34 results found
Katsanos D, Ferrando-Marco M, Razzaq I, et al., 2021, Gene expression profiling of epidermal cell types in C. elegans using Targeted-DamID., Development
The epidermis of Caenorhabditis elegans is an essential tissue for survival as it contributes to the formation of the cuticle barrier, as well as facilitates developmental progression and animal growth. Most of the epidermis consists of the hyp7 hypodermal syncytium, the nuclei of which are largely generated by the seam cells that exhibit stem cell-like behaviour during development. How the seam cell progenitors differ transcriptionally from the differentiated hypodermis is poorly understood. Here, we introduce Targeted DamID (TaDa) in C. elegans as a method for identifying genes expressed within a tissue of interest without cell isolation. We show that TaDa signal enrichment profiles can be used to identify genes transcribed in the epidermis and use this method to resolve differences in gene expression between the seam cells and the hypodermis. We finally predict and functionally validate new transcription and chromatin factors acting in seam cell development. These findings provide insights into cell-type-specific gene expression profiles likely associated with epidermal cell fate patterning.
Koneru SL, Hintze M, Katsanos D, et al., 2021, Cryptic genetic variation in a heat shock protein modifies the outcome of a mutation affecting epidermal stem cell development in C. elegans, NATURE COMMUNICATIONS, Vol: 12, ISSN: 2041-1723
Hintze M, Katsanos D, Shahrezaei V, et al., 2021, Phenotypic Robustness of Epidermal Stem Cell Number in C. elegans Is Modulated by the Activity of the Conserved N-acetyltransferase nath-10/NAT10, FRONTIERS IN CELL AND DEVELOPMENTAL BIOLOGY, Vol: 9, ISSN: 2296-634X
Koneru SL, Quah FX, Ghose R, et al., 2021, A role for the fusogen eff-1 in epidermal stem cell number robustness in Caenorhabditis elegans, SCIENTIFIC REPORTS, Vol: 11, ISSN: 2045-2322
Kalita R, Flanagan W, Lightley J, et al., 2021, Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast
<jats:title>Abstract</jats:title><jats:p>We present a robust, low-cost single-shot implementation of differential phase microscopy utilising a polarisation-sensitive camera to simultaneously acquire 4 images from which the phase gradients and quantitative phase image can be calculated. This polarisation-resolved differential phase contrast (pDPC) microscopy technique can be interleaved with single-shot imaging polarimetry.</jats:p>
Grover M, Barkoulas M, 2021, C. elegans as a new tractable host to study infections by animal pathogenic oomycetes, PLoS Pathogens, Vol: 17, Pages: 1-7, ISSN: 1553-7366
Fasseas M, Grover M, Drury F, et al., 2021, Chemosensory neurons modulate the response to oomycete recognition in Caenorhabditis elegans, Cell Reports, Vol: 34, ISSN: 2211-1247
Understanding how animals detect and respond to pathogen threats is central to dissecting mechanisms of host immunity. The oomycetes represent a diverse eukaryotic group infecting various hosts from nematodes to humans. We have previously shown that Caenorhabditis elegans mounts a defense response consisting of the induction of chitinase-like (chil) genes in the epidermis to combat infection by its natural oomycete pathogen Myzocytiopsis humicola. We provide here evidence that C. elegans can sense the oomycete by detecting an innocuous extract derived from animals infected with M. humicola. The oomycete recognition response (ORR) leads to changes in the cuticle and reduction in pathogen attachment, thereby increasing animal survival. We also show that TAX-2/TAX-4 function in chemosensory neurons is required for the induction of chil-27 in the epidermis in response to extract exposure. Our findings highlight that neuron-to-epidermis communication may shape responses to oomycete recognition in animal hosts.
Adikes RC, Kohrman AQ, Martinez MAQ, et al., 2020, Visualizing the metazoan proliferation-quiescence decision in vivo, ELIFE, Vol: 9, ISSN: 2050-084X
Katsanos D, Barkoulas M, 2020, Targeted DamID in C. elegans reveals a role for LIN-22 and NHR-25 in epidermal cell differentiation
<jats:title>Abstract</jats:title><jats:p>Transcription factors are key players in gene networks controlling cell fate specification during development. In multicellular organisms, they can display complex patterns of expression and binding to their targets, which necessitates tissue-specific characterisation of transcription factor-target interactions. Here, we focus on <jats:italic>C. elegans</jats:italic> seam cell development, which is used as a model of robust epidermal stem cell patterning. Despite our knowledge of multiple transcription factors playing a role in epidermal development, the composition of the gene network underlying cell fate patterning remains largely unknown. We introduce Targeted DamID (TaDa) that allows tissue-specific transcription factor target identification in intact <jats:italic>C. elegans</jats:italic> animals without cell isolation. We employ this method to recover putative targets in the epidermis for two transcription factors, the HES1 homologue LIN-22 and the NR5A1/2 nuclear hormone receptor NHR-25. Using single-molecule FISH (smFISH), we validate TaDa predictions and reveal a role for these transcription factors in promoting cell differentiation, as well as an unusual link between a HES factor and the Wnt signalling pathway.</jats:p><jats:p>Our results expand our understanding of the epidermal gene network and highlight the power of TaDa to dissect the architecture of tissue-specific gene regulatory networks.</jats:p>
Hintze M, Koneru SL, Gilbert SPR, et al., 2020, A Cell Fate Switch in the Caenorhabditis elegans Seam Cell Lineage Occurs Through Modulation of the Wnt Asymmetry Pathway in Response to Temperature Increase, GENETICS, Vol: 214, Pages: 927-939, ISSN: 0016-6731
Reddy KC, Dror T, Underwood RS, et al., 2019, Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans, PLoS Pathogens, Vol: 15, Pages: 1-28, ISSN: 1553-7366
Immune genes are under intense, pathogen-induced pressure, which causes these genes to diversify over evolutionary time and become species-specific. Through a forward genetic screen we recently described a C. elegans-specific gene called pals-22 to be a repressor of “Intracellular Pathogen Response” or IPR genes. Here we describe pals-25, which, like pals-22, is a species-specific gene of unknown biochemical function. We identified pals-25 in a screen for suppression of pals-22 mutant phenotypes and found that mutations in pals-25 suppress all known phenotypes caused by mutations in pals-22. These phenotypes include increased IPR gene expression, thermotolerance, and immunity against natural pathogens, including Nematocida parisii microsporidia and the Orsay virus. Mutations in pals-25 also reverse the reduced lifespan and slowed growth of pals-22 mutants. Transcriptome analysis indicates that pals-22 and pals-25 control expression of genes induced not only by natural pathogens of the intestine, but also by natural pathogens of the epidermis. Indeed, in an independent forward genetic screen we identified pals-22 as a repressor and pals-25 as an activator of epidermal defense gene expression. In summary, the species-specific pals-22 and pals-25 genes act as a switch to regulate a program of gene expression, growth, and defense against diverse natural pathogens in C. elegans.
Barkoulas M, Osman G, Fasseas M, et al., 2018, Natural infection of C. elegans by an oomycete reveals a new pathogen-specific immune response, Current Biology, Vol: 28, Pages: 640-648.e5, ISSN: 1879-0445
In its natural habitat, the nematode Caenorhabditis elegans encounters a plethora of other organisms, including many that are pathogenic [ 1 ; 2]. The study of interactions between C. elegans and various pathogens has contributed to characterizing key mechanisms of innate immunity [ 2; 3 ; 4]. However, how C. elegans recognizes different pathogens to mount pathogen-specific immune responses remains still largely unknown [ 3; 5; 6; 7 ; 8]. Expanding the range of known C. elegans-infecting pathogens and characterizing novel pathogen-specific immune responses are key steps toward answering this question. We report here that the oomycete Myzocytiopsis humicola is a natural pathogen of C. elegans, and we describe its infection strategy. We identify a new host immune response to pathogen exposure that involves induction of members of a previously uncharacterized gene family encoding chitinase-like (CHIL) proteins. We demonstrate that this response is highly specific against M. humicola and antagonizes the infection. We propose that CHIL proteins may diminish the ability of the oomycete to infect by hindering pathogen attachment to the host cuticle. This work expands our knowledge of natural eukaryotic pathogens of C. elegans and introduces a new pathosystem to address how animal hosts recognize and respond to oomycete infections.
Katsanos D, Koneru SL, Mestek Boukhibar L, et al., 2017, Stochastic loss and gain of symmetric divisions in the C. elegans epidermis perturbs robustness of stem cell number., PLoS Biology, Vol: 15, ISSN: 1544-9173
Biological systems are subject to inherent stochasticity. Nevertheless, development is remarkably robust, ensuring the consistency of key phenotypic traits such as correct cell numbers in a certain tissue. It is currently unclear which genes modulate phenotypic variability, what their relationship is to core components of developmental gene networks, and what is the developmental basis of variable phenotypes. Here, we start addressing these questions using the robust number of Caenorhabditis elegans epidermal stem cells, known as seam cells, as a readout. We employ genetics, cell lineage tracing, and single molecule imaging to show that mutations in lin-22, a Hes-related basic helix-loop-helix (bHLH) transcription factor, increase seam cell number variability. We show that the increase in phenotypic variability is due to stochastic conversion of normally symmetric cell divisions to asymmetric and vice versa during development, which affect the terminal seam cell number in opposing directions. We demonstrate that LIN-22 acts within the epidermal gene network to antagonise the Wnt signalling pathway. However, lin-22 mutants exhibit cell-to-cell variability in Wnt pathway activation, which correlates with and may drive phenotypic variability. Our study demonstrates the feasibility to study phenotypic trait variance in tractable model organisms using unbiased mutagenesis screens.
Barkoulas M, Vargas Velazquez AM, Peluffo AE, et al., 2016, Evolution of New cis-Regulatory Motifs Required for Cell-Specific Gene Expression in Caenorhabditis, PLOS Genetics, Vol: 12, ISSN: 1553-7390
Patterning of C. elegans vulval cell fates relies on inductive signaling. In this induction event, a single cell, the gonadal anchor cell, secretes LIN-3/EGF and induces three out of six competent precursor cells to acquire a vulval fate. We previously showed that this developmental system is robust to a four-fold variation in lin-3/EGF genetic dose. Here using single-molecule FISH, we find that the mean level of expression of lin-3 in the anchor cell is remarkably conserved. No change in lin-3 expression level could be detected among C. elegans wild isolates and only a low level of change-less than 30%-in the Caenorhabditis genus and in Oscheius tipulae. In C. elegans, lin-3 expression in the anchor cell is known to require three transcription factor binding sites, specifically two E-boxes and a nuclear-hormone-receptor (NHR) binding site. Mutation of any of these three elements in C. elegans results in a dramatic decrease in lin-3 expression. Yet only a single E-box is found in the Drosophilae supergroup of Caenorhabditis species, including C. angaria, while the NHR-binding site likely only evolved at the base of the Elegans group. We find that a transgene from C. angaria bearing a single E-box is sufficient for normal expression in C. elegans. Even a short 58 bp cis-regulatory fragment from C. angaria with this single E-box is able to replace the three transcription factor binding sites at the endogenous C. elegans lin-3 locus, resulting in the wild-type expression level. Thus, regulatory evolution occurring in cis within a 58 bp lin-3 fragment, results in a strict requirement for the NHR binding site and a second E-box in C. elegans. This single-cell, single-molecule, quantitative and functional evo-devo study demonstrates that conserved expression levels can hide extensive change in cis-regulatory site requirements and highlights the evolution of new cis-regulatory elements required for cell-specific gene expression.
Grimbert S, Tietze K, Barkoulas M, et al., 2016, Anchor cell signaling and vulval precursor cell positioning establish a reproducible spatial context during C. elegans vulval induction, Developmental Biology, Vol: 416, Pages: 123-135, ISSN: 1095-564X
How cells coordinate their spatial positioning through intercellular signaling events is poorly understood. Here we address this topic using Caenorhabditis elegans vulval patterning during which hypodermal vulval precursor cells (VPCs) adopt distinct cell fates determined by their relative positions to the gonadal anchor cell (AC). LIN-3/EGF signaling by the AC induces the central VPC, P6.p, to adopt a 1° vulval fate. Exact alignment of AC and VPCs is thus critical for correct fate patterning, yet, as we show here, the initial AC-VPC positioning is both highly variable and asymmetric among individuals, with AC and P6.p only becoming aligned at the early L3 stage. Cell ablations and mutant analysis indicate that VPCs, most prominently 1° cells, move towards the AC. We identify AC-released LIN-3/EGF as a major attractive signal, which therefore plays a dual role in vulval patterning (cell alignment and fate induction). Additionally, compromising Wnt pathway components also induces AC-VPC alignment errors, with loss of posterior Wnt signaling increasing stochastic vulval centering on P5.p. Our results illustrate how intercellular signaling reduces initial spatial variability in cell positioning to generate reproducible interactions across tissues.
Matus DQ, Lohmer LL, Kelley LC, et al., 2015, Invasive cell fate requires G1 Cell-cycle arrest and histone deacetylase-mediated changes in gene expression, Developmental Cell, Vol: 35, Pages: 162-174, ISSN: 1534-5807
Despite critical roles in development and cancer, the mechanisms that specify invasive cellular behavior are poorly understood. Through a screen of transcription factors in Caenorhabditis elegans, we identified G1 cell-cycle arrest as a precisely regulated requirement of the anchor cell (AC) invasion program. We show that the nuclear receptor nhr-67/tlx directs the AC into G1 arrest in part through regulation of the cyclin-dependent kinase inhibitor cki-1. Loss of nhr-67 resulted in non-invasive, mitotic ACs that failed to express matrix metalloproteinases or actin regulators and lack invadopodia, F-actin-rich membrane protrusions that facilitate invasion. We further show that G1 arrest is necessary for the histone deacetylase HDA-1, a key regulator of differentiation, to promote pro-invasive gene expression and invadopodia formation. Together, these results suggest that invasive cell fate requires G1 arrest and that strategies targeting both G1-arrested and actively cycling cells may be needed to halt metastatic cancer.
Boukhibar LM, Barkoulas M, 2015, The developmental genetics of biological robustness, Annals of Botany, Vol: 117, Pages: 699-707, ISSN: 1095-8290
Background Living organisms are continuously confronted with perturbations, such as environmental changes that include fluctuations in temperature and nutrient availability, or genetic changes such as mutations. While some developmental systems are affected by such challenges and display variation in phenotypic traits, others continue consistently to produce invariable phenotypes despite perturbation. This ability of a living system to maintain an invariable phenotype in the face of perturbations is termed developmental robustness. Biological robustness is a phenomenon observed across phyla, and studying its mechanisms is central to deciphering the genotype–phenotype relationship. Recent work in yeast, animals and plants has shown that robustness is genetically controlled and has started to reveal the underlying mechinisms behind it.Scope and Conclusions Studying biological robustness involves focusing on an important property of developmental traits, which is the phenotypic distribution within a population. This is often neglected because the vast majority of developmental biology studies instead focus on population aggregates, such as trait averages. By drawing on findings in animals and yeast, this Viewpoint considers how studies on plant developmental robustness may benefit from strict definitions of what is the developmental system of choice and what is the relevant perturbation, and also from clear distinctions between gene effects on the trait mean and the trait variance. Recent advances in quantitative developmental biology and high-throughput phenotyping now allow the design of targeted genetic screens to identify genes that amplify or restrict developmental trait variance and to study how variation propagates across different phenotypic levels in biological systems. The molecular characterization of more quantitative trait loci affecting trait variance will provide further insights into the evolution of genes modulating developmental robustness. The
Felix M-A, Barkoulas M, 2015, Pervasive robustness in biological systems, NATURE REVIEWS GENETICS, Vol: 16, Pages: 483-496, ISSN: 1471-0056
van Zon JS, Kienle S, Huelsz-Prince G, et al., 2015, Cells change their sensitivity to an EGF morphogen gradient to control EGF-induced gene expression., Nature Communications, Vol: 6, Pages: 7053-7053, ISSN: 2041-1723
How cells in developing organisms interpret the quantitative information contained in morphogen gradients is an open question. Here we address this question using a novel integrative approach that combines quantitative measurements of morphogen-induced gene expression at single-mRNA resolution with mathematical modelling of the induction process. We focus on the induction of Notch ligands by the LIN-3/EGF morphogen gradient during vulva induction in Caenorhabditis elegans. We show that LIN-3/EGF-induced Notch ligand expression is highly dynamic, exhibiting an abrupt transition from low to high expression. Similar transitions in Notch ligand expression are observed in two highly divergent wild C. elegans isolates. Mathematical modelling and experiments show that this transition is driven by a dynamic increase in the sensitivity of the induced cells to external LIN-3/EGF. Furthermore, this increase in sensitivity is independent of the presence of LIN-3/EGF. Our integrative approach might be useful to study induction by morphogen gradients in other systems.
Hay AS, Pieper B, Cooke E, et al., 2014, Cardamine hirsuta: a versatile genetic system for comparative studies, PLANT JOURNAL, Vol: 78, Pages: 1-15, ISSN: 0960-7412
Barkoulas M, van Zon JS, Milloz J, et al., 2013, Robustness and Epistasis in the C-elegans Vulval Signaling Network Revealed by Pathway Dosage Modulation, DEVELOPMENTAL CELL, Vol: 24, Pages: 64-75, ISSN: 1534-5807
Felix M-A, Barkoulas M, 2012, Robustness and flexibility in nematode vulva development, TRENDS IN GENETICS, Vol: 28, Pages: 185-195, ISSN: 0168-9525
Prasad K, Grigg SP, Barkoulas M, et al., 2011, Arabidopsis PLETHORA Transcription Factors Control Phyllotaxis, CURRENT BIOLOGY, Vol: 21, Pages: 1123-1128, ISSN: 0960-9822
Hoyos E, Kim K, Milloz J, et al., 2011, Quantitative Variation in Autocrine Signaling and Pathway Crosstalk in the Caenorhabditis Vulval Network, CURRENT BIOLOGY, Vol: 21, Pages: 527-538, ISSN: 0960-9822
Bilsborough GD, Runions A, Barkoulas M, et al., 2011, Model for the regulation of Arabidopsis thaliana leaf margin development, PROCEEDINGS OF THE NATIONAL ACADEMY OF SCIENCES OF THE UNITED STATES OF AMERICA, Vol: 108, Pages: 3424-3429, ISSN: 0027-8424
Scarpella E, Barkoulas M, Tsiantis M, 2010, Control of Leaf and Vein Development by Auxin, COLD SPRING HARBOR PERSPECTIVES IN BIOLOGY, Vol: 2, ISSN: 1943-0264
Canales C, Barkoulas M, Galinha C, et al., 2010, Weeds of change: Cardamine hirsuta as a new model system for studying dissected leaf development, JOURNAL OF PLANT RESEARCH, Vol: 123, Pages: 25-33, ISSN: 0918-9440
Jenkins H, Tattersall A, Barkoulas M, et al., 2009, Dissected leaf development in Cardamine hirsuta, 16th Annual Conference of the International-Society-of-Development-Biologists, Publisher: ELSEVIER SCIENCE BV, Pages: S55-S55, ISSN: 0925-4773
Barkoulas M, Bilsborough G, 2009, En passant Family ties, NATURE, Vol: 458, Pages: 1212-1212, ISSN: 0028-0836
Barkoulas M, Hay A, Kougioumoutzi E, et al., 2008, A developmental framework for dissected leaf formation in the Arabidopsis relative Cardamine hirsuta, NATURE GENETICS, Vol: 40, Pages: 1136-1141, ISSN: 1061-4036
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