A genetic framework controlling the differentiation of intestinal stem cells during regeneration in Drosophila

The speed of stem cell differentiation has to be properly coupled with self-renewal, both under basal conditions for tissue maintenance and during regeneration for tissue repair. Using the Drosophila midgut model, we analyze at the cellular and molecular levels the differentiation program required for robust regeneration. We observe that the intestinal stem cell (ISC) and its differentiating daughter, the enteroblast (EB), form extended cell-cell contacts in regenerating intestines. The contact between progenitors is stabilized by cell adhesion molecules, and can be dynamically remodeled to elicit optimal juxtacrine Notch signaling to determine the speed of progenitor differentiation. Notably, increasing the adhesion property of progenitors by expressing Connectin is sufficient to induce rapid progenitor differentiation. We further demonstrate that JAK/STAT signaling, Sox21a and GATAe form a functional relay to orchestrate EB differentiation. Thus, our study provides new insights into the complex and sequential events that are required for rapid differentiation following stem cell division during tissue replenishment.

The Intestinal Epithelium

Luca Pellegrinet

The mammalian intestine is a prototype of a self-renewing organ. The rapid cellular turnover is supported by proliferating progenitors located in the intestinal crypt, which is also the stem cell location. Homeostasis of the intestinal epithelium has to be under stringent control to ensure lifelong self-renewal. Processes like proliferation and differentiation (often deregulated in cancer) seem to be controlled by a relative small number of signaling pathways, including Wnt and Notch. Notch signaling is playing a crucial role in the maintenance of the intestinal homeostasis by controlling progenitors proliferation and their cell fate. In this thesis project we studied Notch signaling and its role in the murine intestine focusing on 1) Its downstream mediators; 2) The identification of the physiological Notch ligands; 3) Its role on the stem cell compartment, and 4) the potential cross talk between Notch and Wnt. Furthermore, 5) we investigated the role of Notch signaling in Wnt induced intestinal tumorigenesis. This study evidenced that Hes1 is not the unique Notch signaling mediator in the intestinal epithelium. Moreover the transcription factor Klf4 is not required for goblet cell differentiation. The in vivo role of the Notch ligands Dll1, Dll4 and Jag1 was assessed deriving inducible intestine specific single and double gene-targeted mice. Inactivation of individual ligands did not show any loss of Notch phenotype with the exception of Dll1 mutant mice. Only by deriving double mutants we identified Dll1 and Dll4 as the physiological Notch ligands in the intestine. We provided evidence for the first time that Notch1 signaling is active in intestinal stem cell. Furthermore we demonstrated that Notch signaling is required for the maintenance of intestinal stem cells, as well as for the transit-amplifying progenitor compartment. Using different genetic gain and loss-off function approaches we investigated the relation between the Notch and Wnt cascade for the maintenance of the proliferative crypt compartment. A synergistic relation was observed between Notch and Wnt in intestinal tumorigenesis. Collectively our data highlight Notch signaling as a key player for the maintenance of intestinal homeostasis.

EPFL2011

Autocrine and paracrine unpaired signalling regulate intestinal stem cell maintenance and division

Sveta Chakrabarti, Yu-Ting Huang, Bruno Lemaitre, Dani Osman, Yu-Chen Tsai

The Janus Kinase (JAK) Signal Transducer and Activator of Transcription (STAT) pathway is involved in the regulation of Intestinal Stem Cell (ISC) activity to ensure a continuous renewal of the adult Drosophila midgut. Three ligands, Unpaired1, 2 and 3 are known to activate the JAK/STAT pathway in Drosophila. Using newly generated upd mutants and cell specific RNAi, we showed that Upd1 is required throughout the fly life to maintain basal turnover of the midgut epithelium by controlling ISC maintenance in an autocrine manner. A role of Upd2 and Upd3 in basal conditions is discernible only in old gut, where they contribute to increased ISC abnormal division. Finally, upon an acute stress such as oral bacterial infection, we showed that Upd3 is released from enterocytes and has an additive effect with Upd2 to promote rapid epithelial regeneration. Taken together, our results show that Upd ligands are required to maintain the midgut homeostasis under both normal and pathological states.

Company Of Biologists Ltd2012

Study of Cell Adhesion and Tracking Methods for in Vivo Cell Localisation in a Tissue-Engineering Scaffold

Thaïs Cléa Laura Séverine Clouet, Tanja Cloé Hausherr

Bone fracture healing problems, because of the loss of tissue regeneration, can affect many old or multi-pathologic patients around the world. Among the therapeutic strategies, bone substitutes, such as poly-(L-lactitve acid) with 5% wt. % ß-tricalcium phosphate (PLA/5%ß-TCP) scaffolds, are promising as a clinical solution. One strategy to tune their osteoinductive properties is the addition of bone progenitor cells. Therefore, cell- free and cell-seeded PLA/5% ß-TCP scaffolds were implanted into femoral condyles of rats to promote bone regeneration. The in vivo study showed that the bone growth inside a cell-seeded scaffold with human bone progenitor cells was delayed compared to cell-free scaffold. What happened to the cells? Did they trigger an immune response, migrate or die? Therefore, the goals of this project are to evaluate the cell adhesion in vitro to the scaffold and to propose a reliable cell labeling method to track them in future in vivo studies. In the first aim, the adhesion of the cells was qualitatively assessed by the cell morphology and the presence of fibronectin using fluorescence imaging. To quantify their adherence, seeded scaffolds were washed up to 3 times with a 0.9 % NaCl solution. The luminescence of the scaffolds and the washing solutions was measured using a cell viability assay. These experiments demonstrated that after 72h of culture, the measured adherent bone progenitor cells on the scaffolds decreased after 3 washes down to 50 % while it stayed stable after 14 days of culture. In the second aim, we focused on methods to label the cells either once implanted or prior to implantation. The first method was to translate the in situ hybridization protocol with the human specific Alu sequence from paraffin embedded samples to resin embedded ones. The obtained results were so far inconclusive. Concerning the labeling of the cells before implantation, the transmembrane label PKH26 was investigated. Its staining efficacy was high just after labeling, while it decreased drastically over time due to cell division.