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.     

Notch signaling respecifies the hemangioblast to a cardiac fate

To efficiently generate cardiomyocytes from embryonic stem (ES) cells in culture it is essential to identify key regulators of the cardiac lineage and to develop methods to control them. Using a tet-inducible mouse ES cell line to enforce expression of a constitutively activated form of the Notch 4 receptor, we show that signaling through the Notch pathway can efficiently respecify hemangioblasts to a cardiac fate, resulting in the generation of populations consisting of >60% cardiomyocytes. Microarray analyses reveal that this respecification is mediated in part through the coordinated regulation of the BMP and Wnt pathways by Notch signaling. Together, these findings have uncovered a potential role for the Notch pathway in cardiac development and provide an approach for generating large numbers of cardiac progenitors from ES cells.     

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. Here, we identify two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers.     

Two Pore Channel 2 Differentially Modulates Neural Differentiation of Mouse Embryonic Stem Cells

Nicotinic acid adenine dinucleotide phosphate (NAADP) is an endogenous Ca²⁺ mobilizing nucleotide presented in various species. NAADP mobilizes Ca²⁺ from acidic organelles through two pore channel 2 (TPC2) in many cell types and it has been previously shown that NAADP can potently induce neuronal differentiation in PC12 cells. Here we examined the role of TPC2 signaling in the neural differentiation of mouse embryonic stem (ES) cells. We found that the expression of TPC2 was markedly decreased during the initial ES cell entry into neural progenitors, and the levels of TPC2 gradually rebounded during the late stages of neurogenesis. Correspondingly, TPC2 knockdown accelerated mouse ES cell differentiation into neural progenitors but inhibited these neural progenitors from committing to neurons. Overexpression of TPC2, on the other hand, inhibited mouse ES cell from entering the early neural lineage. Interestingly, TPC2 knockdown had no effect on the differentiation of astrocytes and oligodendrocytes of mouse ES cells. Taken together, our data indicate that TPC2 signaling plays a temporal and differential role in modulating the neural lineage entry of mouse ES cells, in that TPC2 signaling inhibits ES cell entry to early neural progenitors, but is required for late neuronal differentiation.     

Direct neural fate specification from embryonic stem cells: a primitive mammalian neural stem cell stage acquired through a default mechanism

Little is known about how neural stem cells are formed initially during development. We investigated whether a default mechanism of neural specification could regulate acquisition of neural stem cell identity directly from embryonic stem (ES) cells. ES cells cultured in defined, low-density conditions readily acquire a neural identity. We characterize a novel primitive neural stem cell as a component of neural lineage specification that is negatively regulated by TGFbeta-related signaling. Primitive neural stem cells have distinct growth factor requirements, express neural precursor markers, generate neurons and glia in vitro, and have neural and non-neural lineage potential in vivo. These results are consistent with a default mechanism for neural fate specification and support a model whereby definitive neural stem cell formation is preceded by a primitive neural stem cell stage during neural lineage commitment.     

Notch1-mediated signaling regulates proliferation of porcine satellite cells (PSCs)

Notch signaling is an evolutionarily conserved cell–cell communication mechanism involved in the regulation of cell proliferation, differentiation and fate decisions of mammalian cells. In the present study, we investigated the possible requirement for Notch signaling in the proliferation and differentiation of porcine satellite cells. We show that Notch1, 2 and 3 are expressed in cultured porcine satellite cells. Knock-down of NOTCH1, but not NOTCH2 and NOTCH3, decreases the proliferation of porcine satellite cells. In contrast, enhancement of NOTCH1 expression via treatment of porcine satellite cells with recombinant NF-κB increases the proliferation of porcine satellite cells. The alteration of porcine satellite cell proliferation is associated with significant changes in the expression of cell cycle related genes (cyclin B1, D1, D2, E1 and p21), myogenic regulatory factors (MyoD and myogenin) and the Notch effector Hes5. In addition, alteration of Notch1 expression in porcine satellite cells causes changes in the expression of GSK3β-3. Taken together, these findings suggest that of the four notch-related genes, Notch1is likely to be required for regulating the proliferation and therefore the maintenance of porcine satellite cells in vivo, and do so through activation of the Notch effector gene Hes5.     

Visualization of spatiotemporal activation of Notch signaling: live monitoring and significance in neural development

Notch signaling plays various key roles in cell fate determination during CNS development in a context-dependent fashion. However, its precise physiological role and the localization of its target cells remain unclear. To address this issue, we developed a new reporter system for assessing the RBP-J-mediated activation of Notch signaling target genes in living cells and tissues using a fluorescent protein Venus. Our reporter system revealed that Notch signaling is selectively activated in neurosphere-initiating multipotent neural stem cells in vitro and in radial glia in the embryonic forebrain in vivo. Furthermore, the activation of Notch signaling occurs during gliogenesis and is required in the early stage of astroglial development. Consistent with these findings, the persistent activation of Notch signaling inhibits the differentiation of GFAP-positive astrocytes. Thus, the development of our RBP-J-dependent live reporter system, which is activated upon Notch activation, together with a stage-dependent gain-of-function analysis allowed us to gain further insight into the complexity of Notch signaling in mammalian CNS development.     

Notch activation induces endothelial cell cycle arrest and participates in contact inhibition: role of p21Cip1 repression

Although previous studies demonstrate that appropriate Notch signaling is required during angiogenesis and in vascular homeostasis, the mechanisms by which Notch regulates vascular function remain to be elucidated. Here, we show that activation of the Notch pathway by the ligand Jagged1 reduces the proliferation of endothelial cells. Notch activation inhibits proliferation of endothelial cells in a cell-autonomous manner by inhibiting phosphorylation of the retinoblastoma protein (Rb). During cell cycle entry, p21Cip1 is upregulated in endothelial cells. Activated Notch inhibits mitogen-induced upregulation of p21Cip1 and delays cyclin D-cdk4-mediated Rb phosphorylation. Notch-dependent repression of p21Cip1 prevents nuclear localization of cyclin D and cdk4. The necessity of p21Cip1 for nuclear translocation of cyclin D-cdk4 and S-phase entry in endothelial cells was demonstrated by targeted downregulation of p21Cip1 by using RNA interference. We further demonstrate that when endothelial cells reach confluence, Notch is activated and p21Cip1 is downregulated. Inhibition of the Notch pathway at confluence prevents p21Cip1 downregulation and induces Rb phosphorylation. We suggest that Notch activation contributes to contact inhibition of endothelial cells, in part through repression of p21Cip1 expression.     

The Notch pathway: hair graying and pigment cell homeostasis

The Notch signaling pathway is an essential cell-cell interaction mechanism, which regulates processes such as cell proliferation, cell fate decisions, differentiation or stem cell maintenance. Pigmentation in mammals is provided by melanocytes, which are derived from the neural crest, and by the retinal pigment epithelium (RPE), which is part of the optic cup and hence orginates from neuroectoderm. The importance of functional Notch signaling in melanocytes has been unveiled recently. Here, the pathway is essential for the maintenance of proper hair pigmentation. Deletion of Notch1 and Notch2 or RBP-Jkappa in the melanocyte lineage resulted in a gene dosage-dependent precocious hair graying, due to the elimination of melanoblasts and melanocyte stem cells. Expression data support the idea that Notch signaling might equally be involved in development of the RPE. Furthermore, recent analyses indicate a possible role of Notch signaling in the development of melanoma. In this review, we address the essential role of Notch signaling in the regeneration of the melanocyte population during hair follicle cycles, and discuss data supporting the implication of this signaling pathway in RPE development and melanoma.     

Notch1 Signaling Regulates the Proliferation and Self-Renewal of Human Dental Follicle Cells by Modulating the G1/S Phase Transition and Telomerase Activity

Multipotent human dental follicle cells (HDFCs) have been intensively studied in periodontal regeneration research, yet the role of Notch1 in HDFCs has not been fully understood. The aim of the current study is to explore the role of Notch1 signaling in HDFCs self-renewal and proliferation. HDFCs were obtained from the extracted wisdom teeth from adolescent patients. Regulation of Notch1 signaling in the HDFCs was achieved by overexpressing the exogenous intracellular domain of Notch1 (ICN1) or silencing Notch1 by shRNA. The regulatory effects of Notch1 on HDFC proliferation, cell cycle distribution and the expression of cell cycle regulators were investigated through various molecular technologies, including plasmid construction, retrovirus preparation and infection, qRT-PCR, western blot, RBP-Jk luciferase reporter and cell proliferation assay. Our data clearly show that constitutively activation of Notch1 stimulates the HDFCs proliferation while inhibition of the Notch1 suppresses their proliferation in vitro. In addition, the HDFCs proliferation is associated with the increased expression of cell cycle regulators, e.g. cyclin D1, cyclin D2, cyclin D3, cyclin E1, CDK2, CDK4, CDK6, and SKP2 and the decreased expression of p27 ^kip1. Moreover, our data show that the G1/S phase transition (indicating proliferation) and telomerase activity (indicating self-renewal) can be enhanced by overexpression of ICN1 but halted by inhibition of Notch1. Together, the current study provides evidence for the first time that Notch1 signaling regulates the proliferation and self-renewal capacity of HDFCs through modulation of the G1/S phase transition and the telomerase activity.     

The regulation of Notch signaling in muscle stem cell activation and postnatal myogenesis

The Notch signaling pathway is an evolutionarily conserved pathway that is critical for tissue morphogenesis during development, but is also involved in tissue maintenance and repair in the adult. In skeletal muscle, regulation of Notch signaling is involved in somitogenesis, muscle development, and the proliferation and cell fate determination of muscle stems cells during regeneration. During each of these processes, the spatial and temporal control of Notch signaling is essential for proper tissue formation. That control is mediated by a series of regulatory proteins and protein complexes that enhance or inhibit Notch signaling by regulating protein processing, localization, activity, and stability. In this review, we focus on the regulation of Notch signaling during postnatal muscle regeneration when muscle stem cells ("satellite cells") must activate, proliferate, progress along a myogenic lineage pathway, and ultimately differentiate to form new muscle. We review the regulators of Notch signaling, such as Numb and Deltex, that have documented roles in myogenesis as well as other regulators that may play a role in modulating Notch signaling during satellite cell activation and postnatal myogenesis.     

Notch1 and IL-7 receptor interplay maintains proliferation of human thymic progenitors while suppressing non-T cell fates

Notch signaling is critical for T cell development of multipotent hemopoietic progenitors. Yet, how Notch regulates T cell fate specification during early thymopoiesis remains unclear. In this study, we have identified an early subset of CD34high c-kit+ flt3+ IL-7Ralpha+ cells in the human postnatal thymus, which includes primitive progenitors with combined lymphomyeloid potential. To assess the impact of Notch signaling in early T cell development, we expressed constitutively active Notch1 in such thymic lymphomyeloid precursors (TLMPs), or triggered their endogenous Notch pathway in the OP9-Delta-like1 stroma coculture. Our results show that proliferation vs differentiation is a critical decision influenced by Notch at the TLMP stage. We found that Notch signaling plays a prominent role in inhibiting non-T cell differentiation (i.e., macrophages, dendritic cells, and NK cells) of TLMPs, while sustaining the proliferation of undifferentiated thymocytes with T cell potential in response to unique IL-7 signals. However, Notch activation is not sufficient for inducing T-lineage progression of proliferating progenitors. Rather, stroma-derived signals are concurrently required. Moreover, while ectopic IL-7R expression cannot replace Notch for the maintenance and expansion of undifferentiated thymocytes, Notch signals sustain IL-7R expression in proliferating thymocytes and induce IL-7R up-regulation in a T cell line. Thus, IL-7R and Notch pathways cooperate to synchronize cell proliferation and suppression of non-T lineage choices in primitive intrathymic progenitors, which will be allowed to progress along the T cell pathway only upon interaction with an inductive stromal microenvironment. These data provide insight into a mechanism of Notch-regulated amplification of the intrathymic pool of early human T cell progenitors.     

Hairy is a cell context signal controlling Notch activity

Notch signaling regulates cell fate determination and many developmental processes. Here we report that lateral inhibition, a major mechanism for Notch activity, is modulated by Hairy, a bHLH-WRPW protein. In Xenopus, Notch can have from inhibitory, permissive to enhancing roles in muscle or neural differentiation. These cell context-dependent effects correlate with Hairy expression levels from high to low, respectively, in the cells. Moreover, Notch effects can be altered upon manipulation of Hairy expression. We propose that Hairy provides a cell context in which a cell can interpret Notch and other extrinsic signals by controlling responsiveness of its target genes; this mode of Hairy-Notch interaction may apply in other systems.     

Localization of putative stem cells in dental epithelium and their association with Notch and FGF signaling.

The continuously growing mouse incisor is an excellent model to analyze the mechanisms for stem cell lineage. We designed an organ culture method for the apical end of the incisor and analyzed the epithelial cell lineage by 5-bromo-2'-deoxyuridine and DiI labeling. Our results indicate that stem cells reside in the cervical loop epithelium consisting of a central core of stellate reticulum cells surrounded by a layer of basal epithelial cells, and that they give rise to transit-amplifying progeny differentiating into enamel forming ameloblasts. We identified slowly dividing cells among the Notch1-expressing stellate reticulum cells in specific locations near the basal epithelial cells expressing lunatic fringe, a secretory molecule modulating Notch signaling. It is known from tissue recombination studies that in the mouse incisor the mesenchyme regulates the continuous growth of epithelium. Expression of Fgf-3 and Fgf-10 were restricted to the mesenchyme underlying the basal epithelial cells and the transit-amplifying cells expressing their receptors Fgfr1b and Fgfr2b. When FGF-10 protein was applied with beads on the cultured cervical loop epithelium it stimulated cell proliferation as well as expression of lunatic fringe. We present a model in which FGF signaling from the mesenchyme regulates the Notch pathway in dental epithelial stem cells via stimulation of lunatic fringe expression and, thereby, has a central role in coupling the mitogenesis and fate decision of stem cells.