Biology subjects

Barkoulas, M.

Publications and source records attributed to Barkoulas, M..

15 recordsLinked to original sources

bioRxiv2026

The HSP-90 co-chaperone CDC-37 regulates oomycete immunity in C. elegans through a membrane-associated pseudokinase

Innate immune responses depend on rapid and tightly regulated signalling to ensure effective defence against invading pathogens. In Caenorhabditis elegans, the oomycete recognition response (ORR) is a transcriptional programme that provides protection against lethal infections by oomycetes, such as Myzocytiopsis humicola. The protein tyrosine kinase OLD-1 and the pseudokinase FLOR-1 are essential for activation of this immune response, however, the underlying molecular mechanism is poorly defined. Here, we show that FLOR-1 is regulated by the kinase-specific HSP- 90 co-chaperone CDC-37. Following tandem mass tag-based proteomics, we reveal that FLOR-1 interacts with CDC-37 and that disruption of the CDC-37/HSP-90 complex abolishes ORR gene induction. CDC-37 is required for membrane association of FLOR-1 and mediates its pathogen recognition-induced relocalisation from the plasma membrane to the cytosol, where downstream immune signalling is initiated. Surprisingly, the related active protein tyrosine kinase OLD-1 functions independently of CDC-37. Functional dissection of FLOR-1 localisation demonstrates that membrane-associated FLOR-1 is required for pathogen recognition, whereas cytosolic FLOR-1 is sufficient to activate ORR gene expression. Together, these findings uncover an unexpected role for the CDC-37/HSP-90 complex in regulating innate immunity likely through spatial control of a membrane-associated pseudokinase and reveal a chaperone-mediated mechanism that links pathogen detection to immune activation.

biochemistry↗

bioRxiv2026

Transcriptional feedback targeting Wnt pathway components reveals hidden heterogeneity in C. elegans seam cell lineages.

Asymmetric cell division in the epidermal stem cells of Caenorhabditis elegans, known as seam cells, relies on the Wnt/{beta}-catenin asymmetry pathway to generate daughter cells with distinct fates. However, whether components of this pathway components are transcriptionally regulated during these divisions remains unclear. Here, we employ single molecule fluorescence in situ hybridisation to quantify mRNA distributions of key Wnt pathway components during L2 symmetric and asymmetric seam cell divisions. We find that transcripts encoding the negative regulators pry-1/Axin and apr-1/APC are enriched in posterior daughter cells, while those encoding the positive regulators sys-1/{beta}-catenin, wrm-1/{beta}-catenin, and lit-1/NLK, along with the transcription factor pop-1/TCF, are enriched in anterior daughter cells. Strikingly, molecular asymmetries are already evident following the L2 symmetric division, with anterior and posterior daughters exhibiting distinct levels of Wnt component expression and Wnt pathway activation. These mRNA distributions are surprising considering the established protein localisations that underpin the Wnt asymmetry model and suggest extensive post-divisional transcriptional regulation. We further demonstrate that pop-1 asymmetric expression depends on Wnt signalling activity, supporting a model in which transcriptional feedback reinforces cell fate decisions. Investigation of protein distributions using knock-in reporters for PRY-1 and CAM-1 showed that protein accumulation patterns at L2 are consistent with transcript levels. Our findings uncover pervasive transcriptional feedback within the Wnt pathway that likely contributes to robust fate specification and reveal molecular heterogeneity with potential functional consequences for lineage behaviour.

developmental biology↗

bioRxiv2026

HDAC1/2-mediated repression of Wnt receptor expression orients asymmetric division polarity in C. elegans.

Asymmetric cell division generates distinct daughter cells essential for tissue development, yet the mechanisms orienting division polarity within tissues remain incompletely understood. Here, we uncover a role for chromatin-mediated transcriptional repression in controlling polarity orientation during asymmetric division of C. elegans epidermal stem cells, known as the seam cells. We show that tissue- specific loss of the class I histone deacetylase hda-1, homologous to mammalian HDAC1/2, causes reversals in division polarity and reduces molecular asymmetry between daughter cells. Using Targeted DamID to profile HDA-1 genomic occupancy, we identify the Wnt receptors lin-17/Frizzled and cam-1/Ror as key targets. Single- molecule FISH reveals that these receptors display striking expression gradients along the body axis, with cam-1 enriched anteriorly and lin-17 posteriorly. In hda-1 mutants, both receptors are upregulated so that differences between seam cells are flattened. Overexpression of either receptor alone is sufficient to reproduce the polarity reversals observed in hda-1 mutants, and their co-overexpression produces additive defects. The polarity phenotype is independent of the canonical NuRD and SIN3 complexes, suggesting that HDA-1 acts through an alternative mechanism to regulate Wnt receptor expression. Our findings establish a direct link between histone deacetylase activity and Wnt receptor expression and support a model in which graded expression of Wnt receptors provides positional cues that orient asymmetric division polarity in response to Wnt signals.

developmental biology↗

bioRxiv2024

Paired C-type lectin receptors mediate specific recognition of divergent oomycete pathogens in C. elegans.

Innate immune responses can be initiated through the detection of pathogen or damage-associated molecular patterns by host receptors that are often present on the surface of immune cells. While certain invertebrates like Caenorhabditis elegans lack professional immune cells, they still respond to infection in a pathogen-specific manner. It has been debated for years whether homologues of the canonical pathogen recognition receptors are also functioning in the nematode. Here we show that C-type lectin receptors mediate species-specific recognition of divergent oomycetes in C. elegans. A CLEC-27/CLEC-35 pair is essential for recognition of the oomycete Myzocytiopsis humicola, while a CLEC-26/CLEC-36 pair is required for detection of Haptoglossa zoospora. Both clec pairs are transcriptionally regulated through a shared promoter by the conserved PRD-like homeodomain transcription factor CEH-37/OTX2 and act in sensory neurons and the anterior intestine to trigger a protective immune response in the epidermis. This system enables redundant tissue sensing of oomycete threats through canonical CLEC receptors and host defense via cross-tissue communication. HighlightsO_LIA CLEC-27/CLEC-35 pair is required for recognition of the oomycete Myzocytiopsis humicola C_LIO_LIA CLEC-26/CLEC-36 pair is required for recognition of the oomycete Haptoglossa zoospora C_LIO_LIBoth CLEC pairs are co-regulated by the homeodomain transcription factor CEH-37/OTX2 C_LIO_LIBoth CLEC pairs function redundantly in sensory neurons and the intestine for host defense C_LI

immunology↗

bioRxiv2024

EFL-3/E2F7 modulates Wnt signalling through repressing the LIT-1 Nemo-like kinase during asymmetric epidermal cell division in Caenorhabditis elegans

The E2F family of transcription factors is conserved in higher eukaryotes and plays pivotal roles in controlling gene expression during the cell cycle. Most canonical E2Fs associate with members of the Dimerisation Partner (DP) family to activate or repress target genes. However, atypical repressors, such as E2F7 and E2F8, lack DP interaction domains and their functions are less understood. We report here that EFL-3, the E2F7 homologue of C. elegans, regulates epidermal stem cell differentiation. We show that phenotypic defects in efl-3 mutants depend on the Nemo-like kinase lit-1. EFL-3 represses lit-1 expression through direct binding to a lit-1 intronic element. Increased LIT-1 expression in efl-3 mutants reduces POP-1/TCF nuclear distribution, and consequently alters Wnt pathway activation. Our findings provide a mechanistic link between an atypical E2F family member and NLK during C. elegans asymmetric cell division, which may be conserved in other animals. HighlightsO_LIEFL-3 is enriched in anterior daughter cells following asymmetric seam cell division. C_LIO_LIefl-3 mutants show defects in cell differentiation and seam cell fate maintenance. C_LIO_LIEFL-3 directly represses lit-1/NLK expression. C_LIO_LIEFL-3-mediated lit-1 repression alters POP-1 nuclear levels, linking EFL-3 to Wnt signalling. C_LI

developmental biology↗

bioRxiv2023

Inference of the core gene regulatory network underlying seam cell development in Caenorhabditis elegans

Gene regulatory networks are fundamental in cellular decision-making, yet even in well-studied systems, their topologies are often poorly characterised. The nematode Caenorhabditis elegans contains a population of stem-like cells, known as seam cells. While seam cells are essential to generate the majority of the animal epidermis as well as specific neurons, the architecture of the underlying gene network has not been elucidated. Here, we combine experiments, mathematical modelling and statistical inference to uncover the architecture of the seam cell gene network focusing on three core transcription factors (TFs), the GATA factors ELT-1, EGL-18 and the Engrailed homolog CEH-16. We use single-molecule FISH (smFISH) to quantify TF mRNA abundance in single seam cells in both wild type and mutant backgrounds. We then predict potential TF interactions and their quantitative strengths using a combination of Modular Response Analysis, ordinary differential equations and a Bayesian model discovery approach. Taken together, our findings suggest new relationships between core TFs in seam cells and highlight an approach that can be used to infer quantitative networks from smFISH data.

systems biology↗

bioRxiv2023

Impairment of the SKN-1A/NRF1 proteasome surveillance pathway triggers tissue-specific protective immune responses against distinct natural pathogens in C. elegans.

Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF upregulates proteostasis capacity after blockade of the proteasome, and also promotes resistance against bacterial infection in the nematode C. elegans. SKN-1/NRF has three isoforms, and the SKN-1A/NRF1 isoform in particular regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in skn-1a and its activating enzymes ddi-1 and png-1, show constitutive expression of immune response programmes against natural eukaryotic pathogens of C. elegans. These programmes are the Oomycete Recognition Response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the Intracellular Pathogen Response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, skn-1a mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programmes are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner, against natural eukaryotic pathogens of the C. elegans epidermis and intestine.

immunology↗

bioRxiv2023

A NanoDam toolkit for tissue-specific transcription factor profiling in C. elegans

During development of multicellular organisms, cells must execute precise molecular decisions to achieve cell fate specification and differentiation. These decisions are orchestrated by networks of transcription factors (TFs) which act to regulate gene expression of specific cohorts of genes to ultimately confer identity. Depending on the cellular context, TF expression can vary dramatically both spatially and temporally. These differences in expression patterns can result in tissue-specific differences in TF binding to downstream targets. To identify targets on a tissue-specific basis, Targeted DamID (TaDa) has been recently introduced to generate TF binding profiles in various models including C. elegans. However, TaDa suffers from portability such that a new promoter-TF fusion transgene must be constructed for every new experimental condition of interest. Here, we adapt NanoDam for usage in C. elegans, which relies on endogenous TF-GFP knock-ins, a plethora of which have already been generated by the community. We report that NanoDam single copy transgenes consisting of lowly expressed, tissue-specific GFP nanobody-Dam fusions, when combined with endogenous GFP-tagged alleles of TFs, results in robust, tissue-specific profiling. Using an endogenous GFP-tagged allele of EGL-43/EVI1, we performed NanoDam profiling of two disparate tissue types, the anchor cell (AC) and dopaminergic neurons, and identify targets unique to each and shared by both cell types. We also identify two GATA TFs, ELT-6 and EGL-18, as novel regulators of AC invasion. Together, we demonstrate that NanoDam is capable of profiling endogenous GFP-tagged TFs to identify novel downstream targets in specific cell types of C. elegans.

genetics↗

bioRxiv2022

A receptor tyrosine kinase regulated by the transcription factor VAB-3/PAX6 pairs with a pseudokinase to trigger immune signalling upon oomycete recognition in C. elegans

Oomycetes were recently discovered as natural pathogens of Caenorhabditis elegans and pathogen recognition alone was shown to be sufficient to activate a protective transcriptional program in the host characterised by the expression of multiple chitinase-like (chil) genes. However, the molecular mechanisms underlying oomycete recognition in animals remain fully unknown. We performed here a forward genetic screen to uncover regulators of chil gene induction and found several independent loss-of-function alleles of old-1 and flor-1, which encode receptor tyrosine kinases belonging to the C. elegans-specific KIN-16 family. We present evidence that OLD-1 is an active kinase mounting the immune response, and FLOR-1 a pseudokinase that is also required for the response and regulates the distribution of OLD-1 at the epidermal membrane. Interestingly, the old-1 locus is adjacent to the chil genes in the nematode genome, thereby revealing a genetic cluster important for oomycete-resistance. Furthermore, we identify the VAB-3/PAX-6 transcription factor known for its role in visual system development to regulate old-1 expression, and consequently the spatial pattern of the response to oomycete recognition. Taken together, our study reveals both conserved and species-specific factors shaping the response to oomycete recognition.

immunology↗

bioRxiv2022

A pals-25 gain-of-function allele triggers systemic resistance against natural pathogens of C. elegans

Regulation of immunity throughout an organism is critical for host defense. Previous studies in the nematode Caenorhabditis elegans have described an "ON/OFF" immune switch comprised of the antagonistic paralogs PALS-25 and PALS-22, which regulate resistance against intestinal and epidermal pathogens. Here, we identify and characterize a PALS-25 gain-of-function mutant protein with a premature stop (Q293*), which we find is freed from physical repression by its negative regulator, the PALS-22 protein. PALS-25(Q293*) activates two related gene expression programs, the Oomycete Recognition Response (ORR) against natural pathogens of the epidermis, and the Intracellular Pathogen Response (IPR) against natural intracellular pathogens of the intestine. A subset of ORR/IPR genes is upregulated in pals-25(Q293*) mutants, and they are resistant to oomycete infection in the epidermis, and microsporidia and virus infection in the intestine, but without compromising growth. Surprisingly, we find that activation of PALS-25 seems to primarily stimulate the downstream bZIP transcription factor ZIP-1 in the epidermis, which leads to upregulation of gene expression in both the epidermis and in the intestine. Interestingly, we find that this epidermal-to-intestinal signaling promotes resistance to the N. parisii intestinal pathogen, demonstrating cross-tissue protective immune induction from one epithelial tissue to another in C. elegans. Author summaryMulticellular organisms need to monitor the health and function of multiple tissues simultaneously to respond appropriately to pathogen infection. Here, we study an ON/OFF switch in the roundworm C. elegans that controls immune responses to diverse natural pathogens of the skin and gut. We show a physical association between the ON switch protein PALS-25 and the OFF switch protein PALS-22, and that this association is disrupted in a mutant, activated form of PALS-25. When either PALS-22 is lost, or PALS-25 is activated, a downstream immune regulator ZIP-1 is activated specifically in the skin but not the gut. Excitingly, our findings show that skin-specific loss of PALS-22 or skin-specific activation of PALS-25 can induce immune responses in the worm gut. These findings highlight the coordination of immune responses across different tissues that are commonly infected by microbial pathogens.

immunology↗

bioRxiv2021

Single-shot phase contrast microscopy using polarisation-resolved differential phase contrast

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.

biophysics↗

bioRxiv2020

Tissue-specific transcription factor target identification in the Caenorhabditis elegans epidermis using targeted DamID

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 C. elegans 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 C. elegans 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. 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.

developmental biology↗

bioRxiv2020

Cryptic genetic variation in a heat shock protein shapes the expressivity of a mutation affecting stem cell behaviour in C. elegans.

A fundamental question in medical genetics is how the genetic background modifies the phenotypic outcome of key mutations. We address this question by focusing on the epidermal seam cells, which display stem cell properties in Caenorhabditis elegans. We demonstrate that a null mutation in the GATA transcription factor egl-18, which is involved in seam cell fate maintenance, is more tolerated and thus has lower expressivity in the divergent CB4856 isolate from Hawaii than the lab reference strain N2 from Bristol. We identify multiple quantitative trait loci (QTLs) underlying the difference in mutation expressivity between the two isolates. These QTLs reveal cryptic genetic variation, which acts to reinforce seam cell fate through potentiating Wnt signalling. Within one QTL region, a single amino acid deletion in the heat shock protein HSP-110 in CB4856 lowers egl-18 mutation expressivity. Our work underscores that natural variation in conserved heat shock proteins can shape mutation expressivity.

genetics↗

bioRxiv2019

A cell fate switch in the C. elegans seam cell lineage occurs through modulation of the Wnt asymmetry pathway in response to temperature increase

Populations often display consistent developmental phenotypes across individuals despite the inevitable biological stochasticity. Nevertheless, developmental robustness has limits and systems can fail upon change in the environment or the genetic background. We use here the seam cells, a population of epidermal stem cells in Caenorhabditis elegans, to study the influence of temperature change and genetic variation on cell fate. Seam cell development has mostly been studied so far in the lab reference strain (N2), grown at 20{degrees} temperature. We demonstrate that an increase in culture temperature to 25{degrees}, introduces variability in the wild-type seam cell lineage with a proportion of animals showing an increase in seam cell number. We map this increase to lineage-specific symmetrisation events of normally asymmetric cell divisions at the final larval stage, leading to the retention of seam cell fate in both daughter cells. Using genetics and single molecule imaging, we demonstrate that this symmetrisation occurs via changes in the Wnt asymmetry pathway, leading to aberrant Wnt target activation in anterior cell daughters. We find that intrinsic differences in the Wnt asymmetry pathway already exist between seam cells at 20{degrees} and this may sensitise cells towards a cell fate switch at increased temperature. Finally, we demonstrate that wild isolates of C. elegans display variation in seam cell sensitivity to increased culture temperature, although seam cell numbers are comparable when raised at 20{degrees}. Our results highlight how temperature can modulate cell fate decisions in an invertebrate model of stem cell patterning.

developmental biology↗

bioRxiv2018

Antagonistic paralogs control a switch between growth and pathogen resistance in C. elegans

Immune genes are under intense pressure from pathogens, which cause 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. 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. These phenotypic and evolutionary features of pals-22 and pals-25 are strikingly similar to species-specific R gene pairs in plants that control immunity against co-evolved pathogens.

genetics↗