In adult organisms, tissues are maintained and repaired by stem cells, which divide and differentiate to generate more specialized progeny. The mechanisms that control the balance between self-renewal and differentiation promise fundamental insights into the origin and design of multi-cellular organisms. A tissue that is particularly suited to approach these questions is the intestinal epithelium, as it consists of a monolayer of epithelial cells that endlessly divide, migrate while differentiating and are replaced to ensure continuous and fast cell renewal throughout adult life. In this tissue, somatic stem cells represent crucial elements that govern tissue remodeling and homeostasis and the combined work of several laboratories has provided evidence for the existence of at least two different stem cell populations in the small intestinal crypt. In this program, we focus on the Notch signaling pathway as a new promising marker to study gut stem cell physiology. Our observations indicate that the Notch1 receptor is expressed in both crypt stem cell populations, implying that it can mark either one of the two in vivo and providing a precious tool to dissect the hierarchy between these different stem cells. Notch signaling plays an evolutionarily conserved role in metazoans, which basically consists in mediating cell coordination in development and during adult tissue homeostasis, through the profound effects it has on stem cells. The precise identity of the cells in which Notch signaling is active is still unclear, mainly due to the lack of reliable tools to investigate Notch expression in vivo. For this purpose, we have recently developed and characterized a novel roster of unique transgenic mice that permit to assess Notch expression and activation in vivo in an unprecedented fashion. Thanks to these novel mice, we have been able to formally show that the expression of the Notch1 receptor and of its transcriptional target Hes1 identifies crypt stem cells. We are now using these mouse models to dissect the hierarchy between stem cell populations in the small intestine, while expanding our knowledge on colonic stem cells, that remain poorly studied. In addition, we study the dynamic behavior of stem cells during regeneration upon injury. The critical role played by Notch signaling in intestinal renewal and differentiation, as well as in tumorigenesis in this tissue, is exemplified by the vast number of reports on this topic appeared in recent years (i.e. [1-6]). In the past five years, the Notch signaling pathway has emerged as an essential regulator of intestinal homeostasis; indeed Notch signals can control the segregation of each mature lineage from undifferentiated progenitor cells, and they are instrumental for maintaining the proliferating intestinal cell pool. When Notch signaling is inhibited, all crypt cells cease to proliferate and differentiate into secretory cells [4]. Reciprocally, we have shown that gain of Notch function in the developing intestinal epithelium dramatically impairs cell differentiation and increases the proportion of dividing cells [2]. The role of Notch in promoting intestinal proliferation requires Wnt signals, whereas it specifies cell fate independently of Wnt [5]. Importantly, our work has shown that Notch acts in synergy with the Wnt pathway to induce intestinal polyps [5]. The accumulated evidence in both systems supports a role for Notch in expanding a potentially malignant cell population and hence increasing the chances of a tumorigenic event. It is indeed widely accepted that the primary events in tumourigenesis are linked to stem cell transformation: the process of tumor development is thought to initially affect normal stem cells or closely related early progenitors. In order to establish the identity of Notch1-expressing cells within a tumor and to follow their fate in vivo, we have crossed N1-CreERT2/GFP mice to Apc heterozygous animals, which give rise to intestinal tumors by stochastic loss of heterozygosity (LOH) at the Apc locus. Our novel genetic fate-mapping system will consent to identify which cells within a tumor express the Notch1 receptor, as well as to follow the progeny of the Notch-expressing tumor cells. Our preliminary results show that only a small fraction of tumor cells expresses the N1cre-driven GFP reporter. These experiments will allow us to highlight in vivo which and how many cells within a tumor present Notch activity. Our research aims at understanding the molecular features of adult stem cells, as well as the mechanisms by which Notch signaling controls the fate of specific cell populations during regeneration and tumourigenesis. We believe that the identification of common organizational principles of tissue architecture is an essential step in order to develop safe and efficacious applications of stem cells in regenerative medicine.