Summary Dendritic cells (DCs) in tissues and lymphoid organs comprise distinct functional subsets that differentiate in situ from circulating progenitors. Tissue-specific signals that regulate DC subset differentiation are poorly understood. We report that DC-specific deletion of the Notch2 receptor caused a reduction of DC populations in the spleen. Within the splenic CD11b+ DC subset, Notch signaling blockade ablated a distinct population marked by high expression of the adhesion molecule Esam. The Notch-dependent Esamhi DC subset required lymphotoxin beta receptor signaling, proliferated in situ and facilitated CD4+ T cell priming. The Notch-independent Esamlo DCs expressed monocyte-related genes and showed superior cytokine responses. In addition, Notch2 deletion led to the loss of CD11b+ CD103+ DCs in the intestinal lamina propria and to a corresponding decrease of IL-17-producing CD4+ T cells in the intestine. Thus, Notch2 is a common differentiation signal for T cell-priming CD11b+ DC subsets in the spleen and intestine.

Notch signaling is a central regulator of differentiation in a variety of organisms and tissue types1. Its activity is controlled by the multi-subunit γ–secretase complex (γSE) complex2. Although Notch signaling can play both oncogenic and tumor suppressor roles in solid tumors, in the hematopoietic system, it is exclusively oncogenic, notably in T cell acute lymphoblastic leukemia (T-ALL), a disease characterized by Notch1 activating mutations3. Here we identify novel somatic inactivating Notch pathway mutations in a fraction of chronic myelomonocytic leukemia (CMML) patients. Inactivation of Notch signaling in mouse hematopoietic stem cells (HSC) resulted in an aberrant accumulation of granulocyte/monocyte progenitors (GMP), extramedullary hematopoieisis and the induction of CMML-like disease. Transcriptome analysis revealed that Notch signaling regulates an extensive myelomonocytic-specific gene signature, through the direct suppression of gene transcription by the Notch target Hes1. Our studies identify a novel role for Notch signaling during early hematopoietic stem cell differentiation and suggest that the Notch pathway can play both tumor-promoting and suppressive roles within the same tissue.

T-cell acute lymphoblastic leukaemia (T-ALL) is a blood malignancy afflicting mainly children and adolescents1. T-ALL patients present at diagnosis with increased white cell counts and hepatosplenomegaly, and are at an increased risk of central nervous system (CNS) relapse2,3. For that reason, T-ALL patients usually receive cranial irradiation in addition to intensified intrathecal chemotherapy. The marked increase in survival is thought to be worth the considerable side-effects associated with this therapy. Such complications include secondary tumours, neurocognitive deficits, endocrine disorders and growth impairment3. Little is known about the mechanism of leukaemic cell infiltration of the CNS, despite its clinical importance4. Here we show, using T-ALL animal modelling and gene-expression profiling, that the chemokine receptor CCR7 (ref. 5) is the essential adhesion signal required for the targeting of leukaemic T-cells into the CNS. Ccr7 gene expression is controlled by the activity of the T-ALL oncogene Notch1 and is expressed in human tumours carrying Notch1-activating mutations. Silencing of either CCR7 or its chemokine ligand CCL19 (ref. 6) in an animal model of T-ALL specifically inhibits CNS infiltration. Furthermore, murine CNS-targeting by human T-ALL cells depends on their ability to express CCR7. These studies identify a single chemokine–receptor interaction as a CNS ‘entry’ signal, and open the way for future pharmacological targeting. Targeted inhibition of CNS involvement in T-ALL could potentially decrease the intensity of CNS-targeted therapy, thus reducing its associated short- and long-term complications.

Much of the information about the function of D. melanogaster genes has come from P-element mutagenesis. The major drawback of the P element, however, is its strong bias for insertion into some genes (hotspots) and against insertion into others (coldspots). Within genes, 59-UTRs are preferential targets. For the successful completion of the Drosophila Genome Disruption Project, the use of transposon vectors other than P will be necessary. We examined here the suitability of the Minos element from Drosophila hydei as a tool for Drosophila genomics. Previous work has shown that Minos, a member of the Tc1/mariner family of transposable elements, is active in diverse organisms and cultured cells; it produces stable integrants in the germ line of several insect species, in the mouse, and in human cells. We generated and analyzed 96 Minos integrations into the Drosophila genome and devised an efficient ''jumpstarting'' scheme for production of single insertions. The ratio of insertions into genes vs. intergenic DNA is consistent with a random distribution. Within genes, there is a statistically significant preference for insertion into introns rather than into exons. About 30% of all insertions were in introns and 55% of insertions were into or next to genes that have so far not been hit by the P element. The insertion sites exhibit, in contrast to other transposons, little sequence requirement beyond the TA dinucleotide insertion target. We further demonstrate that induced remobilization of Minos insertions can delete nearby sequences. Our results suggest that Minos is a useful tool complementing the P element for insertional mutagenesis and genomic analysis in Drosophila. O NE of the main goals of modern genetics is to link the many thousands of genes identified through the sequencing of whole genomes of model organisms to gene function. The most powerful technique for this purpose so far has been transgenesis with mobile elements. This technique is a means to disrupt, overexpress, or misexpress single genes to identify expression patterns and also to characterize genetic pathways and their interactions. One of the main advantages of insertional mutagenesis over the classical method of chemical mutagenesis is the ease with which the targeted gene can be identified, since it carries an inserted tag.The P element was the first mobile element that enabled germ-line transformation of an insect species (Rubin and Spradling 1982). Since then, thousands of single P-element insertions causing lethality, semilethality, sterility, semisterility, and visible phenotypes have been created and analyzed in Drosophila (Cooley et al.

To explore the potential involvement of aberrant Notch1 signaling in breast cancer pathogenesis, we have used a transgenic mouse model. In these animals, mouse mammary tumor virus LTR-driven expression of the constitutively active intracellular domain of the Notch1 receptor (N1 IC ) causes development of lactation-dependent mammary tumors that regress upon gland involution but progress to nonregressing, invasive adenocarcinomas in subsequent pregnancies. Up-regulation of Myc in these tumors prompted a genetic investigation of a potential Notch1͞Myc functional relationship in breast carcinogenesis. Conditional ablation of Myc in the mammary epithelium prevented the induction of regressing N1 IC neoplasms and also reduced the incidence of nonregressing carcinomas, which developed with significantly increased latency. Molecular analyses revealed that both the mouse and human Myc genes are direct transcriptional targets of N1 IC acting through its downstream Cbf1 transcriptional effector. Consistent with this mechanistic link, Notch1 and Myc expression is positively correlated by immunostaining in 38% of examined human breast carcinomas.breast cancer ͉ mouse model

The urothelium is a specialized epithelium that lines the urinary tract. It consists of three different cell types, namely, basal, intermediate and superficial cells arranged in relatively distinct cell layers. Normally, quiescent, it regenerates fast upon injury, but the regeneration process is not fully understood. Although several reports have indicated the existence of progenitors, their identity and exact topology, as well as their role in key processes such as tissue regeneration and carcinogenesis have not been clarified. Here we show that a minor subpopulation of basal cells, characterized by the expression of keratin 14, possesses self-renewal capacity and also gives rise to all cell types of the urothelium during natural and injury-induced regeneration. Moreover, these cells represent cells of origin of urothelial cancer. Our findings support the hypothesis of basally located progenitors with profound roles in urothelial homoeostasis.

The kidney papilla contains a population of cells with several characteristics of adult stem cells, including the retention of proliferation markers during long chase periods (i.e., they are label-retaining cells [LRCs]). To determine whether the papillary LRCs generate new cells in the normal adult kidney, we examined cell proliferation throughout the kidney and found that the upper papilla is a site of enhanced cell cycling. Using genetically modified mice that conditionally expressed green fluorescence protein fused to histone 2B, we observed that the LRCs of the papilla proliferated only in its upper part, where they associate with "chains" of cycling cells. The papillary LRCs decreased in number with age, suggesting that the cells migrated to the upper papilla before entering the cell cycle. To test this directly, we marked papillary cells with vital dyes in vivo and found that some cells in the kidney papilla, including LRCs, migrated toward other parts of the kidney. Acute kidney injury enhanced both cell migration and proliferation. These results suggest that during normal homeostasis, LRCs of the kidney papilla (or their immediate progeny) migrate to the upper papilla and form a compartment of rapidly proliferating cells, which may play a role in repair after ischemic injury. Adult stem cells contribute to organ repair after injury 1-5 ; however, their contribution to normal tissue homeostasis by the generation of a continuous supply of new cells has not been readily apparent, except in tissues with relatively simple architecture and high rate of cell turnover, such as the skin and the intestinal epithelia. 1,6 In addition, recent studies have shown that in some organs, precursor cells responsible for homeostatic cell turnover are different from those responsible for cell replacement after injury. For example, stem cells in the bulge of the hair follicle contribute to wound repair, 5 but normal cell turnover of the epidermis is maintained by a distinct progenitor population located in the interfollicular epidermis. 7 In the olfactory neuroepithelium, a site of continuous neurogenesis under normal conditions in the adult, new neurons are generated by the globose basal cells that reside in the basal germinal zone of the pseudostratified olfactory neuroepithelium; however, after extensive tissue damage, another population of cells (horizontal basal cells) starts proliferating and generates several cell types. 8 Finally, ␤ cells of the pancreas are maintained by proliferation of terminally differentiated ␤ cells, 9,10 but in injured pancreas, multipotent progenitor cells can give rise to ␤ cells. 11 The normal adult kidney has a very low rate of cell turnover, 12 but it is capable of responding quickly to injury by generating new cells to replace

The Notch signaling pathway controls cell fates through interactions between neighboring cells by positively or negatively affecting the processes of proliferation, differentiation and apoptosis in a context-dependent manner. This pathway has been implicated in human cancer as both an oncogene and a tumor suppressor. Here we report new inactivating mutations in Notch pathway components in over 40% of human bladder cancers examined. Bladder cancer is the fourth most commonly diagnosed malignancy in the male population of the United States. Thus far, driver mutations in fibroblast growth factor receptor 3 (FGFR3) and, less commonly, in RAS proteins have been identified. We show that Notch activation in bladder cancer cells suppresses proliferation both in vitro and in vivo by directly upregulating dual-specificity phosphatases (DUSPs), thus reducing the phosphorylation of ERK1 and ERK2 (ERK1/2). In mouse models, genetic inactivation of Notch signaling leads to Erk1/2 phosphorylation, resulting in tumorigenesis in the urinary tract. Collectively our findings show that loss of Notch activity is a driving event in urothelial cancer.