Lluis Espinosa

Jagged1 is the pathological link between Wnt and Notch pathways in colorectal cancer.

Notch has been linked to β-catenin-dependent tumorigenesis; however, the mechanisms leading to Notch activation and the contribution of the Notch pathway to colorectal cancer is not yet understood. By microarray analysis, we have identified a group of genes downstream of Wnt/β-catenin (down-regulated when blocking Wnt/β-catenin) that are directly regulated by Notch (repressed by γ-secretase inhibitors and up-regulated by active Notch1 in the absence of β-catenin signaling). We demonstrate that Notch is downstream of Wnt in colorectal cancer cells through β-catenin-mediated transcriptional activation of the Notch-ligand Jagged1. Consistently, expression of activated Notch1 partially reverts the effects of blocking Wnt/β-catenin pathway in tumors implanted s.c. in nude mice. Crossing APCMin/+ with Jagged1+/Δ mice is sufficient to significantly reduce the size of the polyps arising in the APC mutant background indicating that Notch is an essential modulator of tumorigenesis induced by nuclear β-catenin. We show that this mechanism is operating in human tumors from Familial Adenomatous Polyposis patients. We conclude that Notch activation, accomplished by β-catenin-mediated up-regulation of Jagged1, is required for tumorigenesis in the intestine. The Notch-specific genetic signature is sufficient to block differentiation and promote vasculogenesis in tumors whereas proliferation depends on both pathways.

Dll1- and Dll4-Mediated Notch Signaling Are Required for Homeostasis of Intestinal Stem Cells

BACKGROUND & AIMS Ablation of Notch signaling within the intestinal epithelium results in loss of proliferating crypt progenitors due to their conversion into postmitotic secretory cells. We aimed to confirm that Notch was active in stem cells (SCs), investigate consequences of loss of Notch signaling within the intestinal SC compartment, and identify the physiologic ligands of Notch in mouse intestine. Furthermore, we investigated whether the induction of goblet cell differentiation that results from loss of Notch requires the transcription factor Krüppel-like factor 4 (Klf4). METHODS Transgenic mice that carried a reporter of Notch1 activation were used for lineage tracing experiments. The in vivo functions of the Notch ligands Jagged1 (Jag1), Delta-like1 (Dll1), Delta-like4 (Dll4), and the transcription factor Klf4 were assessed in mice with inducible, gut-specific gene targeting (Vil-Cre-ER(T2)). RESULTS Notch1 signaling was found to be activated in intestinal SCs. Although deletion of Jag1 or Dll4 did not perturb the intestinal epithelium, inactivation of Dll1 resulted in a moderate increase in number of goblet cells without noticeable effects of progenitor proliferation. However, simultaneous inactivation of Dll1 and Dll4 resulted in the complete conversion of proliferating progenitors into postmitotic goblet cells, concomitant with loss of SCs (Olfm4(+), Lgr5(+), and Ascl2(+)). Klf4 inactivation did not interfere with goblet cell differentiation in adult wild-type or in Notch pathway-deficient gut. CONCLUSIONS Notch signaling in SCs and progenitors is activated by Dll1 and Dll4 ligands and is required for maintenance of intestinal progenitor and SCs. Klf4 is dispensable for goblet cell differentiation in intestines of adult Notch-deficient mice.

RBPjκ-dependent Notch function regulates Gata2 and is essential for the formation of intra-embryonic hematopoietic cells

Definitive hematopoiesis in the mouse embryo originates from the aortic floor in the P-Sp/AGM region in close association with endothelial cells. An important role for Notch1 in the control of hematopoietic ontogeny has been recently established, although its mechanism of action is poorly understood. Here, we show detailed analysis of Notch family gene expression in the aorta endothelium between embryonic day (E) 9.5 and E10.5. Since Notch requires binding to RBPjκ transcription factor to activate transcription, we analyzed the aorta of the para-aortic splanchnopleura/AGM in RBPjκ mutant embryos. We found specific patterns of expression of Notch receptors, ligands and Hes genes that were lost in RBPjκ mutants. Analysis of these mutants revealed the absence of hematopoietic progenitors, accompanied by the lack of expression of the hematopoietic transcription factors Aml1/Runx1, Gata2 and Scl/Tal1. We show that in wild-type embryos, a few cells lining the aorta endothelium at E9.5 simultaneously expressed Notch1 and Gata2, and demonstrate by chromatin immunoprecipitation that Notch1 specifically associated with the Gata2 promoter in E9.5 wild-type embryos and 32D myeloid cells, an interaction lost in RBPjκ mutants. Consistent with a role for Notch1 in regulating Gata2, we observe increased expression of this gene in 32D cells expressing activated Notch1. Taken together, these data strongly suggest that activation of Gata2 expression by Notch1/RBPjκ is a crucial event for the onset of definitive hematopoiesis in the embryo.

RBPJκ-Dependent Signaling Is Essential for Long-Term Maintenance of Neural Stem Cells in the Adult Hippocampus

The generation of new neurons from neural stem cells in the adult hippocampal dentate gyrus contributes to learning and mood regulation. To sustain hippocampal neurogenesis throughout life, maintenance of the neural stem cell pool has to be tightly controlled. We found that the Notch/RBPJκ-signaling pathway is highly active in neural stem cells of the adult mouse hippocampus. Conditional inactivation of RBPJκ in neural stem cells in vivo resulted in increased neuronal differentiation of neural stem cells in the adult hippocampus at an early time point and depletion of the Sox2-positive neural stem cell pool and suppression of hippocampal neurogenesis at a later time point. Moreover, RBPJκ-deficient neural stem cells displayed impaired self-renewal in vitro and loss of expression of the transcription factor Sox2. Interestingly, we found that Notch signaling increases Sox2 promoter activity and Sox2 expression in adult neural stem cells. In addition, activated Notch and RBPJκ were highly enriched on the Sox2 promoter in adult hippocampal neural stem cells, thus identifying Sox2 as a direct target of Notch/RBPJκ signaling. Finally, we found that overexpression of Sox2 can rescue the self-renewal defect in RBPJκ-deficient neural stem cells. These results identify RBPJκ-dependent pathways as essential regulators of adult neural stem cell maintenance and suggest that the actions of RBPJκ are, at least in part, mediated by control of Sox2 expression.

The Notch/Hes1 Pathway Sustains NF-κB Activation through CYLD Repression in T Cell Leukemia

It was previously shown that the NF-κB pathway is downstream of oncogenic Notch1 in T cell acute lymphoblastic leukemia (T-ALL). Here, we visualize Notch-induced NF-κB activation using both human T-ALL cell lines and animal models. We demonstrate that Hes1, a canonical Notch target and transcriptional repressor, is responsible for sustaining IKK activation in T-ALL. Hes1 exerts its effects by repressing the deubiquitinase CYLD, a negative IKK complex regulator. CYLD expression was found to be significantly suppressed in primary T-ALL. Finally, we demonstrate that IKK inhibition is a promising option for the targeted therapy of T-ALL as specific suppression of IKK expression and function affected both the survival of human T-ALL cells and the maintenance of the disease in vivo.

Impaired embryonic haematopoiesis yet normal arterial development in the absence of the Notch ligand Jagged1.

Specific deletion of Notch1 and RBPjκ in the mouse results in abrogation of definitive haematopoiesis concomitant with the loss of arterial identity at embryonic stage. As prior arterial determination is likely to be required for the generation of embryonic haematopoiesis, it is difficult to establish the specific haematopoietic role of Notch in these mutants. By analysing different Notch‐ligand‐null embryos, we now show that Jagged1 is not required for the establishment of the arterial fate but it is required for the correct execution of the definitive haematopoietic programme, including expression of GATA2 in the dorsal aorta. Moreover, successful haematopoietic rescue of the Jagged1‐null AGM cells was obtained by culturing them with Jagged1‐expressing stromal cells or by lentiviral‐mediated transduction of the GATA2 gene. Taken together, our results indicate that Jagged1‐mediated activation of Notch1 is responsible for regulating GATA2 expression in the AGM, which in turn is essential for definitive haematopoiesis in the mouse.

Hematopoietic stem cells: to be or Notch to be

Notch is a well-conserved signaling pathway and its function in cell fate determination is crucial in embryonic development and in the maintenance of tissue homeostasis during adult life. Notch activation depends on cell-cell interactions that are essential for the generation of cell diversity from initially equivalent cell populations. In the adult hematopoiesis, Notch is undoubtedly a very efficient promoter of T-cell differentiation, and this has masked for a long time the effects of Notch on other blood lineages, which are gradually being identified. However, the adult hematopoietic stem cell (HSC) remains mostly refractory to Notch intervention in experimental systems. In contrast, Notch is essential for the generation of the HSCs, which takes place during embryonic development. This review summarizes the knowledge accumulated in recent years regarding the role of the Notch pathway in the different stages of HSC ontology from embryonic life to fetal and adult bone marrow stem cells. In addition, we briefly examine other systems where Notch regulates specific stem cell capacities, in an attempt to understand how Notch functions in stem cell biology.

Nuclear IKK activity leads to dysregulated Notch-dependent gene expression in colorectal cancer

Nuclear functions for IκB kinase (IKK), including phosphorylation of histone H3 and nuclear corepressors, have been recently described. Here, we show that IKK is activated in colorectal tumors concomitant with the presence of phosphorylated SMRT (silencing mediator of retinoic acid and thyroid hormone receptor) corepressor that is aberrantly localized in the cytoplasm. In these tumors, IKKα associates to the chromatin of specific Notch targets, leading to the release of SMRT. Abrogation of IKK activity by BAY11-7082 or by expressing dominant negative IKKα restores the association of SMRT with Notch target genes, resulting in specific gene repression. Finally, BAY11-7082 significantly reduces tumor size in colorectal cancer xenografts (CRC-Xs) implanted in nude mice.

Hematopoietic stem cell development requires transient Wnt/β-catenin activity

Deletion of β-catenin from mouse embryonic endothelium, but not embryonic hematopoietic cells, prevents hematopoietic differentiation; thus Wnt/β-catenin signaling is needed for emergence but not maintenance of HSCs.

The Notch pathway in the developing hematopoietic system.

The main function of the Notch signaling pathway is to generate cell diversity during both embryonic development and adult tissue homeostasis. The extended use of this pathway, together with its conservation during evolution, is indicative of its importance. During embryonic development, the vascular and hematopoietic systems are intimately associated and Notch signals are responsible for the correct specification of both systems. More explicitly, Notch is required for the induction of the arterial program; however, it is simultaneously or consecutively also involved in the generation of hematopoietic stem cells. Although both genetic programs are different, they are both implemented in endothelial cells of the dorsal aorta in the midgestation embryo. This close association during the development of arteries and blood has hindered our understanding of Notch function in the generation of hematopoietic stem cells. Here, we will review the work from recent years showing how Notch participates in the embryonic development of hematopoiesis in the mouse, but also in other organisms such as chick, zebrafish and flies.