Entry into the thymic microenvironment triggers Notch activation in the earliest migrant T cell progenitors

Interactions between T cell precursors and thymic stromal cells are essential during thymocyte development. However, the role of the thymus in initial commitment of lymphoid progenitors to the T lineage remains controversial, with data providing evidence for both extra- and intrathymic commitment mechanisms. In this context, it is clear that Notch1 is an important mediator during initiation of T cell development. Here we have analyzed the mechanisms regulating Notch activation in lymphoid precursors at extrathymic sites and in the thymus, including stages representing the first wave of embryonic thymus colonization on embryonic day 12 of gestation. We show that Notch activation in migrant lymphoid precursors requires entry into the thymic microenvironment where they are exposed to Notch ligands expressed by immature thymic epithelial cells. Moreover, continued Notch signaling in such precursors requires sustained interactions with Notch ligands. Collectively, these findings suggest a role for Notch in an intrathymic mechanism of T cell lineage commitment involving sustained interactions with Notch ligand bearing thymic epithelium.     

Normal immune system development in mice lacking the Deltex-1 RING finger domain

The Notch signaling pathway controls several cell fate decisions during lymphocyte development, from T-cell lineage commitment to the peripheral differentiation of B and T lymphocytes. Deltex-1 is a RING finger ubiquitin ligase which is conserved from Drosophila to humans and has been proposed to be a regulator of Notch signaling. Its pattern of lymphoid expression as well as gain-of-function experiments suggest that Deltex-1 regulates both B-cell lineage and splenic marginal-zone B-cell commitment. Deltex-1 was also found to be highly expressed in germinal-center B cells. To investigate the physiological function of Deltex-1, we generated a mouse strain lacking the Deltex-1 RING finger domain, which is essential for its ubiquitin ligase activity. Deltex-1(Delta/Delta) mice were viable and fertile. A detailed histological analysis did not reveal any defects in major organs. T- and B-cell development was normal, as were humoral responses against T-dependent and T-independent antigens. These data indicate that the Deltex-1 ubiquitin ligase activity is dispensable for mouse development and immune function. Possible compensatory mechanisms, in particular those from a fourth Deltex gene identified during the course of this study, are also discussed.     

Surface expression of Notch1 on thymocytes: correlation with the double-negative to double-positive transition

Notch1 plays a critical role in regulating T lineage commitment during the differentiation of lymphoid precursors. The physiological relevance of Notch1 signaling during subsequent stages of T cell differentiation has been more controversial. This is due in part to conflicting data from studies examining the overexpression or targeted deletion of Notch1 and to difficulties in distinguishing between the activities of multiple Notch family members and their ligands, which are expressed in the thymus. We employed a polyclonal antiserum against the extracellular domain of Notch1 to study surface expression during thymopoiesis. We found high levels of Notch1 on the cell surface only on double negative (DN) stage 2 through the immature single-positive stage of thymocyte development, before the double-positive (DP) stage. The Notch signaling pathway, as read out by Deltex1 expression levels, is highly active in DN thymocytes. When an active Notch1 transgene, Notch1IC, is exogenously introduced into thymocytes of recombinase-activating gene 2-deficient mice, it promotes proliferation and development to the DP stage following anti-CD3 treatment without apparently affecting the intensity of pre-TCR signaling. In addition, a stromal cell line expressing the Notch ligand, Delta-like-1, promotes the in vitro expansion of wild-type DN3 thymocytes in vitro. Consistent with other recent reports, these data suggest a role for Notch1 during the DN to DP stage of thymocyte maturation and suggest a cellular mechanism by which Notch1IC oncogenes could contribute to thymoma development and maintenance.     

Notch1 signaling regulates chondrogenic lineage determination through Sox9 activation

Notch signaling is involved in several cell lineage determination processes during embryonic development. Recently, we have shown that Sox9 is most likely a primary target gene of Notch1 signaling in embryonic stem cells (ESCs). By using our in vitro differentiation protocol for chondrogenesis from ESCs through embryoid bodies (EBs) together with our tamoxifen-inducible system to activate Notch1, we analyzed the function of Notch signaling and its induction of Sox9 during EB differentiation towards the chondrogenic lineage. Temporary activation of Notch1 during early stages of EB, when lineage determination occurs, was accompanied by rapid and transient Sox9 upregulation and resulted in induction of chondrogenic differentiation during later stages of EB cultivation. Using siRNA targeting Sox9, we knocked down and adjusted this early Notch1-induced Sox9 expression peak to non-induced levels, which led to reversion of Notch1-induced chondrogenic differentiation. In contrast, continuous Notch1 activation during EB cultivation resulted in complete inhibition of chondrogenic differentiation. Furthermore, a reduction and delay of cardiac differentiation observed in EBs after early Notch1 activation was not reversed by siRNA-mediated Sox9 knockdown. Our data indicate that Notch1 signaling has an important role during early stages of chondrogenic lineage determination by regulation of Sox9 expression.     

Notch signaling augments T cell responsiveness by enhancing CD25 expression

Notch receptors signal through a highly conserved pathway to influence cell fate decisions. Notch1 is required for T lineage commitment; however, a role for Notch signaling has not been clearly defined for the peripheral T cell response. Notch gene expression is induced, and Notch1 is activated in primary CD4(+) T cells following specific peptide-Ag stimulation. Notch activity contributes to the peripheral T cell response, as inhibition of endogenous Notch activation decreases the proliferation of activated T cells in a manner associated with the diminished production of IL-2 and the expression of the high affinity IL-2R (CD25). Conversely, forced expression of a constitutively active Notch1 in primary T cells results in increased surface expression of CD25, and renders these cells more sensitive to both cognate Ag and IL-2, as measured by cell division. These data suggest an important role for Notch signaling during CD4(+) T cell responses, which operates through augmenting a positive feedback loop involving IL-2 and its high affinity receptor.     

Impairment of thymocyte development by dominant-negative Kuzbanian (ADAM-10) is rescued by the Notch ligand, delta-1

Although Notch plays a crucial role in T cell development, regulation of Notch signaling in the thymus is not well understood. Kuzbanian, an ADAM protease, has been implicated in the cleavage of both Notch receptors and the Notch ligand, Delta. In this study we show that the expression of a dominant-negative form of Kuzbanian (dnKuz) leads to reduced TCRbeta expression in double-negative thymocytes and to a partial block between the double-negative to double-positive stages of development. These defects were rescued by overexpression of Delta-1 on thymocytes. Mixed chimeras showed a cell-autonomous block by dnKuz, but non-cell-autonomous rescue by Delta-1. This suggests that dnKuz impairs Notch signaling in receiving cells, and increasing Delta-1 on sending cells overcomes this defect. Interestingly, the expression of an activated form of Notch-1 rescued some, but not all, the defects in dnKuz Tg mice. Our data suggest that multiple Notch-dependent steps in early thymocyte development require Kuzbanian, but differ in the involvement of other Notch signaling components.     

Notch signaling regulates mammary stem cell function and luminal cell-fate commitment

The recent identification of mouse mammary stem cells (MaSCs) and progenitor subpopulations has enhanced the prospect of investigating the genetic control of their lineage specification and differentiation. Here we have explored the role of the Notch pathway within the mammary epithelial hierarchy. We show that knockdown of the canonical Notch effector Cbf-1 in the MaSC-enriched population results in increased stem cell activity in vivo as well as the formation of aberrant end buds, implying a role for endogenous Notch signaling in restricting MaSC expansion. Conversely, Notch was found to be preferentially activated in the ductal luminal epithelium in vivo and promoted commitment of MaSCs exclusively along the luminal lineage. Notably, constitutive Notch signaling specifically targeted luminal progenitor cells for expansion, leading to hyperplasia and tumorigenesis. These findings reveal key roles for Notch signaling in MaSCs and luminal cell commitment and further suggest that inappropriate Notch activation promotes the self-renewal and transformation of luminal progenitor cells.     

Activated Notch2 potentiates CD8 lineage maturation and promotes the selective development of B1 B cells

Although studies have shown that the Notch2 family member is critical for embryonic development, little is known concerning its role in hematopoiesis. In this study, we show that the effects of an activated form of Notch2 (N2IC) on the T-cell lineage are dosage related. High-level expression of N2IC results in the development of T-cell leukemias. In contrast, lower-level expression of N2IC does not lead to transformation but skews thymocyte development to the CD8 lineage. Underlying this skew is a dramatic enhancement in positive selection and CD8SP maturation. N2IC permits early B-cell development but blocks the maturation of conventional B2 cells at the pre-B stage, which is the limit of endogenous Notch2 protein expression in developing B cells. Most strikingly, while B2 B cell development is blocked at the pre-B-cell stage, N2IC promotes the selective development of LPS-responsive B1 B cells. This study implicates a role for Notch2 in the maturation of the CD8 lineage and suggests a novel function for Notch2 in the development of the B1 B-cell subset.     

Conservation of the Notch1 signaling pathway in gastrointestinal carcinoid cells

Gastrointestinal (GI) carcinoid cells secrete multiple neuroendocrine (NE) markers and hormones including 5-hydroxytryptamine and chromogranin A. We were interested in determining whether activation of the Notch1 signal transduction pathway in carcinoid cells could modulate production of NE markers and hormones. Human pancreatic carcinoid cells (BON cells) were stably transduced with an estrogen-inducible Notch1 construct, creating BON-NIER cells. In the present study, we found that Notch1 is not detectable in human GI carcinoid tumor cells. The induction of Notch1 in human BON carcinoid cells led to high levels of functional Notch1, as measured by CBF-1 binding studies, resulting in activation of the Notch1 pathway. Similar to its developmental role in the GI tract, Notch1 pathway activation led to an increase in hairy enhancer of split 1 (HES-1) protein and a concomitant silencing of human Notch1/HES-1/achaete-scute homolog 1. Furthermore, Notch1 activation led to a significant reduction in NE markers. Most interestingly, activation of the Notch1 pathway caused a significant reduction in 5-hydroxytryptamine, an important bioactive hormone in carcinoid syndrome. In addition, persistent activation of the Notch1 pathway in BON cells led to a notable reduction in cellular proliferation. These results demonstrate that the Notch1 pathway, which plays a critical role in the differentiation of enteroendocrine cells, is highly conserved in the gut. Therefore, manipulation of the Notch1 signaling pathway may be useful for expanding the targets for therapeutic and palliative treatment of patients with carcinoid tumors.     

Notch promotes neural lineage entry by pluripotent embryonic stem cells

A central challenge in embryonic stem (ES) cell biology is to understand how to impose direction on primary lineage commitment. In basal culture conditions, the majority of ES cells convert asynchronously into neural cells. However, many cells resist differentiation and others adopt nonneural fates. Mosaic activation of the neural reporter Sox-green fluorescent protein suggests regulation by cell-cell interactions. We detected expression of Notch receptors and ligands in mouse ES cells and investigated the role of this pathway. Genetic manipulation to activate Notch constitutively does not alter the stem cell phenotype. However, upon withdrawal of self-renewal stimuli, differentiation is directed rapidly and exclusively into the neural lineage. Conversely, pharmacological or genetic interference with Notch signalling suppresses the neural fate choice. Notch promotion of neural commitment requires parallel signalling through the fibroblast growth factor receptor. Stromal cells expressing Notch ligand stimulate neural specification of human ES cells, indicating that this is a conserved pathway in pluripotent stem cells. These findings define an unexpected and decisive role for Notch in ES cell fate determination. Limiting activation of endogenous Notch results in heterogeneous lineage commitment. Manipulation of Notch signalling is therefore likely to be a key factor in taking command of ES cell lineage choice.     

Notch1 intracellular domain increases cytoplasmic EZH2 levels during early megakaryopoiesis

Notch pathway is a well-known factor in the development of lymphoid lineage. However, its role in the myeloid lineage has remained ambiguous. We looked into the effect of Notch1 on the megakaryocytic lineage commitment and found an increase in megakaryocyte-specific lineage markers upon transfection with Notch1 intracellular domain (NICD). This effect was mediated by Akt whereby constitutive activation of Akt increased the megakaryocyte markers, whereas inhibition of Akt signalling reduced these marker levels. Along with the change in differentiation status, NICD-induced initiation of early megakaryopoiesis was accompanied by an increased cytoplasmic enhancer of zeste homolog-2 (EZH2) expression. This process was found to be Akt-dependent, and inhibition or overexpression of Akt lead to concurrent changes in EZH2 levels. To elucidate the function of EZH2 in the cytoplasm, novel cytoplasmic interactors of EZH2 were identified by co-immunoprecipitation followed by matrix-assisted laser desorption ionization MS/MS-based protein identification, and thus, PDIA1 and LIM domain kinase-1 (LIMK1) were identified. Interaction of EZH2 with LIMK1 changed the activity of cofilin (a downstream target of LIMK1) towards actin filaments, thereby leading to lower filamentous actin content within these cells. Thus, Notch1 not only induces early megakaryopoiesis but also prepares these cells for subsequent morphological changes.     

Critical regulation of bone morphogenetic protein-induced osteoblastic differentiation by Delta1/Jagged1-activated Notch1 signaling

Functional involvement of the Notch pathway in osteoblastic differentiation has been previously investigated using the truncated intracellular domain, which mimics Notch signaling by interacting with the DNA-binding protein CBF-1. However, it is unclear whether Notch ligands Delta1 and Jagged1 also induce an identical cellular response in osteoblastic differentiation. We have shown that both Delta1 and Jagged1 were expressed concomitantly with Notch1 in maturating osteoblastic cells during bone regeneration and that overexpressed and immobilized recombinant Delta1 and Jagged1 alone did not alter the differentiated state of MC3T3-E1 and C2C12 cells. However, they augmented bone morphogenetic protein-2 (BMP2)-induced alkaline phosphatase activity and the expression of several differentiation markers, except for osteocalcin, and ultimately enhanced calcified nodule and in vivo ectopic bone formation of MC3T3-E1. In addition, both ligands transmitted signal through the CBF-1-dependent pathway and stimulated the expression of HES-1, a direct target of Notch pathway. To test the necessity of Notch signaling in BMP2-induced differentiation, Notch signaling was inhibited by the dominant negative extracellular domain of Notch1, specific inhibitor, or small interference RNA. These treatments decreased alkaline phosphatase activity as well as the expression of other differentiation markers and inhibited the promoter activity of Id-1, a target gene of the BMP pathway. These results indicate the functional redundancy between Delta1 and Jagged1 in osteoblastic differentiation whereby Delta1/Jagged1-activated Notch1 enhances BMP2-induced differentiation through the identical signaling pathway. Furthermore, our data also suggest that functional Notch signaling is essential not only for BMP2-induced osteoblast differentiation but also for BMP signaling itself.     

Notch1 perturbation of hemopoiesis involves non-cell- autonomous modifications

To study the effects of Notch on hemopoiesis we used a bone marrow transduction/transplantation model and compared the transduced and nontransduced populations in reconstituted mice. While cells expressing a constitutively active form of murine Notch1 (Notch1IC) completely lacked B cells, a profound suppression of the B lineage was also seen in the nontransduced compartment. Experiments performed with retroviral supernatants of varying titers showed that the perturbations of B cell development among the nontransduced population correlated with the percentage of Notch1IC-transduced cells inoculated into the mice. The myeloid lineage of the Notch1IC-transplanted mice was altered as well, and this also affected the nontransduced population that had features of excessive maturation. To explore the basis of these non-cell-autonomous modifications we prepared conditioned medium from ex vivo cultures of Notch1IC-transplanted mice bone marrow and showed that it inhibited B cell maturation and promoted myeloid differentiation in a dose-dependent manner. Finally, we found that the T cell leukemia/lymphomas that occur in Notch1IC-transplanted mice were accompanied by abnormal maturation of nontransduced T cells in the bone marrow. These findings indicate that modifications of neighboring cells through non-cell-autonomous modifications take part in multiple facets of the activity of Notch on hemopoiesis.     

Activation dynamics and signaling properties of Notch3 receptor in the developing pulmonary artery

Notch3 signaling is fundamental for arterial specification of systemic vascular smooth muscle cells (VSMCs). However, the developmental role and signaling properties of the Notch3 receptor in the mouse pulmonary artery remain unknown. Here, we demonstrate that Notch3 is expressed selectively in pulmonary artery VSMCs, is activated from late fetal to early postnatal life, and is required to maintain the morphological characteristics and smooth muscle gene expression profile of the pulmonary artery after birth. Using a conditional knock-out mouse model, we show that Notch3 receptor activation in VSMCs is Jagged1-dependent. In vitro VSMC lentivirus-mediated Jagged1 knockdown, confocal localization analysis, and co-culture experiments revealed that Notch3 activation is cell-autonomous and occurs through the physical engagement of Notch3 and VSMC-derived Jagged1 in the interior of the same cell. Although the current models of mammalian Notch signaling involve a two-cell system composed of a signal-receiving cell that expresses a Notch receptor on its surface and a neighboring signal-sending cell that provides membrane-bound activating ligand, our data suggest that pulmonary artery VSMC Notch3 activation is cell-autonomous. This unique mechanism of Notch activation may play an important role in the maturation of the pulmonary artery during the transition to air breathing.     

Activation of Notch1 promotes development of human CD8 single positive T cells in humanized mice

Notch1 mutations are found in more than 50% of human T cell acute lymphoblastic leukemia (T-ALL) cells. However, the functions of Notch1 for human T cell development and leukemogenesis are not well understood. To examine the role of Notch1, human hematopoietic stem cells (HSCs), which had been transduced with a constitutively active form of Notch1 (ICN1), were transplanted into severely immunodeficient NOD/Shi-scid-IL2rγ^null (NOG) mice. We found that the great majority of the ICN1-expressing hematopoietic cells in the bone marrow expressed surface markers for T cells, such as CD3, CD4, and CD8, and that this T cell development was independent of the thymus. Accordingly, phenotypically mature CD8⁺ single positive (SP) T cells were observed in the spleen. Furthermore, T-ALL developed in one NOG recipient mouse out of 26 that had been secondary transferred with the T cells developed in the first NOG mice. These results indicate that Notch1 signaling in HSCs promotes CD8⁺ SP T cell development, and that T cell leukemogenesis may require additional oncogenic factors other than Notch1 activation.