Mapping spatio-temporal activation of Notch signaling during neurogenesis and gliogenesis in the developing mouse brain

Notch1 plays various important roles including the maintenance of the stem cell state as well as the promotion of glial fates in mammalian CNS development. However, because of the very low amount of the activated form of Notch1 present in vivo, its precise activation pattern has remained unknown. In this study, we mapped the active state of this signaling pathway in situ in the developing mouse brain using a specific antibody that recognizes the processed form of the intracellular domain of Notch1 cleaved by presenilin/gamma-secretase activity. By using this antibody, active state of Notch1 came to be detectable with a higher sensitivity than using conventional antibody against Notch1. We found that activated Notch1 was mainly detected in the nuclei of a subpopulation of radial glial cells, the majority of proliferating precursor cells in the ventricular zone (VZ). However, Notch1 activation was not detected in neuronal precursor cells positive for neuronal basic helix-loop-helix proteins or in differentiating neurons in the embryonic forebrain. Interestingly, we found that Notch1 was transiently activated in the astrocytic lineage during perinatal CNS development. Taken together, the present method has enabled us to determine the timing, gradients, and boundaries of the activation of Notch signaling.     

XNAP, a conserved ankyrin repeat-containing protein with a role in the Notch pathway during Xenopus primary neurogenesis.

The Notch signaling pathway plays an important role in many cell-fate decisions during development. Here we investigate the regulation and function of the conserved gene XNAP, which is a member of the Delta-Notch synexpression group in Xenopus. XNAP encodes a small protein with two C-terminal tandem ankyrin repeats which is expressed in the neurectoderm and in the presomitic mesoderm in a pattern that resembles that of other component of the Notch pathway. When a myc-tag form of XNAP is overexpressed in Xenopus or Hela cells, XNAP protein is detected both in the nucleus and the cytoplasm. In embryos and in animal cap assays, XNAP expression is activated, perhaps directly, by the Notch pathway and this activation appears to be Su(H) dependent. Overexpression of XNAP in embryos decreases Notch signaling, which leads to an increase in the number of primary neurons that form within the domains of the neural plate where neurogenesis normally occurs. In culture Hela cells, XNAP overexpression interferes with ICD activation of a Notch regulated reporter gene. Together, these data indicate that XNAP is a novel target of the Notch pathway that may, in a feedback loop, modulate its activity.     

Notch induces cyclin-D1-dependent proliferation during a specific temporal window of neural differentiation in ES cells

The Notch signaling pathway controls cell fate choices at multiple steps during cell lineage progression. To produce the cell fate choice appropriate for a particular stage in the cell lineage, Notch signaling needs to interpret the cell context information for each stage and convert it into the appropriate cell fate instruction. The molecular basis for this temporal context-dependent Notch signaling output is poorly understood, and to study this, we have engineered a mouse embryonic stem (ES) cell line, in which short pulses of activated Notch can be produced at different stages of in vitro neural differentiation. Activation of Notch signaling for 6 h specifically at day 3 during neural induction in the ES cells led to significantly enhanced cell proliferation, accompanied by Notch-mediated activation of cyclin D1 expression. A reduction of cyclin-D1-expressing cells in the developing CNS of Notch signaling-deficient mouse embryos was also observed. Expression of a dominant negative form of cyclin D1 in the ES cells abrogated the Notch-induced proliferative response, and, conversely, a constitutively active form of cyclin D1 mimicked the effect of Notch on cell proliferation. In conclusion, the data define a novel temporal context-dependent function of Notch and a critical role for cyclin D1 in the Notch-induced proliferation in ES cells.     

The autonomous notch signal pathway is activated by baicalin and baicalein but is suppressed by niclosamide in K562 cells

The Notch signaling pathway plays important roles in a variety of cellular processes. Aberrant transduction of Notch signaling contributes to many diseases and cancers in humans. The Notch receptor intracellular domain, the activated form of Notch receptor, is extremely difficult to detect in normal cells. However, it can activate signaling at very low protein concentration to elicit its biological effects. In the present study, a cell based luciferase reporter gene assay was established in K562 cells to screen drugs which could modulate the endogenous CBF1‐dependent Notch signal pathway. Using this system, we found that the luciferase activity of CBF1‐dependent reporter gene was activated by baicalin and baicalein but suppressed by niclosamide in both dose‐ and time‐dependent manners. Treatment with these drugs modulated endogenous Notch signaling and affected mRNA expression levels of Notch1 receptor and Notch target genes in K562 cells. Additionally, erythroid differentiation of K562 cells was suppressed by baicalin and baicalein yet was promoted by niclosamide. Colony‐forming ability in soft agar was decreased after treatment with baicalin and baicalein, but was not affected in the presence of niclosamide. Thus, modulation of Notch signaling after treatment with any of these three drugs may affect tumorigenesis of K562 cells suggesting that these drugs may have therapeutic potential for those tumors associated with Notch signaling. Taken together, this system could be beneficial for screening of drugs with potential to treat Notch signal pathway‐associated diseases. J. Cell. Biochem. 106: 682–692, 2009. © 2009 Wiley‐Liss, Inc.     

Activated Notch1 alters differentiation of embryonic stem cells into mesodermal cell lineages at multiple stages of development

Signals of Notch transmembrane receptors function to regulate a wide variety of developmental cell fates. Here we investigate the role of Notch signaling in the development of mesodermal cell types by expressing a tamoxifen-inducible, activated form of Notch1 in embryonic stem cells (ESC). For differentiation of ESC into first mesodermal progenitor cells and then endothelial, mural, cardiac muscle and hematopoietic cells, the OP9 stroma co-culture system was used. Timed activation of Notch signaling by the addition of tamoxifen at various stages during differentiation of ESC into mesodermal cell lineages results in profound alterations in the generation of all of these cells. Differentiation of ESC into Flk1(+) mesodermal cells is inhibited by activated Notch. When Notch signaling is activated in mesodermal cells, generation of cardiac muscle, endothelial and hematopoietic cells is inhibited, favoring the generation of mural cells. Activation of Notch signaling in hematopoietic cells reduces colony formation and maintenance of hematopoiesis. These data suggest that Notch signaling plays a regulatory role in mesodermal development, cardiomyogenesis, the balanced generation of endothelial versus mural cells of blood vessels and hematopoietic development.     

Notch inhibits Ptf1 function and acinar cell differentiation in developing mouse and zebrafish pancreas

Notch signaling regulates cell fate decisions in a variety of adult and embryonic tissues, and represents a characteristic feature of exocrine pancreatic cancer. In developing mouse pancreas, targeted inactivation of Notch pathway components has defined a role for Notch in regulating early endocrine differentiation, but has been less informative with respect to a possible role for Notch in regulating subsequent exocrine differentiation events. Here, we show that activated Notch and Notch target genes actively repress completion of an acinar cell differentiation program in developing mouse and zebrafish pancreas. In developing mouse pancreas, the Notch target gene Hes1 is co-expressed with Ptf1-P48 in exocrine precursor cells, but not in differentiated amylase-positive acinar cells. Using lentiviral delivery systems to induce ectopic Notch pathway activation in explant cultures of E10.5 mouse dorsal pancreatic buds, we found that both Hes1 and Notch1-IC repress acinar cell differentiation, but not Ptf1-P48 expression, in a cell-autonomous manner. Ectopic Notch activation also delays acinar cell differentiation in developing zebrafish pancreas. Further evidence of a role for endogenous Notch in regulating exocrine pancreatic differentiation was provided by examination of zebrafish embryos with homozygous mindbomb mutations, in which Notch signaling is disrupted. mindbomb-deficient embryos display accelerated differentiation of exocrine pancreas relative to wild-type clutchmate controls. A similar phenotype was induced by expression of a dominant-negative Suppressor of Hairless [Su(H)] construct, confirming that Notch actively represses acinar cell differentiation during zebrafish pancreatic development. Using transient transfection assays involving a Ptf1-responsive reporter gene, we further demonstrate that Notch and Notch/Su(H) target genes directly inhibit Ptf1 activity, independent of changes in expression of Ptf1 component proteins. These results define a normal inhibitory role for Notch in the regulation of exocrine pancreatic differentiation.     

The regulation of apoptosis by Numb/Notch signaling in the serotonin lineage of Drosophila

Apoptosis is prevalent during development of the central nervous system (CNS), yet very little is known about the signals that specify an apoptotic cell fate. In this paper, we examine the role of Numb/Notch signaling in the development of the serotonin lineage of Drosophila and show that it is necessary for regulating apoptosis. Our results indicate that when Numb inhibits Notch signaling, cells undergo neuronal differentiation, whereas cells that maintain Notch signaling initiate apoptosis. The apoptosis inhibitor p35 can counteract Notch-mediated apoptosis and rescue cells within the serotonin lineage that normally undergo apoptosis. Furthermore, we observe tumor-like overproliferation of cells in the CNS when Notch signaling is reduced. These data suggest that the distribution of Numb during terminal mitotic divisions of the CNS can distinguish between a neuronal cell fate and programmed cell death.     

Notch: from neural development to neurological disorders

Notch is an integral membrane protein that functions as receptor for ligands such as jagged and delta that are associated with the surface of neighboring cells. Upon ligand binding, notch is proteolytically cleaved within its transmembrane domain by presenilin‐1 (the enzymatic component of the γ‐secretase complex) resulting in the release of a notch intracellular domain which translocates to the nucleus where it regulates gene expression. Notch signaling plays multiple roles in the development of the CNS including regulating neural stem cell (NSC) proliferation, survival, self‐renewal and differentiation. Notch is also present in post‐mitotic neurons in the adult CNS wherein its activation influences structural and functional plasticity including processes involved in learning and memory. Recent findings suggest that notch signaling in neurons, glia, and NSCs may be involved in pathological changes that occur in disorders such as stroke, Alzheimer’s disease and CNS tumors. Studies of animal models suggest the potential of agents that target notch signaling as therapeutic interventions for several different CNS disorders.     

Notch1 signaling stimulates proliferation of immature cardiomyocytes

The identification of the molecular mechanisms controlling cardiomyocyte proliferation during the embryonic, fetal, and early neonatal life appears of paramount interest in regard to exploiting this information to promote cardiac regeneration. Here, we show that the proliferative potential of neonatal rat cardiomyocytes is powerfully stimulated by the sustained activation of the Notch pathway. We found that Notch1 is expressed in proliferating ventricular immature cardiac myocytes (ICMs) both in vitro and in vivo, and that the number of Notch1-positive cells in the heart declines with age. Notch1 expression in ICMs paralleled the expression of its Jagged1 ligand on non-myocyte supporting cells. The inhibition of Notch signaling in ICMs blocked their proliferation and induced apoptosis; in contrast, its activation by Jagged1 or by the constitutive expression of its activated form using an adeno-associated virus markedly stimulated proliferative signaling and promoted ICM expansion. Maintenance or reactivation of Notch signaling in cardiac myocytes might represent an interesting target for innovative regenerative therapy.     

Notch Pathway Activation Contributes to Inhibition of C2C12 Myoblast Differentiation by Ethanol

The loss of muscle mass in alcoholic myopathy may reflect alcohol inhibition of myogenic cell differentiation into myotubes. Here, using a high content imaging system we show that ethanol inhibits C2C12 myoblast differentiation by reducing myogenic fusion, creating smaller and less complex myotubes compared with controls. Ethanol administration during C2C12 differentiation reduced MyoD and myogenin expression, and microarray analysis identified ethanol activation of the Notch signaling pathway target genes Hes1 and Hey1. A reporter plasmid regulated by the Hes1 proximal promoter was activated by alcohol treatment in C2C12 cells. Treatment of differentiating C2C12 cells with a gamma secretase inhibitor (GSI) abrogated induction of Hes1. On a morphological level GSI treatment completely rescued myogenic fusion defects and partially restored other myotube parameters in response to alcohol. We conclude that alcohol inhibits C2C12 myoblast differentiation and the inhibition of myogenic fusion is mediated by Notch pathway activation.     

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 regulates gastric antral LGR5 stem cell function

The major signaling pathways regulating gastric stem cells are unknown. Here we report that Notch signaling is essential for homeostasis of LGR5⁺ antral stem cells. Pathway inhibition reduced proliferation of gastric stem and progenitor cells, while activation increased proliferation. Notch dysregulation also altered differentiation, with inhibition inducing mucous and endocrine cell differentiation while activation reduced differentiation. Analysis of gastric organoids demonstrated that Notch signaling was intrinsic to the epithelium and regulated growth. Furthermore, in vivo Notch manipulation affected the efficiency of organoid initiation from glands and single Lgr5‐GFP stem cells, suggesting regulation of stem cell function. Strikingly, constitutive Notch activation in LGR5⁺ stem cells induced tissue expansion via antral gland fission. Lineage tracing using a multi‐colored reporter demonstrated that Notch‐activated stem cells rapidly generate monoclonal glands, suggesting a competitive advantage over unmanipulated stem cells. Notch activation was associated with increased mTOR signaling, and mTORC1 inhibition normalized NICD‐induced increases in proliferation and gland fission. Chronic Notch activation induced undifferentiated, hyper‐proliferative polyps, suggesting that aberrant activation of Notch in gastric stem cells may contribute to gastric tumorigenesis.     

Generation and characterization of a Notch1 signaling-specific reporter mouse line

Signaling through the Notch1 receptor is essential for the control of numerous developmental processes during embryonic life as well as in adult tissue homeostasis and disease. Since the outcome of Notch1 signaling is highly context‐dependent, and its precise physiological and pathological role in many organs is unclear, it is of great interest to localize and identify the cells that receive active Notch1 signals in vivo. Here, we report the generation and characterization of a BAC‐transgenic mouse line, N1‐Gal4VP16, that when crossed to a Gal4‐responsive reporter mouse line allowed the identification of cells undergoing active Notch1 signaling in vivo. Analysis of embryonic and adult N1‐Gal4VP16 mice demonstrated that the activation pattern of the transgene coincides with previously observed activation patterns of the endogenous Notch1 receptor. Thus, this novel reporter mouse line provides a unique tool to specifically investigate the spatial and temporal aspects of Notch1 signaling in vivo. genesis 50:700–710, 2012. © 2012 Wiley Periodicals, Inc.     

Hyper-Activation of Notch3 Amplifies the Proliferative Potential of Rhabdomyosarcoma Cells

Rhabdomyosarcoma (RMS) is a pediatric myogenic-derived soft tissue sarcoma that includes two major histopathological subtypes: embryonal and alveolar. The majority of alveolar RMS expresses PAX3-FOXO1 fusion oncoprotein, associated with the worst prognosis. RMS cells show myogenic markers expression but are unable to terminally differentiate. The Notch signaling pathway is a master player during myogenesis, with Notch1 activation sustaining myoblast expansion and Notch3 activation inhibiting myoblast fusion and differentiation. Accordingly, Notch1 signaling is up-regulated and activated in embryonal RMS samples and supports the proliferation of tumor cells. However, it is unable to control their differentiation properties. We previously reported that Notch3 is activated in RMS cell lines, of both alveolar and embryonal subtype, and acts by inhibiting differentiation. Moreover, Notch3 depletion reduces PAX3-FOXO1 alveolar RMS tumor growth in vivo. However, whether Notch3 activation also sustains the proliferation of RMS cells remained unclear. To address this question, we forced the expression of the activated form of Notch3, Notch3IC, in the RH30 and RH41 PAX3-FOXO1-positive alveolar and in the RD embryonal RMS cell lines and studied the proliferation of these cells. We show that, in all three cell lines tested, Notch3IC over-expression stimulates in vitro cell proliferation and prevents the effects of pharmacological Notch inhibition. Furthermore, Notch3IC further increases RH30 cell growth in vivo. Interestingly, knockdown of Notch canonical ligands JAG1 or DLL1 in RMS cell lines decreases Notch3 activity and reduces cell proliferation. Finally, the expression of Notch3IC and its target gene HES1 correlates with that of the proliferative marker Ki67 in a small cohort of primary PAX-FOXO1 alveolar RMS samples. These results strongly suggest that high levels of Notch3 activation increase the proliferative potential of RMS cells.