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 signaling represses the glial fate in fly PNS

By using gain-of-function mutations it has been proposed that vertebrate Notch promotes the glial fate. We show in vivo that glial cells are produced at the expense of neurons in the peripheral nervous system of flies lacking Notch and that constitutively activated Notch produces the opposite phenotype. Notch acts as a genetic switch between neuronal and glial fates by negatively regulating glial cell deficient/glial cells missing, the gene required in the glial precursor to induce gliogenesis. Moreover, Notch represses neurogenesis or gliogenesis, depending on the sensory organ type. Numb, which is asymmetrically localized in the multipotent cell that produces the glial precursor, induces glial cells at the expense of neurons. Thus, a cell-autonomous mechanism inhibits Notch signaling.     

Oh no, Notch again!

The Notch receptor signaling pathway is important for morphogenesis and development of many organs and tissues in most if not all multicellular species. The classical view holds that Notch signaling keeps cells in an undifferentiated state. Recently, however, this notion has been challenged in the nervous system by two sets of observations: Notch plays an active role in the differentiation of glial cells,(1-4) and Notch influences the length and organisation of neuronal processes.(5-7) In this review, we analyse these recent data and discuss how Notch signaling may be able to perform such quite different tasks during nervous system development. BioEssays 23:3-7, 2001.     

Activated Notch1 prevents differentiation of pancreatic acinar cells and attenuate endocrine development

Mice carrying loss-of-function mutations in certain Notch pathway genes display increased and accelerated pancreatic endocrine development, leading to depletion of precursor cells followed by pancreatic hypoplasia. Here, we have investigated the effect of expressing a constitutively active form of the Notch1 receptor (Notch1(ICD)) in the developing pancreas using the pdx1 promoter. At e10.5 to e12.5, we observe a disorganized pancreatic epithelium with reduced numbers of endocrine cells, confirming a repressive activity of Notch1 upon the early differentiation program. Subsequent branching morphogenesis is impaired and the pancreatic epithelium forms cyst-like structures with ductal phenotype containing a few endocrine cells but completely devoid of acinar cells. The endocrine cells that do form show abnormal expression of cell type-specific markers. Our observations show that sustained Notch1 signaling not only significantly represses endocrine development, but also fully prevents pancreatic exocrine development, suggesting that a possible role of Notch1 is to maintain the undifferentiated state of common pancreatic precursor cells.     

Context-dependent utilization of Notch activity in Drosophila glial determination

During Drosophila neurogenesis, glial differentiation depends on the expression of glial cells missing (gcm). Understanding how glial fate is achieved thus requires knowledge of the temporal and spatial control mechanisms directing gcm expression. A recent report showed that in the adult bristle lineage, gcm expression is negatively regulated by Notch signaling ( Van De Bor, V. and Giangrande, A. (2001). Development 128, 1381-1390). Here we show that the effect of Notch activation on gliogenesis is context-dependent. In the dorsal bipolar dendritic (dbd) sensory lineage in the embryonic peripheral nervous system (PNS), asymmetric cell division of the dbd precursor produces a neuron and a glial cell, where gcm expression is activated in the glial daughter. Within the dbd lineage, Notch is specifically activated in one of the daughter cells and is required for gcm expression and a glial fate. Thus Notch activity has opposite consequences on gcm expression in two PNS lineages. Ectopic Notch activation can direct gliogenesis in a subset of embryonic PNS lineages, suggesting that Notch-dependent gliogenesis is supported in certain developmental contexts. We present evidence that POU-domain protein Nubbin/PDM-1 is one of the factors that provide such context.     

Notch1 and the activated NOTCH1 intracellular domain are expressed in differentiated trophoblast cells

The Notch signalling pathway regulates proliferation, cell death and cell type specification that is critical for organogenesis. Mouse models carrying mutations in the Notch signalling pathway display defects in development of the placenta, suggesting that this pathway is required for placental development. In particular, Notch1 mutant embryos exhibit abnormal placental morphogenesis and arrest early in development. However, expression of Notch1 gene has not been detected during placental development. Trophoblast stem cells are derived from the precursor of the placenta and express Notch1. We report that Notch1 is also expressed in differentiated trophoblast cells. Under standard differentiation conditions, Notch1 expression ceases by day 6. Furthermore, the activated NOTCH1 intracellular domain is enriched at the nucleolus of trophoblast stem cells and differentiated trophoblast cells. Our results suggest that NOTCH1 is active in both trophoblast stem cells and differentiated trophoblast cells.     

An instructive function for Notch in promoting gliogenesis in the zebrafish retina

The Gal4-UAS technique has been used to misexpress a constitutively active Notch receptor variant (notch1a-intra) in the developing zebrafish retina. This is the first study to use this technique to misexpress genes and assess their function in neural development of the zebrafish. Expression of activated Notch1a either ubiquitously, driven by a heat-shock70 promoter, or in a spatially regulated manner, controlled by the deltaD promoter, causes a block in neuronal differentiation that affects all cell types. Developing cells take on either a glial fate or remain undifferentiated. A large number of cells eventually undergo apoptosis. These phenotypic effects of activated Notch1a are expressed cell autonomously. Cells within central regions of the retina adopt a glial fate if they express activated Notch1a in a time window that extends from 27 to 48 hours postfertilization. This period corresponds mainly to the time of origin of ganglion cells in the normal retina. Activation of notch1a at later stages results in defects in cell type specification that remain restricted to the ciliary marginal zone, whereas neuronal types are specified normally within the central region. These observations indicate that glial differentiation is initiated by Notch1a-intra expressing cells, which become postmitotic in the same time window. Our results strongly suggest that Notch1a instructs a certain cell population to enter gliogenesis, and keeps the remaining cells in an undifferentiated state. Some or all of these cells will eventually succumb to apoptosis.     

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.     

Gamma-secretase activity is dispensable for mesenchyme-to-epithelium transition but required for podocyte and proximal tubule formation in developing mouse kidney

Notch signaling is involved in pronephros development in Xenopus and in glomerulogenesis in mice. However, owing to early lethality in mice deficient for some Notch pathway genes and functional redundancy for others, a role for Notch signaling during early stages of metanephric development has not been defined. Using an antibody specific to the N-terminal end of gamma-secretase-cleaved Notch1, we found evidence for Notch1 activation in the comma and S-shaped bodies of the mouse metanephros. We therefore cultured mouse metanephroi in the presence of a gamma-secretase inhibitor, N-S-phenyl-glycine-t-butyl ester (DAPT), to block Notch signaling. We observed slightly reduced ureteric bud branching but normal mesenchymal condensation and expression of markers indicating that mesenchyme induction had occurred. However, fewer renal epithelial structures were observed, with a severe deficiency in proximal tubules and glomerular podocytes, which are derived from cells in which activated Notch1 is normally present. Distal tubules were present but in reduced numbers, and this was accompanied by an increase in intervening, non-epithelial cells. After a transient 3-day exposure to DAPT, proximal tubules expanded, but podocyte differentiation failed to recover after removal of DAPT. These observations suggest that gamma-secretase activity, probably through activation of Notch, is required for maintaining a competent progenitor pool as well as for determining the proximal tubule and podocyte fates.     

Physiological Notch signaling promotes gliogenesis in the developing peripheral and central nervous systems

Constitutive activation of the Notch pathway can promote gliogenesis by peripheral (PNS) and central (CNS) nervous system progenitors. This raises the question of whether physiological Notch signaling regulates gliogenesis in vivo. To test this, we conditionally deleted Rbpsuh (Rbpj) from mouse PNS or CNS progenitors using Wnt1-Cre or Nestin-Cre. Rbpsuh encodes a DNA-binding protein (RBP/J) that is required for canonical signaling by all Notch receptors. In most regions of the developing PNS and spinal cord, Rbpsuh deletion caused only mild defects in neurogenesis, but severe defects in gliogenesis. These resulted from defects in glial specification or differentiation, not premature depletion of neural progenitors, because we were able to culture undifferentiated progenitors from the PNS and spinal cord despite their failure to form glia in vivo. In spinal cord progenitors, Rbpsuh was required to maintain Sox9 expression during gliogenesis, demonstrating that Notch signaling promotes the expression of a glial-specification gene. These results demonstrate that physiological Notch signaling is required for gliogenesis in vivo, independent of the role of Notch in the maintenance of undifferentiated neural progenitors.     

Long-range, nonautonomous effects of activated Notch1 on tissue homeostasis in the nail

Using an antibody directed against gamma-secretase-generated antigen unique to activated Notch1, we mapped Notch1 activation strictly to suprabasal cells in epidermis, nail matrix, and other skin appendages during normal development. The consequences of Notch1 activation in keratinizing nail cells were investigated in a transgenic mouse model. Ectopic activation of Notch1 in postmitotic cells within the nail keratogenous zone resulted in longer nails. BrdU labeling revealed an increased number of mitotic cells in transgenic nails. The matrix and keratogenous zone expanded distally due to the increase in cell numbers. The mitosis-promoting effects by a gene product expressed exclusively in postmitotic cells indicates a long-range effect of transgenic Notch1 on regulation of nail homeostasis. We demonstrate that activation of Notch1 in the keratogenous zone resulted in ectopic activation of Wnt signaling, the first such evidence in vertebrates. However, we detected little or no beta-catenin activation in proliferating matrix cells, indicating that Wnt is at most an indirect mediator of Notch-induced proliferation. These data support the existence of a novel, cell-nonautonomous role for Notch in maintaining homeostasis of stratified squamous epithelia by indirectly promoting mitosis in basally located cells.     

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.     

Activation of the Notch pathway in the hair cortex leads to aberrant differentiation of the adjacent hair-shaft layers

Little is known about the mechanisms underlying the generation of various cell types in the hair follicle. To investigate the role of the Notch pathway in this process, transgenic mice were generated in which an active form of Notch1 (Notch(DeltaE)) was overexpressed under the control of the mouse hair keratin A1 (MHKA1) promoter. MHKA-Notch(DeltaE) is expressed only in one precursor cell type of the hair follicle, the cortex. Transgenic mice could be easily identified by the phenotypes of curly whiskers and wavy, sheen pelage hair. No effects of activated Notch on proliferation were detected in hair follicles of the transgenic mice. We find that activating Notch signaling in the cortex caused abnormal differentiation of the medulla and the cuticle, two neighboring cell types that did not express activated Notch. We demonstrate that these non-autonomous effects are likely caused by cell-cell interactions between keratinocytes within the hair follicle and that Notch may function in such interactions either by directing the differentiation of follicular cells or assisting cells in interpreting a gradient emanating from the dermal papilla.     

Notch promotes survival of neural precursor cells via mechanisms distinct from those regulating neurogenesis

During development of the mammalian brain, many neural precursor cells (NPCs) undergo apoptosis. The regulation of such cell death, however, is poorly understood. We now show that the survival of mouse embryonic NPCs in vitro was increased by culture at a high cell density and that this effect was attributable to activation of Notch signaling. Expression of an active form of Notch1 thus markedly promoted NPC survival. Hes proteins, key effectors of Notch signaling in inhibition of neurogenesis, were not sufficient for the survival-promoting effect of Notch1. This effect of Notch1 required a region of the protein containing the RAM domain and was accompanied by up-regulation of the anti-apoptotic proteins Bcl-2 and Mcl-1. Moreover, knockdown of these proteins by RNA interference resulted in blockade of the Notch1-induced survival. These results reveal a new function of Notch, the promotion of NPC survival.     

Different downstream pathways for Notch signaling are required for gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells

We examined the roles of Notch signaling and fibroblast growth factors (FGFs) in the gliogenesis of mouse mesencephalic neural crest cells. The present study demonstrated that Notch activation or FGF treatment promotes the differentiation of glia expressing glial fibrillary acidic protein. Notch activation or FGF2 exposure during the first 48 h in culture was critical for glial differentiation. The promotion of gliogenesis by FGF2 was significantly suppressed by the inhibition of Notch signaling using Notch-1 siRNA. These data suggest that FGFs activate Notch signaling and that this activation promotes the gliogenic specification of mouse mesencephalic neural crest cells. Notch activation and FGF treatment have been shown to participate in the chondrogenic specification of these cells [Nakanishi, K., Chan, Y.S., Ito, K., 2007. Notch signaling is required for the chondrogenic specification of mouse mesencephalic neural crest cells. Mech. Dev. 124, 190–203]. Therefore, we analyzed whether or not there were differences between gliogenic and chondrogenic specifications in the downstream pathway of the Notch receptor. Whereas the activation of only the Deltex-mediated pathway was sufficient to promote glial specification, the activation of both RBP-J- and Deltex-dependent pathways was required for chondrogenic specification. These results suggest that the different downstream pathways of the Notch receptor participate in the gliogenic and chondrogenic specification of mouse mesencephalic neural crest cells.