The use of in vitro oligodendrocyte differentiation for transplantation of stem cells to treat demyelinating diseases is an important consideration. In this study, we investigated the effects of serum on glia and oligodendrocyte differentiation from human mesenchymal stem cells (KP-hMSCs). We found that serum deprivation resulted in a reversible downregulation of glial- and oligodendrocyte-specific markers. Serum stimulated expression of oligodendrocyte markers, such as galactocerebroside, as well as Notch1 and JAK1 transcripts. Inhibition of Notch1 activation by the Notch inhibitor, MG132, led to enhanced expression of a serum-stimulated oligodendrocyte marker. This marker was undetectable in serum-deprived KP-hMSCs treated with MG132, suggesting that inhibition of Notch1 function is additive to serum-stimulated oligodendrocyte differentiation. Furthermore, a dominant-negative mutant RBP-J protein also inhibited Notch1 function and led to upregulation of oligodendrocyte-specific markers. Our results demonstrate that serum-stimulated oligodendrocyte differentiation is enhanced by the inhibition of Notch1-associated functions.
Because of the importance of voltage-activated K(+) channels during embryonic development and in cell proliferation, we present here the first description of these channels in E15 rat embryonic neural progenitor cells derived from the subventricular zone (SVZ). Activation, inactivation, and single-channel conductance properties of recorded progenitor cells were compared with those obtained by others when these Kv gene products were expressed in oocytes.Neural progenitor cells derived from the subventricular zone of E15 embryonic rats were cultured under conditions that did not promote differentiation. Immunocytochemical and Western blot assays for nestin expression indicated that almost all of the cells available for recording expressed this intermediate filament protein, which is generally accepted as a marker for uncommitted embryonic neural progenitor cells. However, a very small numbers of the cells expressed GFAP, a marker for astrocytes, O4, a marker for immature oligodendrocytes, and betaIII-tubulin, a marker for neurons. Using immunocytochemistry and Western blots, we detected consistently the expression of Kv2.1, and 4.3. In whole-cell mode, we recorded two outward currents, a delayed rectifier and an A-type current.We conclude that Kv2.1, and 4.3 are expressed in E15 SVZ neural progenitor cells, and we propose that they may be associated with the delayed-rectifier and the A-type currents, respectively, that we recorded. These results demonstrate the early expression of delayed rectifier and A-type K(+) currents and channels in embryonic neural progenitor cells prior to the differentiation of these cells.
The mechanisms that regulate peripheral nervous system (PNS) gliogenesis are incompletely understood. For example, gut neural crest stem cells (NCSCs) do not respond to known gliogenic factors, suggesting that yet-unidentified factors regulate gut gliogenesis. To identify new mechanisms, we performed gene expression profiling to identify factors secreted by gut NCSCs during the gliogenic phase of development. These cells highly expressed leucine-rich glioma inactivated 4 (Lgi4) despite the fact that Lgi4 has never been implicated in stem cell function or enteric nervous system development. Lgi4 is known to regulate peripheral nerve myelination (having been identified as the mutated gene in spontaneously arising claw paw mutant mice), but Lgi4 is not known to play any role in PNS development outside of peripheral nerves. To systematically analyze Lgi4 function, we generated gene-targeted mice. Lgi4-deficient mice exhibited a more severe phenotype than claw paw mice and had gliogenic defects in sensory, sympathetic, and enteric ganglia. We found that Lgi4 is required for the proliferation and differentiation of glial-restricted progenitors throughout the PNS. Analysis of compound-mutant mice revealed that the mechanism by which Lgi4 promotes enteric gliogenesis involves binding the ADAM22 receptor. Our results identify a new mechanism regulating enteric gliogenesis as well as novel functions for Lgi4 regulating the proliferation and maturation of glial lineage cells throughout the PNS.
Glioblastoma (GBM) is a highly lethal brain tumour presenting as one of two subtypes with distinct clinical histories and molecular profiles. The primary GBM subtype presents acutely as a high-grade disease that typically harbours mutations in EGFR, PTEN and INK4A/ARF (also known as CDKN2A), and the secondary GBM subtype evolves from the slow progression of a low-grade disease that classically possesses PDGF and TP53 events. Here we show that concomitant central nervous system (CNS)-specific deletion of p53 and Pten in the mouse CNS generates a penetrant acute-onset high-grade malignant glioma phenotype with notable clinical, pathological and molecular resemblance to primary GBM in humans. This genetic observation prompted TP53 and PTEN mutational analysis in human primary GBM, demonstrating unexpectedly frequent inactivating mutations of TP53 as well as the expected PTEN mutations. Integrated transcriptomic profiling, in silico promoter analysis and functional studies of murine neural stem cells (NSCs) established that dual, but not singular, inactivation of p53 and Pten promotes an undifferentiated state with high renewal potential and drives increased Myc protein levels and its associated signature. Functional studies validated increased Myc activity as a potent contributor to the impaired differentiation and enhanced renewal of NSCs doubly null for p53 and Pten (p53(-/-) Pten(-/-)) as well as tumour neurospheres (TNSs) derived from this model. Myc also serves to maintain robust tumorigenic potential of p53(-/-) Pten(-/-) TNSs. These murine modelling studies, together with confirmatory transcriptomic/promoter studies in human primary GBM, validate a pathogenetic role of a common tumour suppressor mutation profile in human primary GBM and establish Myc as an important target for cooperative actions of p53 and Pten in the regulation of normal and malignant stem/progenitor cell differentiation, self-renewal and tumorigenic potential.
Although neurogenic pathways have been described in the developing neocortex, less is known about mechanisms ensuring correct neuronal differentiation thus also preventing tumor growth. We have shown that RP58 (aka zfp238 or znf238) is highly expressed in differentiating neurons, that its expression is lost or diminished in brain tumors, and that its reintroduction blocks their proliferation. Mice with loss of RP58 die at birth with neocortical defects. Using a novel conditional RP58 allele here we show that its CNS-specific loss yields a novel postnatal phenotype: microencephaly, agenesis of the corpus callosum and cerebellar hypoplasia that resembles the chr1qter deletion microcephaly syndrome in human. RP58 mutant brains maintain precursor pools but have reduced neuronal and increased glial differentiation. Well-timed downregulation of pax6, ngn2 and neuroD1 depends on RP58 mediated transcriptional repression, ngn2 and neuroD1 being direct targets. Thus, RP58 may act to favor neuronal differentiation and brain growth by coherently repressing multiple proneurogenic genes in a timely manner.
B-cell regulator of immunoglobulin heavy chain transcription (Bright)/ARID3a, an A+T-rich interaction domain protein, was originally discovered in B lymphocyte lineage cells. However, expression patterns and high lethality levels in knockout mice suggested that it had additional functions. Three independent lines of evidence show that functional inhibition of Bright results in increased developmental plasticity. Bright-deficient cells from two mouse models expressed a number of pluripotency-associated gene products, expanded indefinitely, and spontaneously differentiated into cells of multiple lineages. Furthermore, direct knockdown of human Bright resulted in colonies capable of expressing multiple lineage markers. These data suggest that repression of this single molecule confers adult somatic cells with new developmental options.
During development of the central nervous system, the apical-basal polarity of neuroepithelial cells is critical for homeostasis of proliferation and differentiation of neural stem cells. While adherens junctions at the apical surface of neuroepithelial cells are important for maintaining the polarity, the molecular mechanism regulating integrity of these adherens junctions remains largely unknown. Given the importance of actin cytoskeleton in adherens junctions, we have analyzed the role of mDia, an actin nucleator and a Rho effector, in the integrity of the apical adherens junction. Here we show that mDia1 and mDia3 are expressed in the developing brain, and that mDia3 is concentrated in the apical surface of neuroepithelium. Mice deficient in both mDia1 and mDia3 develop periventricular dysplastic mass widespread throughout the developing brain, where neuroepithelial cell polarity is impaired with attenuated apical actin belts and loss of apical adherens junctions. In addition, electron microscopic analysis revealed abnormal shrinkage and apical membrane bulging of neuroepithelial cells in the remaining areas. Furthermore, perturbation of Rho, but not that of ROCK, causes loss of the apical actin belt and adherens junctions similarly to mDia-deficient mice. These results suggest that actin cytoskeleton regulated by Rho-mDia pathway is critical for the integrity of the adherens junctions and the polarity of neuroepithelial cells, and that loss of this signaling induces aberrant, ectopic proliferation and differentiation of neural stem cells.
Abnormalities in the migration of cortical neurons to ectopic sites can be caused by prenatal exposure to ethanol. In extreme cases, cells migrate past the pial surface and form suprapial heterotopias or 'warts'. We used organotypic slice cultures from 17-day-old rat fetuses to examine structural and molecular changes that accompany wart formation. Cultures were exposed to ethanol (0, 200, 400 or 800 mg/dl) and maintained for 2-32 h. Fixed slices were sectioned and immunolabeled with antibodies directed against calretinin, reelin, nestin, GFAP, doublecortin, MAP-2 and NeuN. Ethanol promoted the widespread infiltration of the marginal zone (MZ) with neurons and the focal formation of warts. The appearance of warts is time- and concentration-dependent. Heterotopias comprised migrating neurons and were not detected in control slices. Warts were associated with breaches in the array of Cajal-Retzius cells and with translocation of reelin-immunoexpression from the MZ to the outer limit of the wart. Ethanol also altered the morphology of the radial glia. Thus, damage to the integrity of superficial cortex allows neurons to infiltrate the MZ, and if the pial-subpial glial barrier is also compromised these ectopic neurons can move beyond the normal cerebral limit to form a wart.