Induction of cardiogenesis in embryonic stem cells via downregulation of Notch1 signaling

Embryonic stem cells represent an attractive source of cardiomyocytes for cell-replacement therapies. However, before embryonic stem cells can be successfully used for the treatment of cardiac diseases, the precise molecular mechanisms that underlie their cardiogenic differentiation must be identified. A network of intrinsic and extrinsic factors regulates embryonic stem cell self-renewal and differentiation into a variety of different cell lineages. Here, we show that Notch signaling takes place in some but not all embryonic stem cells and that the Notch pathway is shut down during the course of differentiation concomitantly with downregulation of Notch receptor and ligand expression. Moreover, gain- and loss-of-function experiments for Notch signaling components show that this pathway is a crucial regulator of cardiomyocyte differentiation within ES cells. Differentiation of ES cells into cardiomyocytes is favored by inactivation of the Notch1 receptor, whereas endogenous Notch signaling promotes differentiation of ES cells into the neuronal lineage. We conclude that Notch signaling influences the cell fate decision between mesodermal and the neuroectodermal cell fates during embryonic stem cell differentiation. These findings should help to optimize the production of specific cell types via modulation of the Notch pathways and, in particular, to improve the production of embryonic stem cell-derived cardiomyocytes.

Mechanisms underlying cell-fate determination in the mammary gland development

Duje Buric

Notch signaling pathway is an important developmental pathway and has been implicated in both mammary gland development and tumorigenesis. Several studies investigating Notch signaling in mouse mammary gland implied that Notch signaling is an important factor in the determination of the luminal cell type. Clinical studies on breast tumors as well as studies in transgenic mouse models overexpressing active Notch receptors suggested an oncogenic function of Notch in the mammary gland connecting it to the poor breast cancer prognosis. However the physiological role of Notch signaling and its downstream mechanisms remain unclear. Analysis of Transgenic-EGFP Notch reporter mouse mammary epithelium revealed that Notch signaling is active specifically in a subset of hormone receptor positive cells. More sensitive FACS analysis consistently shows EGFP expression in HR+ luminal cells and reveals a weaker signal in a subset of basal (CD24lo) cells. Additionally mRNA analysis revealed that Notch active luminal cells are expressing Wnt4 ligand. To unveil the role of the Notch signaling in the Wnt4 expressing HR+ cells, we conditionally deleted RBP-Jκ, Notch signaling mediator, in Wnt4 expressing subpopulation of hormone receptor positive cells via Wnt4Cre. Abrogation of RBP-Jκ resulted in loss of progesterone receptor expression in 90% of cells bearing the RBP-Jκ deletion while estrogen receptor expression remained intact, implicating RBP-Jκ in regulation of progesterone receptor expression. To test whether Notch signaling regulates PR expression in the entire HR+ population, we analyzed mammary epithelium in which RBP-Jκ has been conditionally deleted via MMTV-Cre in progenitor cells revealing the presence of a subpopulation of luminal hormone receptor positive cells that differentiate independently of Notch signaling. Time directed deletion of RBP-Jκ via intraductally injected Adeno-Cre virus into the adult mouse mammary ductal system through the nipple additionally confirmed that RBP-Jκ is required for progesterone receptor expression. Chromatin immunoprecipitation assay showed that RBP-Jκ binds two out of four putative RBPJκ binding sites in the progesterone receptor promoter. Inhibition of Notch signaling in vivo via intraductal injection of γ-secretase inhibitor, DAPT, resulted in reduced progesterone receptor expression strongly suggesting that Notch-related RBP-Jκ signaling is responsible for PR expression. In this project we are proposing that there are two different populations of HR+ cells: one defined as Wnt4 expressing subpopulation of HR+ cells in which PR expression is dependent of Notch signaling, and another one defined as Wnt4 non expressing subpopulation of HR+ cells which is independent of Notch signaling. Since Notch signaling is able to replace ER signaling and activate ER target genes in the endocrine therapy resistant cell lines, Wnt4 expressing population of HR+ luminal cells might play a crucial role in the acquiring of the resistance.

EPFL2015

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

Jagged1 is the major regulator of notch-dependent cell fate in proximal airways

Freddy Radtke

Background: The Notch signaling pathway plays complex roles in developing lungs, including regulation of proximodistal fates, airway cell specification and differentiation. However, the specific Notch-mediated signals involved in lung development remain unclear. Results: Here we report that Jagged1 is expressed in a subset of bronchial and bronchiolar epithelial cells, where it controls proximal airway cell fate and differentiation. In agreement with previous studies involving disruption of all Notch signaling, we found that deletion of Jagged1 in airway epithelium increased the number of ciliated cells at the expense of Clara cells, a phenotype associated with downregulation of Hes1. Deletion of Jagged1 also led to an increased number of pulmonary neuroendocrine cells (PNEC), suggesting that Jagged1/Notch signaling inhibits PNEC cell fate. As expected, Jagged1 deletion did not affect alveolar cell differentiation, although alveolar septation was impaired, likely an indirect effect of proximal airway defects. Finally, in the postnatal lung, Jagged1 deletion induced mucous metaplasia, accompanied by downregulation of Hes1 and Hes5. Conclusions: Our results demonstrate that Jagged1-mediated Notch signaling regulates multiple cell fate decisions as well as differentiation in the respiratory system to coordinate lung development and to maintain a balance of airway cell types in adult life. Developmental Dynamics 242:678-686, 2013. (c) 2013 Wiley Periodicals, Inc.