REGULATION DE LA PROLIFERATION DES CELLULES SOUCHES/PROGENITRICES DES CRYPTES INTESTINALES – HOMEOSTEM

The intestinal epithelium produces 10E10 cells daily, and this proliferating activity is restricted to a specialised compartment of the epithelium: the crypt. The second compartment of this epithelium is the villus, which contains terminally differentiated cells. This project aims at obtaining an integrated view of the intestinal turnover, containing the bottom-up aspect of cell production, as well as the top-down aspect of the turnover rate according to the physiological state of the villus compartment. On the one hand, we will generate innovative tools to analyse the activity of the driving forces of the intestinal epithelium cell proliferation, i.e. the Wnt and Notch pathways. Indeed, in spite of intensive research efforts during the last decade, important questions remain on the actual functions of the Wnt and Notch pathways: (i) signalling activities have never been observed in real time in an intact tissue, (ii) although most of the researchers agree that the two pathways cooperate to ensure crypt homeostasis, such an interaction has never been visualised, (iii) most of the functional data available on the Wnt and Notch pathways in the intestinal epithelium have been obtained from mouse models, the physiology of which was altered by gene deletion or over-expression approaches, and (iv) several functions have been assigned to the Wnt and Notch pathways, including proliferation, lineage commitment, terminal differentiation, migration, but little is known about how Wnt-stimulated cells adopt the appropriate behaviour among these possibilities. To address these issues, we are developing a system of short-lived fluorescent reporters to visualise the cells in which Wnt or Notch activities occur, in a minimally perturbed, physiologically relevant, system. The signalling activities will be analysed in cultured, immobilised, crypts and in intact tissue of transgenic mouse containing the reporters. The mice containing the fluorescent reporters will also be instrumental to isolate cells with Wnt, Notch, or both activities from cultured crypts or from tissue, and functionally characterise them. This should provide clues about the respective roles of the Wnt and Notch pathways in the self-renewal potential of intestinal epithelial cells. We will also exploit our recent finding that Sox9 constitutes a ramification point of the cellular response to Wnt signals. We have developed an attractive set of mouse models and protocols to identify Sox9 target genes in each Sox9-expressing cell type, individually. This should facilitate the identification of the role played by Sox9 in the stem cells of the intestinal epithelium, as a part of, or independently of, Wnt signalling, particularly regarding pluripotency and self-renewal, two essential properties of stem cells. On the other hand, we ask how the crypt stem cell proliferation is regulated in response to villus environmental cues. Here, we focus on the function of tuft cells, a minor, poorly characterised cell type. Although the function of tuft cells has not been validated in the intestine, their unique morphology, with a very developed apical border (tuft) protruding in the intestinal lumen (hence the name) and their expression of taste-like receptors and of multiple components of the prostanoid biosynthesis pathway make them likely sensor cells that use prostanoids as messengers. We recently found that Sox9 is strongly expressed in tuft cells, might regulates their abundance, and might be involved in the regulation of PGDS, a key enzyme for prostaglandin D2 synthesis. Prostanoids, in turn, are widely implicated in the physiopathology of the intestinal epithelium. Here, we will first make an inventory of the prostanoid biosynthesis pathway components in the intestine and then address the role of Sox9 in the regulation of prostanoid production by tuft cells. Eventually, we will analyse the impact of various prostanoids on crypt stem cell proliferation in cultured crypts and intact tissue, with the hypothesis that tuft cells function as sensors and signal in a top-down pathway to regulate stem cell proliferation activity in crypts. We believe that this project is ambitious, novel, and addresses key questions underlying the understanding of stem cells properties such as self-renewal and pluripotency, as well as how these properties are regulated to ensure tissue homeostasis. The use of adapted animal models and robust techniques should facilitate the production of physiologically relevant data. Risks are present in all three tasks, most notably in tasks 1 and 3, but strong indications from the literature and preliminary data substantiate the working hypotheses.