Proliferating neural progenitors in the developing CNS of zebrafish require Jagged2 and Jagged1b

In the central nervous system (CNS), giving rise to the diversity and the complexity of neurons is the spatial and temporal differentiation of neural stem cells and/or neural precursors. Here, we investigated the role of Jagged-mediated Notch signaling in the maintenance and differentiation of progenitor cells during late neurogenesis by analyzing the expression patterns of zebrafish jagged homologues, and by injecting their morpholinos. Expression of both jagged2 and jagged1b mRNA in the CNS suggested that they might be involved in control of differentiating neural progenitors in which they are involved later in development. In Jagged2 and Jagged1b knock-down embryos, the overall rate of cell division dramatically decreased, and the ectopic VeMe neurons were generated. The results suggest that Jagged-Notch signaling plays a critical role in the maintenance of proliferating neural precursors, and that the generation of late-born neurons, especially VeMe neurons, is regulated by the interplay between Jagged2 and Jagged1b.     

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.     

Multiple roles of mouse Numb in tuning developmental cell fates.

BACKGROUND: Notch signaling regulates multiple differentiation processes and cell fate decisions during both invertebrate and vertebrate development. Numb encodes an intracellular protein that was shown in Drosophila to antagonize Notch signaling at binary cell fate decisions of certain cell lineages. Although overexpression experiments suggested that Numb might also antagonize some Notch activity in vertebrates, the developmental processes in which Numb is involved remained elusive. RESULTS: We generated mice with a homozygous inactivation of Numb. These mice died before embryonic day E11.5, probably because of defects in angiogenic remodeling and placental dysfunction. Mutant embryos had an open anterior neural tube and impaired neuronal differentiation within the developing cranial central nervous system (CNS). In the developing spinal cord, the number of differentiated motoneurons was reduced. Within the peripheral nervous system (PNS), ganglia of cranial sensory neurons were formed. Trunk neural crest cells migrated and differentiated into sympathetic neurons. In contrast, a selective differentiation anomaly was observed in dorsal root ganglia, where neural crest--derived progenitor cells had migrated normally to form ganglionic structures, but failed to differentiate into sensory neurons. CONCLUSIONS: Mouse Numb is involved in multiple developmental processes and required for cell fate tuning in a variety of lineages. In the nervous system, Numb is required for the generation of a large subset of neuronal lineages. The restricted requirement of Numb during neural development in the mouse suggests that in some neuronal lineages, Notch signaling may be regulated independently of Numb.     

Identification of c-Kit receptor as a regulator of adult neural stem cells in the mammalian eye: interactions with Notch signaling

Neural stem cells are present in specific regions of the adult central nervous system (CNS). Recent evidence suggests that the ciliary epithelium (CE), a CNS derivative, in the adult mammalian eye, harbors a quiescent population of neural stem cells. Here, we report the identification of c-Kit signaling as one of the regulators of adult CE neural stem cells in vitro. c-Kit receptors are expressed in proliferating adult CE neural stem cells and colocalized with neural progenitor markers. Perturbation of c-Kit signaling influences the self-renewal and differentiation of CE neural stem cells, thus demonstrating the role of c-Kit signaling in the maintenance of these cells. In addition, we observed an influence of c-Kit-mediated signaling on the expression of Notch1, another critical regulator of neural stem cells. Our observations suggest that, given the importance of preservation of a stem cell pool for generating different cell types at different times, multiple signaling pathways act in concert for the maintenance of neural stem cells.     

Function of RNA-binding protein Musashi-1 in stem cells

Musashi is an evolutionarily conserved family of RNA-binding proteins that is preferentially expressed in the nervous system. The first member of the Musashi family was identified in Drosophila. This protein plays an essential role in regulating the asymmetric cell division of ectodermal precursor cells known as sensory organ precursor cells through the translational regulation of target mRNA. In the CNS of Drosophila larvae, however, Musashi is expressed in proliferating neuroblasts and likely has a different function. Its probable mammalian homologue, Musashi-1, is a neural RNA-binding protein that is strongly expressed in fetal and adult neural stem cells (NSCs). Mammalian Musashi-1 augments Notch signaling through the translational repression of its target mRNA, m-Numb, thereby contributing to the self-renewal of NSCs. In addition to its functions in NSCs, the role of mammalian Musashi-1 protein in epithelial stem cells, including intestinal and mammary gland stem cells, is attracting increasing interest.     

miR-124 function during Ciona intestinalis neuronal development includes extensive interaction with the Notch signaling pathway

The nervous system-enriched microRNA miR-124 is necessary for proper nervous system development, although the mechanism remains poorly understood. Here, through a comprehensive analysis of miR-124 and its gene targets, we demonstrate that, in the chordate ascidian Ciona intestinalis, miR-124 plays an extensive role in promoting nervous system development. We discovered that feedback interaction between miR-124 and Notch signaling regulates the epidermal-peripheral nervous system (PNS) fate choice in tail midline cells. Notch signaling silences miR-124 in epidermal midline cells, whereas in PNS midline cells miR-124 silences Notch, Neuralized and all three Ciona Hairy/Enhancer-of-Split genes. Furthermore, ectopic expression of miR-124 is sufficient to convert epidermal midline cells into PNS neurons, consistent with a role in modulating Notch signaling. More broadly, genome-wide target extraction with validation using an in vivo tissue-specific sensor assay indicates that miR-124 shapes neuronal progenitor fields by downregulating non-neural genes, notably the muscle specifier Macho-1 and 50 Brachyury-regulated notochord genes, as well as several anti-neural factors including SCP1 and PTBP1. 3'UTR conservation analysis reveals that miR-124 targeting of SCP1 is likely to have arisen as a shared, derived trait in the vertebrate/tunicate ancestor and targeting of PTBP1 is conserved among bilaterians except for ecdysozoans, while extensive Notch pathway targeting appears to be Ciona specific. Altogether, our results provide a comprehensive insight into the specific mechanisms by which miR-124 promotes neuronal development.     

Apoptosis regulates notochord development in Xenopus

The notochord is the defining characteristic of the chordate embryo and plays critical roles as a signaling center and as the primitive skeleton. In this study we show that early notochord development in Xenopus embryos is regulated by apoptosis. We find apoptotic cells in the notochord beginning at the neural groove stage and increasing in number as the embryo develops. These dying cells are distributed in an anterior to posterior pattern that is correlated with notochord extension through vacuolization. In axial mesoderm explants, inhibition of this apoptosis causes the length of the notochord to approximately double compared to controls. In embryos, however, inhibition of apoptosis decreases the length of the notochord and it is severely kinked. This kinking also spreads from the anterior with developmental stage such that, by the tadpole stage, the notochord lacks any recognizable structure, although notochord markers are expressed in a normal temporal pattern. Extension of the somites and neural plate mirrors that of the notochord in these embryos, and the somites are severely disorganized. These data indicate that apoptosis is required for normal notochord development during the formation of the anterior-posterior axis, and its role in this process is discussed.     

A requirement for Notch in the genesis of a subset of glial cells in the Drosophila embryonic central nervous system which arise through asymmetric divisions

In the Drosophila central nervous system (CNS) glial cells are known to be generated from glioblasts, which produce exclusively glia or neuroglioblasts that bifurcate to produce both neuronal and glial sublineages. We show that the genesis of a subset of glial cells, the subperineurial glia (SPGs), involves a new mechanism and requires Notch. We demonstrate that the SPGs share direct sibling relationships with neurones and are the products of asymmetric divisions. This mechanism of specifying glial cell fates within the CNS is novel and provides further insight into regulatory interactions leading to glial cell fate determination. Furthermore, we show that Notch signalling positively regulates glial cells missing (gcm) expression in the context of SPG development.     

Myelin-associated glycoprotein-mediated signaling in central nervous system pathophysiology

The myelin-associated glycoprotein (MAG) is a type I membrane-spanning protein expressed exclusively in oligoden drocytes and Schwann cells. It has two generally known pathophysiological roles in the central nervous system (CNS): maintenance of myelin integrity and inhibition of CNS axonal regeneration. The subtle CNS phenotype resulting from genetic ablation of MAG expression has made mechanistic analysis of its functional role in these difficult. However, the past few years have brought some major revelations, particularly in terms of mechanisms of MAG signaling through the Nogo-66 receptor (NgR) complex. Although apparently converging through NgR, a readily noticeable fact is that the neuronal growth inhibitory effect of MAG differs from that of Nogo-66. This may result from the influence of coreceptors in the form of gangliosides or from MAG-specific neuronal receptors such as NgR2. MAG has several other neuronal binding partners, and some of these may modulate its interaction with the NgR complex or downstream signaling. This article discusses new findings in MAG-forward and-reverse signaling and its role in CNS pathophysiology.     

Characterization of Brachyury-downstream notochord genes in the Ciona intestinalis embryo

The notochord has two major roles during chordate embryogenesis, as a source of inductive signals for the patterning of neural tube and paraxial mesoderm and as a supportive organ of the larval tail. Despite the recent identification of mutations that affect the notochord development in vertebrate embryos, little is known about genes that are expressed in the differentiating notochord itself. In the urochordate ascidian Ciona intestinalis, Brachyury (Ci-Bra) plays a key role in notochord differentiation. In a previous study, we isolated cDNA clones for nearly 40 potential Ci-Bra target genes that are expressed in notochord cells (H. Takahashi et al., 1999, Genes Dev. 13, 1519-1523). Here we characterized 20 of them by determining the complete nucleotide sequences of the cDNAs. These genes encode a broad spectrum of divergent proteins associated with notochord formation and function. Two genes encode ascidian homologs of the Drosophila Prickle LIM domain proteins and another encodes the ERM protein, all 3 of which appear to be involved in the control of cytoskeletal architecture. In addition, genes for netrin, leprecan, cdc45, ATP:citrate lyase, ATP sulfurylase/APS kinase, protein tyrosine phosphatase, beta4-galactosyltransferase, fibrinogen-like protein, divergent tropomyosin-like proteins, and Drosophila Pellino-like protein were identified. The observation of the netrin gene expression in the notochord may provide the first molecular evidence that the ascidian notochord is a source of signals as in vertebrates. In addition, the present information should be used to identify nonchordate deuterostome tissues homologous to the notochord as well as genes which are expressed in the notochord cells of vertebrate embryos.     

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.     

The function of cholesterol in embryogenesis

Cholesterol is critical in embryonic development. Inhibition of cholesterol synthesis in experimental animals has caused a birth defect called holoprosencephaly (HPE), which is evidenced by cyclopia (one eye in the middle of the face), monorhinia (protruding single nose above the eye), absence of the pituitary gland, and central nervous system (CNS) abnormalities. In humans, an inherited defect in the cholesterol-synthesizing enzyme 7-dehydrocholesterol reductase depletes cholesterol and results in human HPE, termed Smith-Lemli-Opitz syndrome. In its most severe form, the syndrome leads to cyclopia, monorhinia, and lack of separation of cerebral hemispheres. The cause of the syndrome is a defect in a protein coded by the gene Sonic hedgehog (SHH). The protein SHH is expressed in the notochord of the CNS in the early embryo and is activated by being cleaved autocatalytically, with simultaneous covalent attachment of cholesterol to the N-terminal fragment, which is secreted by cells of the mesoderm layer, signaling the establishment of the neural midline cells. Thus, cholesterol is essential for proper signaling in the development of the normal embryo.     

HIV-1 Tat Activates Neuronal Ryanodine Receptors with Rapid Induction of the Unfolded Protein Response and Mitochondrial Hyperpolarization

Neurologic disease caused by human immunodeficiency virus type 1 (HIV-1) is ultimately refractory to highly active antiretroviral therapy (HAART) because of failure of complete virus eradication in the central nervous system (CNS), and disruption of normal neural signaling events by virally induced chronic neuroinflammation. We have previously reported that HIV-1 Tat can induce mitochondrial hyperpolarization in cortical neurons, thus compromising the ability of the neuron to buffer calcium and sustain energy production for normal synaptic communication. In this report, we demonstrate that Tat induces rapid loss of ER calcium mediated by the ryanodine receptor (RyR), followed by the unfolded protein response (UPR) and pathologic dilatation of the ER in cortical neurons in vitro. RyR antagonism attenuated both Tat-mediated mitochondrial hyperpolarization and UPR induction. Delivery of Tat to murine CNS in vivo also leads to long-lasting pathologic ER dilatation and mitochondrial morphologic abnormalities. Finally, we performed ultrastructural studies that demonstrated mitochondria with abnormal morphology and dilated endoplasmic reticulum (ER) in brain tissue of patients with HIV-1 inflammation and neurodegeneration. Collectively, these data suggest that abnormal RyR signaling mediates the neuronal UPR with failure of mitochondrial energy metabolism, and is a critical locus for the neuropathogenesis of HIV-1 in the CNS.     

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.