Barkoulas, Michalis et al. (2011) International Worm Meeting "Towards a quantitative understanding of the vulva cell fate patterning network."

Barkoulas, Michalis, Felix, Marie-Anne

[International Worm Meeting, 2011]

The C. elegans vulva is the egg-laying and copulatory organ of the adult hermaphrodite that is specified during the third larval stage of postembryonic development from a row of six vulva precursor cells (P3.p-P8.p). These six cells are all competent to adopt vulva fates, but only three (P5.p-7.p) will normally generate vulva tissues, by adopting two distinct cell fates: the 1 deg cell fate, which is acquired by P6.p, and the 2 deg fate which is adopted by P5.p and P7.p. Two major signalling pathways have been shown to be critical in specifying the vulva precursor cell fate pattern. Firstly, an inductive signal from the uterine anchor cell activates the 1 deg cell fate in P6.p through EGF/LIN-3 -Ras-MAP kinase signalling. Secondly, a Delta-Notch/LIN-12 pathway is involved in lateral signalling among the Pn.p cells, resulting in acquisition of the 2 deg cell fate in P5.p and P7.p. Extensive genetic analyses over the last thirty years have revealed both the key molecular components of these two pathways, and also how these two pathways interact within a single or neighbouring Pn.p cells. Nonetheless, even in such a well-studied system, a quantitative understanding of the molecular network is still lacking. For example, although the vulva cell fate pattern is known to be very robust to environmental and genetic variation, the exact range of this robustness with regard to changes of EGF or Notch pathway activity remains unclear. We have started addressing such questions experimentally by systematically varying the doses of EGF and Notch pathway activities in the vulva precursor cells by using tissue-specific transgenic manipulations. We present here our results on the first errors of the vulva cell fate pattern that we observed in these transgenic lines. Our aim is to establish complete EGF and Notch pathway dose-response curves and study their interaction, combining our experimental results with a computational model of the vulva cell fate network that we recently developed in the lab. Finally, we would like to transfer the quantitative knowledge of the C. elegans network to other wild isolates and species to better understand the evolution of the vulva patterning system.

Barkoulas, Michalis et al. (2010) C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany "Towards a quantitative understanding of the vulva cell fate patterning network"

Felix, Marie-Anne, Barkoulas, Michalis

[C. elegans: Development and Gene Expression, EMBL, Heidelberg, Germany, 2010]

The C. elegans vulva is the egg-laying and copulatory organ of the adult hermaphrodite that is specified during the third larval stage of postembryonic development from a row of six vulva precursor cells (P3.p-P8.p). These six cells are all competent to adopt vulva fates, but only three of these cells (P5-7.p) will normally generate vulva tissues, by adopting two distinct cell fates; the 1 cell fate, which is acquired by P6.p, or the 2 fate which is adopted by P5.p and P7.p. Two major signalling pathways have been shown to be critical in specifying the vulva precursor cell fate pattern. Firstly, an inductive signal from the uterine anchor cell activates the 1 cell fate in P6.p through EGF-RAS-Map kinase signalling. Secondly, Notch signalling is involved in lateral signalling among the Pn.p cells, resulting in acquisition o f the 2 cell fate in P5.p and P7.p. Extensive genetic analyses over the last thirty years have revealed both the key molecular components of these two pathways, and also the ways via which these pathways positively or negatively interact within a single or neighbouring Pn.p cells. Nonetheless, even in such a well-studied system, a quantitative understanding of the molecular network is still lacking. We have started addressing this question experimentally by systematically varying the doses of Ras and Notch pathway activities using transgenic manipulations and we present here our first, preliminary results. Our aim is to establish Ras and Notch pathway dose-response curves and study their interaction, combining our experimental results with a computational model of the vulva cell fate network that we have recently developed. We would like eventually to extend the quantitative knowledge of the C. elegans network to other Caenorhabditis species in order to understand the evolu tion of the vulva.

Barkoulas, Michalis et al. (2014) Evolutionary Biology of Caenorhabditis and Other Nematodes "Quantitative single cell evo-devo:turnover of the lin-3 cis-regulatory module in Caenorhabditis"

Barkoulas, Michalis, Felix, Marie-Anne

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2014]

A classical problem in the field of "evo-devo" is to address how gene expression and activity of critical developmental regulators evolve. However, this field has generally made use of qualitative expression patterns. Yet the advent of quantitative methods, such as single- molecule RNA FISH hybridisation (smFISH), makes it now possible to quantitatively compare gene expression of homologs across species. Here we use the newest tools to address the evolution of the vulval inducer gene lin-3/egf. Using smFISH, we show that lin-3 expression in the anchor cell of the somatic gonad is quantitatively conserved within C. elegans isolates, other Caenorhabditis species and Oscheius tipulae. Using RNAi and pharmalogical inhibition of the downstream MAP kinase, we demonstrate that this pathway plays a key role in vulval induction in these species. The conservation of lin-3 gene expression is not trivial, as we do not find in several species, such as C. angaria, known cis- regulatory elements that are required in C. elegans to drive lin-3 expression in the anchor cell. Using CRISPR-Cas9 genome editing and cross-species MosSCI transgenesis, we identify 300 bp of non-coding DNA from C. elegans and C. angaria that are substantially divergent from one another, yet can still drive lin-3 expression in the anchor cell of C. elegans. These results suggest extensive cis-evolution despite quantitative stasis in lin-3 anchor cell expression.

Barkoulas, Michalis et al. (2013) International Worm Meeting "Robustness of the vulval cell fate pattern to pathway dosage modulation and cryptic evolution of lin-3 regulatory sequences."

Barkoulas, Michalis, Peluffo, Alexandre, Felix, Marie-Anne

[International Worm Meeting, 2013]

Biological systems can perform reproducibly to generate invariant outcomes, despite external or internal noise. Over the last few years, the C. elegans vulva has become a model system to explore phenotypic robustness, as the cell fate pattern is highly invariable and the underlying developmental mechanisms well described. Although most of the key molecular factors underpinning vulval formation have now been identified, still very little is understood in quantitative terms. Through experimental perturbations of the two main pathways (EGF and Notch) involved in C. elegans vulval patterning we investigated some quantitative aspects of developmental robustness and system behaviour. We established a dose-response curve for the EGF pathway in the vulva and show that the system can tolerate a four-fold variation in genetic dose of the upstream signalling molecule, lin-3/egf, without phenotypic change in cell fate pattern. Using comparative single molecule FISH hybridisation, we show that levels of lin-3 expression are conserved within C. elegans isolates and C. briggsae, but differ in more distant nematodes, such as C. angaria. We demonstrate that LIN-3 plays a key role in vulval induction in other Caenorhabditis species despite substantial evolution of some cis-regulatory elements required for correct spatiotemporal expression in the C. elegans context.

Michalis Barkoulas et al. (2010) Evolutionary Biology of Caenorhabditis and Other Nematodes "TOWARDS A QUANTITATIVE UNDERSTANDING OF THE VULVA CELL FATE PATTERNING NETWORK"

Michalis Barkoulas, Marie-Anne FELIX

[Evolutionary Biology of Caenorhabditis and Other Nematodes, 2010]

The C. elegans vulva is the egg-laying and copulatory organ of the adult hermaphrodite that is specified during the third larval stage of postembryonic development from a row of six vulva precursor cells (P3.p-P8.p). These six cells are all competent to adopt vulva fates, but only three of these cells (P5-7.p) will normally generate vulva tissues, by adopting two distinct cell fates; the 1 cell fate, which is acquired by P6.p, or the 2 fate which is adopted by P5.p and P7.p. Two major signalling pathways have been shown to be critical in specifying the vulva precursor cell fate pattern. Firstly, an inductive signal from the uterine anchor cell activates the 1 cell fate in P6.p through EGF-RAS-Map kinase signalling. Secondly, Notch signalling is involved in lateral signalling among the Pn.p cells, resulting in acquisition of the 2 cell fate in P5.p and P7.p. Extensive genetic analyses over the last thirty years have revealed both the key molecular components of these two pathways, and also the ways via which these pathways positively or negatively interact within a single or neighbouring Pn.p cells. Nonetheless, even in such a well-studied system, a quantitative understanding of the molecular network is still lacking. We have started addressing this question experimentally by systematically varying the doses of Ras and Notch pathway activities using transgenic manipulations and we present here our first, preliminary results. Our aim is to establish Ras and Notch pathway dose-response curves and study their interaction, combining our experimental results with a computational model of the vulva cell fate network that we have recently developed. The final vulva cell fate pattern is conserved across many Caenorhabditis species, although differences can be revealed upon experimental manipulations. These results suggest that within a shared vulva molecular network among closely related species, the quantitative strengths of the lateral and inductive pathways may differ. We would like eventually to extend the quantitative knowledge of the C. elegans network to other species to understand the evolution of the vulva.

Koneru, Sneha Latha et al. (2017) International Worm Meeting "The effect of the genetic background on seam cell patterning."

Barkoulas, Michalis, Koneru, Sneha Latha

[International Worm Meeting, 2017]

The seam cells are lateral epidermal cells that show intriguing stem cell-like properties. However, seam cell development has only been studied so far in the laboratory reference strain N2, thus it remains unclear whether what we currently know about seam cell patterning is indeed representative for the species. As a first step towards addressing this question, we have introgressed a seam cell marker (wIs51) into divergent C. elegans isolates. We found that the terminal seam cell number is robust to standing genetic variation. It has previously been shown that cryptic genetic variation can be uncovered by sensitizing a developmental system by introducing mutations. We have used CRISPR/Cas9-mediated genome editing and genetic introgressions to generate de novo or transfer existing mutations in known seam cell gene network components into divergent C. elegans isolates. We found that mutations in lin-22, eff-1 and bro-1 did not show consistent differences in mutation expressivity in the different isolates tested. However, we discovered that both the expression of the GATA transcription factor egl-18 in the seam and the expressivity of a null mutation in this factor vary significantly among isolates. We will discuss here these first experiments and our planned strategy to identify the genetic modifiers involved. Taken together, these results provide the first evidence linking the composition of the genetic background to seam cell patterning.

Grover, Manish et al. (2021) International Worm Meeting "Neuronal C-type lectin receptors mediate recognition of oomycete pathogens in C. elegans"

Grover, Manish, Liu, Kenneth, Barkoulas, Michalis

[International Worm Meeting, 2021]

Innate immune responses rely on signaling events triggered by the detection of pathogen-associated molecular patterns or damage related host biomolecules by specific host receptors in specialized immune cells, such as macrophages and dendritic cells. While such cells are missing in C. elegans, there is some evidence of pathogen recognition, which points towards existence of detection strategies that are yet unknown. We have previously shown that C. elegans can respond to an innocuous extract made from oomycete infected animals by triggering a recognition response characterized by the induction of chitinase-like (chil) genes in the hypodermis. Through a forward genetic screen aimed at identifying suppressors of chil gene induction upon treatment with this extract, we recovered loss-of-function mutations in ceh-37, clec-27 and clec-35 genes which led to complete loss of oomycete recognition response in C. elegans and made animals more susceptible to infection by the oomycete Myzocytiopsis humicola. Using smFISH and fluorescent reporters, we found clec-27 and clec-35 to be expressed in neurons while RNA-seq analysis revealed changes in the expression of these genes in a ceh-37 mutant background. Furthermore, neuronal rescue of clec-27 function specifically in ceh-37 expressing neurons, and particularly in AWA, was sufficient to restore chil gene induction in clec-27 mutants upon exposure to oomycete extract. Interestingly, clec-27 and clec-35 are neighboring genes sharing a bidirectional promoter, an organization which indicates coregulation of the two genes and a possible requirement to produce the two proteins in stoichiometrically equal amounts. These observations suggest that CLEC-27 and CLEC-35 could be forming a heterodimeric receptor involved in oomycete recognition in AWA neurons. Additionally, when these mutants were exposed to the phylogenetically distinct oomycete Haptoglossa zoospora, both clec-27 and clec-35 mutant animals showed chil gene induction, whereas ceh-37 mutants did not. This suggests that CLEC-27 and CLEC-35 are receptors specifically involved in the detection of M. humicola and a different receptor(s) in ceh-37 expressing neurons mediates detection of H. zoospora. Overall, our study provides evidence for neuronally expressed C-type lectins as pathogen recognition receptors in C. elegans which mediate detection of a newly identified class of natural pathogens of C. elegans in a pathogen-specific way.

hintze, mark et al. (2021) International Worm Meeting "Uncovering highly conserved factors that contribute to phenotypic robustness of seam cell patterning in C. elegans"

Hintze, Mark, Katsanos , Dimitris, Barkoulas, Michalis

[International Worm Meeting, 2021]

Biological systems experience various perturbations but are able to buffer these to produce consistent phenotypes, a property known as robustness. Genes contributing to phenotypic robustness have been largely examined in unicellular organisms, such as S. cerevisiae, as opposed to multicellular animals. In our lab, we study phenotypic robustness using the C. elegans seam cells as a model. Seam cells are stem-like lateral epithelial cells, which undergo asymmetric and symmetric divisions to produce the hypodermis and neurons. Similar to other cell lineages, the number of seam cells is quite invariant between animals in the wild-type population. To this end, a forward genetic screen was performed to identify mutants with greater levels of population variance in seam cell number. Our screen has identified mutations in previously uncharacterised genes involved in the regulation seam cell pattering, some of which are highly conserved throughout the animal kingdom. Key examples are a mutation in nath-10, an N-acetyltransferase, and bus-19, an ancient transmembrane protein. Both mutations can be phenocopied by RNAi knockdown, rescued through seam cell specific expression of the wild-type gene and result in stochastic gains and losses of seam cells during development. Interestingly, both genes can be linked to the role of Wnt signalling in seam cell patterning, as revealed by studying the localisation of Wnt components and the expression of downstream targets via RNA-seq analysis, single molecule FISH and reporter gene expression. We also find that mutations in nath-10 lead to an increase in mRNA transcript variability, which may also influence phenotypic variability. These results provide insights for novel and highly conserved regulators of seam cell patterning, which may influence phenotypic robustness through modulation of Wnt signalling.

Katsanos, Dimitris et al. (2021) International Worm Meeting "Dissecting the functional genomic landscape of epidermal patterning in C. elegans using Targeted-DamID."

Ferrando Marco, Mar, Barkoulas, Michalis, Katsanos, Dimitris

[International Worm Meeting, 2021]

Development is driven by complex patterning events such as cell division, cell fate maintenance and differentiation. Strict transcriptional and epigenetic control permits the selective decoding of batteries of genes that assemble within gene regulatory networks underlying these developmental decisions. In our lab, we focus on the patterning of the stem cell-like seam cells of the C. elegans epidermis as a model to dissect how gene regulatory networks, epigenomic regulation and the resulting expression profiles bring about robust developmental outcomes. Here, we tackle these questions by employing the multifaceted methodology of Targeted-DamID (TaDa) for the first time in C. elegans. We find that TaDa is capable of effectively identifying protein-DNA interactions of interest within a tissue of interest, requiring little starting material and preventing Dam-associated toxicity. Using TaDa we identify putative targets of the transcription factors LIN-22 and NHR-25 within the epidermis, thereby expanding our understanding of the underlying gene regulatory network and proposing new biological functions for these epidermal regulators. We further identify by assaying RNApol occupancy gene expression profiles for specific cell types of the epidermis, such as the seam cells and the major hypodermal syncytium hyp7, which reveals novel regulators of epidermal fate. Lastly, for those same cell types we probe chromatin accessibility to uncover aspects of the epigenomic regulation and identify tissue-specific regulatory elements. Our findings expand our current understanding of the genetic regulation of epidermal cell fate patterning and underline the potential value of TaDa to address diverse questions in C. elegans. Keywords: cell fate and patterning, gene regulation, genomics, novel genetic technologies

Brozek, Alicja et al. (2021) International Worm Meeting "Towards a quantitative gene network underlying robustness of seam cell fate"

Shahrezaei, Vahid, Barkoulas, Michalis, Hintze, Mark, Brozek, Alicja

[International Worm Meeting, 2021]

Among all multicellular organisms, Caenorhabditis elegans offers a unique experimental system to study phenotypic robustness because of its remarkably reproducible development. In our lab, we focus on seam cells, which show stem cell-like properties being able to divide both symmetrically and asymmetrically in order to self-renew or generate daughter cells that differentiate into hypodermis and neurons, and this happens in a fairly invariant manner. To improve our understanding of the seam cell gene network, we have started focusing on key transcription factors that are thought to be its core members, such as elt-1, egl-18 and ceh-16. We have used single molecule FISH to quantify the expression levels of these factors in wild-type and mutant backgrounds and characterise their putative interactions. Preliminary data suggest that individual seam cells may be subject to different quantitative relationships based on their position along the anterior-posterior axis. In order to evaluate the stability of these interactions we have built a preliminary Boolean network model based on our smFISH data. In silico simulations have been used to evaluate the robustness of various network architectures upon perturbation. Our aim is to integrate experimental and theoretical work to understand the structure of the seam cell fate gene network and its resilience to perturbation.