The epithelium-mesenchyme transition (EMT) is an essential process broadly used in development, wound healing and disease (e.g. fibrosis, cancer). It is activated by a handful of transcription factors (EMT-TFs) collectively controlling cell-cell adhesion and cell motility. Although numerous studies have analyzed these factors individually, our current knowledge includes significant gaps, with key open questions about EMT either in development or in cancer [1]. This project addresses one of them: what is the spatial and temporal dynamics of EMT-TFs co-activation and what is its functional significance for the process of EMT at the scale of cells and tissue and for cell migration? We explore if and when EMT-TFs are activated simultaneously in a given cell, if there are specific characteristics for these expressions, a precise combinatorial logic and a temporal dynamics, that can be related to a specific cell state; or alternatively if there is a degree of stochastic activation of these genes in a given tissue. Secondly, when the EMT-TFs are co-activated, do they cooperate molecularly in the same cell and for which outcome on the modalities of EMT? By combining approaches of cell biology, molecular embryology and bioinformatics, this project explores EMT from single cell scale to tissue level, in the classical model of the embryonic neural crest. Neural crest EMT is a well-understood and extensively explored model of a stereotypical EMT, accessible for studies in vivo and ex-vivo, providing a framework for more general understanding of EMT in pathological contexts. We will describe the spatial and temporal dynamics of EMT-TFs co- expression and compare with expression of their transcriptional regulators during neural border development. Using single cell transcriptomes and single cell multiplexed in situ hybridization, we will provide the first in vivo map of EMT-TFs relative expression during development with individual cell resolution. We will push limits of the current analyses, (e.g. low depth of single cell transcriptomes, biostatistics issues for evaluation cell-to-cell heterogeneity of a given gene...): we will prepare novel, deeper, and neural crest-focused datasets and novel analysis tools with the two partners specialized in single cell transcriptomics, L. Peshkin (Partner 3, Harvard Medical School, Dept Systems Biology, [2]) et I. Adameyko (Partner 4, U. Vienna [3]). We will develop quantitative in situ hybridization (with single cell and single molecule resolution) with external collaborator T. Walter (Mines ParisTech, [4]) to produce a quantitative map during neural crest development. To understand the functional impact of EMT-TFs cooperation, we will experimentally manipulate the gene regulatory network of neural crest EMT using a suite of dedicated and custom made molecular tools, used to measure the fine- tuned parameters of EMT and cell migration in vivo and ex vivo. A.H. Monsoro-Burq (Coordinator, Partner 1, I. Curie, U. Paris Saclay, [5, 6]) et E. Théveneau (Partner 2, U. Toulouse, [7, 8]) will thus set-up an integrated series of assays testing the developmental and cellular consequences of EMT- TFs conjoined expressions. Our project will bring a new dimension in the landscape of EMT control and mechanism and will advance significantly our understanding of EMT both in normal and in pathological contexts. [1] Stemmler et al, Nat.Cell.Biol 2019; 10.1038/s41556-018-0196-y [2] Briggs et al., Science 2018, 10.1126/science.aar5780 [3] Soldatov et al., Science 2019; 10.1126/science.aas9536 [4] Tsanov et al., Nucl Ac.Res.2016;10.1093/nar/gkw784 [5] Plouhinec et al., PLOS Biology 2017; DOI:10.1371/journal.pbio.2004045. [6] Scerbo and Monsoro-Burq 2020; Science Advances, DOI: 10.1126/sciadv.aaz1469 [7] Bajanca et al., Nature Communications 2019; DOI:10.1038/s41467-019-09548-5 [8] Yang et al, Nat Rev Mol Cell Biol. 2020, DOI: 10.1038/s41580-020-0237-9.