be a useful system for the assay of temporal regulation of transcription during later stages of C. elegans development. References: Terskikh A, Fradkov A, Ermakova G, Zaraisky A, Tan P, Kajava AV, Zhao X, Lukyanov S, Matz M, Kim S, Weissman I, Siebert P. 2000, "Fluorescent timer": protein that changes color with time. Science. Nov 24;290(5496):1585-8. Hobert O. 2002, PCR fusion-based approach to create reporter gene constructs for expression analysis in transgenic C. elegans. Biotechniques. Apr;32(4):728-30.