Katsanos, Dimitris et al. (2017) International Worm Meeting "Deconstructing eutely: mechanisms of phenotypic robustness in the seam."

Poole, Richard J., van Zon, Jeroen, Barkoulas, Michalis, Koneru, Sneha, Woollard, Alison, Mestek Boukhibar, Lamia, Katsanos, Dimitris, Doitsidou, Maria, Gritti, Nicola, Ghose, Ritobrata, Appleford, Peter J.

[International Worm Meeting, 2017]

Biological systems constantly face perturbations both in the form of environmental changes and intrinsic stochasticity. Nevertheless, they are still capable of producing constant phenotypic outcomes. Development in particular is a remarkably robust process ensuring the reproducible generation of key phenotypic traits, such as tissues with the correct cell number. However, very little is known about the mechanisms governing developmental robustness. We use the epidermal stem cells of C. elegans, known as seam cells, as a model to identify factors modulating cell number variability. We employ a pipeline consisting of chemical mutagenesis, phenotype selection and mapping by next generation sequencing to identify genes modulating seam cell number variance. To establish a proof of concept, we present here that mutations in the Hes-related bHLH transcription factor lin-22 increase the variability of terminal seam cell number. Using time-lapse imaging of post-embryonic seam cell divisions, we pinned down the developmental basis of phenotypic variability. We show that stage and lineage-specific gain or loss of cell fate symmetry in stem cell daughters underlies the variable phenotype and that these developmental errors occur within the same animal or even a single cell lineage in a stochastic manner. We provide evidence by smFISH and genetics that LIN-22 acts in the epidermis to antagonise Wnt signalling. In contrast to the seam, lin-22 mutants also demonstrate loss of stochasticity when it comes to P3.p division frequency, emphasising the context-specific manifestation of phenotypic variability within a whole organism. Taken together, our work identifies a factor involved in phenotype construction as a modulator of developmental robustness and highlights the feasibility to map genes influencing developmental variability.

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

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.