Heterogeneity of glial progenitor cells during the neurogenesis-to-gliogenesis switch in the developing human cerebral cortex

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Chromatin and gene-regulatory dynamics of the developing human cerebral cortex at single-cell resolution

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Stem cells in the embryonic cerebral cortex: Their role in histogenesis and patterning

The cytoarchitectural simplicity of the cerebral cortex makes it an attractive system to study central nervous system (CNS) histogenesis—the process whereby diverse cells are generated in the right numbers at the appropriate place and time. Recently, multipotent stem cells have been implicated in this process, as progenitor cells for diverse types of cortical neurons and glia. Continuous analysis of stem cell clone development reveals stereotyped division patterns within their lineage trees, highly reminiscent of neural lineage trees in arthropods and Caenorhabditis elegans. Given that these division patterns play a critical part in generating diverse neural types in invertebrates, we speculate that they play a similar role in the cortex. Because stereotyped lineage trees can be observed from cells growing at clonal density, cell-intrinsic factors are likely to have a key role in stem cell behavior. Cortical stem cells also respond to environmental signals to alter the types of cells they generate, providing the means for feedback regulation on the germinal zone. Evidence is accumulating that cortical stem cells, influenced by intrinsic programs and environmental signals, actually change with development—for example, by reducing the number and types of neurons they produce. Age-related changes in the stem cell population may have a critical role in orchestrating development; whether these cells truly self-renew is a point of discussion. In summary, we propose that cortical stem cells are the focus of regulatory mechanisms central to the development of the cortical cytoarchitecture. © 1998 John Wiley & Sons, Inc. J Neurobiol 36: 162–174, 1998     

Effect of nonionizing radiation on the purkinje cells of the uvula in squirrel monkey cerebellum

Pregnant squirrel monkeys were exposed to 2450-MHz (CW) microwaves at an equivalent power density of 10 mW/cm² (SAR 3.4 mW/g) for three hours daily in a cavity-cage module. The exposure began when pregnancy was determined by a hormonal method, and continued through the offspring's first 9.5 months. After irradiation, the brains of the offspring were fixed with formaldehyde, and the inferior vermis of each cerebella was removed and processed for histologic observations. Purkinje cell density in the uvula was determined in sagittal serial sections. There was no significant difference between control and experimental animals in the number of Purkinje cells per mm of Purkinje cell line (linear density), as well as in the density of Purkinje cells in the Purkinje cell layer.     

Synthesis of Lewis A trisaccharide analogues in which D-glucose and L-rhamnose replace D-galactose and L-fucose, respectively

In our effort to design a safe anti-cancer vaccine based on the tumor associated carbohydrate antigen Le(a)Le(x), we are studying the cross-reactivity between the Le(a) natural trisaccharide antigen and analogues in which the L-fucose, D-galactose, and/or D-glucosamine residues are replaced by L-rhamnose or D-glucose, respectively. We describe here the chemical synthesis of two such Le(a) trisaccharide analogues. In one trisaccharide, D-glucose replaces D-galactose and in the second analogue L-rhamnose and D-glucose replace L-fucose and D-galactose, respectively. Introduction of the rhamnose and fucose moiety onto the poorly reactive 4-OH group of the N-acetylglucosamine residue in a disaccharide acceptor was successful after bis-N-acetylation of the amine group. These analogues will be used in competitive binding experiments with anti-Le(a) antibodies and their solution conformations will be studied.     

Expression of glial antigens C1 and M1 in developing and adult neurologically mutant mice

The distribution of two glial antigens (C1 and M1) has been studied by indi-rect immunofluorescence during postnatal development of the cerebella of normal and neurologically mutant mice (weaver, staggerer, reeler, Purkinje cell degeneration, and wobbler). During the first postnatal week of normal development, C1 antigen is expressed in ependyma, Bergmann glial fibers (BG), and astrocytes of the internal granular layer and white matter. After day 10, C1 antigen is restricted to BG and ependymal cells. During the sec-ond and third week, BG undergo a transient loss of C1 antigen that starts in medioventral areas and spreads in a gradient dorsally and laterally. In reeler, weaver, and staggerer, C1 antigen expression is normal during the first postnatal week, and subsides in BG in a similar spatial gra- dient as described for the normal littermates. However, the loss of C1 anti-gen in BG occurs earlier (first in reeler, then in weaver, and last in staggerer) and is not reversible as it is in normal mice. In Purkinje cell de-generation, C1 antigen expression is diminished in BG after the onset of be-havioral abnormalities. Wobbler is normal with respect to C1 antigen ex-pression at adult ages. M1 antigen is detectable in white matter astrocytes from postnatal day 7 on, and persists in these cells into adulthood. Astrocytes of the internal granular layer and BG express M1 antigen only transiently in normal mice during the second and third weeks. The appearance of M1 antigen in BG occurs in a spatiotemporal gradient, matching the one in which C1 antigen disappears. M1 antigen expression is abnormally maintained in BG of reeler, staggerer, and weaver. In Purkinje cell degeneration, M1 antigen is ex-pressed abnormally at the onset of behavioral abnormalities first in.astro-cytes of the internal granular layer and, with growing age, increasingly also in BG. In wobbler, BG do not express M1 antigen. However, astrocytes of the granular layer are abnormally M1 antigen-positive.     

Spatiotemporal expression and functional implication of CXCL14 in the developing mice cerebellum

Cerebellar granule neurons migrate from the external granule cell layer (EGL) to the internal granule cell layer (IGL) during postnatal morphogenesis. This migration process through 4 different layers is a complex mechanism which is highly regulated by many secreted proteins. Although chemokines are well-known peptides that trigger cell migration, but with the exception of CXCL12, which is responsible for prenatal EGL formation, their functions have not been thoroughly studied in granule cell migration. In the present study, we examined cerebellar CXCL14 expression in neonatal and adult mice. CXCL14 mRNA was expressed at high levels in adult mouse cerebellum, but the protein was not detected. Nevertheless, Western blotting analysis revealed transient expression of CXCL14 in the cerebellum in early postnatal days (P1, P8), prior to the completion of granule cell migration. Looking at the distribution of CXCL14 by immunohistochemistry revealed a strong immune reactivity at the level of the Purkinje cell layer and molecular layer which was absent in the adult cerebellum. In functional assays, CXCL14 stimulated transwell migration of cultured granule cells and enhanced the spreading rate of neurons from EGL microexplants. Taken together, these results revealed the transient expression of CXCL14 by Purkinje cells in the developing cerebellum and demonstrate the ability of the chemokine to stimulate granule cell migration, suggesting that it must be involved in the postnatal maturation of the cerebellum.     

Localization of tissue plasminogen activator mRNA in the developing rat cerebellum and effects of inhibiting tissue plasminogen activator on granule cell migration

Tissue plasminogen activator (tPA) mRNA was localized in the developing cerebellum and the potentials role of tPA in migration of cerebellar granule cells was investigated. Proteolytic assays and Northern blots showed little variation in levels of tPA proteolytic activity or tPA mRNA expression in the developing cerebellum. The distribution of cerebellar tPA mRNA at different ages was visualized by in situ hybridization histochemistry. At postnatal day 7 (P7), most labeled cells were in the internal granule layer or developing white matter, and very few if any premigratory granule cells contained tPA mRNA. Although the molecular layer contained labeled cells at all ages, cell counts indicated that a greater percentage of cells in the molecular layer contained tPA mRNA during adulthood than during the period of granule cell migration. The most striking change in tPA mRNA expression was in Purkinje neurons, most of which began to express tPA mRNA between P7 and P14. The potential role of tPA in granule cell migration was investigated by performing migration assays in cerebellar slice explants in the presence or absence of protease inhibitors. The presence of inhibitors did not affect the distance that granule cells migrated. Data in the present study do not support a role for tPA in granule neuron migration; however, they do indicate that tPA is both spatially and temporally regulated during cerebellar development. Possible functions of tPA in the cerebellum are discussed. © 1995 John Wiley & Sons, Inc.     

Interindividual methylomic variation across blood,cortex, and cerebellum: implications for epigenetic studies of neurological and neuropsychiatric phenotypes

Given the tissue-specific nature of epigenetic processes, the assessment of disease-relevant tissue is an important consideration for epigenome-wide association studies (EWAS). Little is known about whether easily accessible tissues, such as whole blood, can be used to address questions about interindividual epigenomic variation in inaccessible tissues, such as the brain. We quantified DNA methylation in matched DNA samples isolated from whole blood and 4 brain regions (prefrontal cortex, entorhinal cortex, superior temporal gyrus, and cerebellum) from 122 individuals. We explored co-variation between tissues and the extent to which methylomic variation in blood is predictive of interindividual variation identified in the brain. For the majority of DNA methylation sites, interindividual variation in whole blood is not a strong predictor of interindividual variation in the brain, although the relationship with cortical regions is stronger than with the cerebellum. Variation at a subset of probes is strongly correlated across tissues, even in instances when the actual level of DNA methylation is significantly different between them. A substantial proportion of this co-variation, however, is likely to result from genetic influences. Our data suggest that for the majority of the genome, a blood-based EWAS for disorders where brain is presumed to be the primary tissue of interest will give limited information relating to underlying pathological processes. These results do not, however, discount the utility of using a blood-based EWAS to identify biomarkers of disease phenotypes manifest in the brain. We have generated a searchable database for the interpretation of data from blood-based EWAS analyses (http://epigenetics.essex.ac.uk/bloodbrain/).     

Effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of neural stem cells derived from the cerebral cortex of embryonic mice

Salvianolic acid B is isolated from Salvia miltiorrhiza,the root of which is widely used as a traditional Chinese medicine to treat stroke.However,little is known about how salvianolic acid B influences growth characteristics of neural stem cells (NSCs).The purpose of the present study was to evaluate the effects of salvianolic acid B on proliferation,neurite outgrowth and differentiation of NSCs derived from the cerebral cortex of embryonic mice using MTT,flow cytometry,immunofluorescence and RT-PCR.It was found that 20 μg mL·1 and 40 μg mL·1 salvianolic acid B had similar effects on proliferation of NSCs,and a suitable concentration of salvianolic acid B increased the number of NSCs and their derivative neurospheres.The growth-promoting activity of salvianolic acid B was dependent on and associated with an accumulation in the G2/S-phase cell population.Salvianolic acid B also promoted the neurite outgrowth of NSCs and their differentiation into neurons.The mRNA for tau,GFAP and nestin were present in differentiating neurospheres induced by salvianolic acid B.However,high-level expression of tau mRNA and low-level expression of GFAP mRNA was detected in differentiated cells,in contrast to the control conditions.This collective evidence indicates that exogenous salvianolic acid B is capable of promoting proliferation of neurospheres and differentiation towards the neuronal lineage in vitro and may act in the proliferation of NSCs and may promote NSC differentiation into neuronal cells.     

Specific labeling of climbing fibers shows early synaptic interactions with immature Purkinje cells in the prenatal cerebellum

During development, growing axons must locate target cells to form synapses. This is not easy, since target cells are also growing and even actively migrating. In some brain regions, such axons have been reported to wait for the timing when target cells become mature, without invading their target region. However, in the cerebellum climbing fibers (CFs), major afferent axons, arrive near their target neurons, Purkinje cells, when the neurons are still actively migrating. We, therefore, examined whether synaptic contacts are established at such early stages. To specifically label CFs, we introduced by in utero electroporation a mixture of genes encoding for Ptf1a‐enhancer‐driven Cre recombinase and Cre‐dependent fluorescent protein into the mouse hindbrain at embryonic day (E) 10.5 and observed them during development. The earliest stages at which labeled CFs were observed in the cerebellar primordium were E15.5–E16.5. These fibers were fasciculated in the dorsal region and entered the cerebellar primordium. Some fibers defasciculated and reached the caudal region. At E17.5 and E18.5, fasciculated fibers were also found in the mantle region, and some grew toward the surface of the primordium to penetrate a mass of Purkinje cells. Interestingly, as early as E16.5, labeled fibers were found to run in close apposition to Purkinje cell dendrites and to express a presynaptic marker. These observations suggest that CFs form synapses with Purkinje cells as soon as the fibers enter the cerebellum. © 2014 Wiley Periodicals, Inc. Develop Neurobiol 75: 927–934, 2015     

Identification of genes mediating thyroid hormone action in the developing mouse cerebellum

Despite the indispensable role thyroid hormone (TH) plays in brain development, only a small number of genes have been identified to be directly regulated by TH and its precise mechanism of action remains largely unknown, partly because most of the previous studies have been carried out at postnatal day 15 or later. In the present study, we screened for TH-responsive genes in the developing mouse cerebellum at postnatal day 4 when morphological alterations because of TH status are not apparent. Among the new candidate genes selected by comparing gene expression profiles of experimentally hypothyroid, hypothyroid with postnatal thyroxine replacement, and control animals using oligoDNA microarrays, six genes were confirmed by real-time PCR to be positively ( orc1l, galr3, sort1, nlgn3, cdk5r2 , and zfp367 ) regulated by TH. Among these, sort1 , cdk5r2, and zfp367 were up-regulated already at 1 h after a single injection of thyroxine to the hypothyroid or control animal, suggesting them to be possible primary targets of the hormone. Cell proliferation and apoptosis examined by BrdU incorporation and terminal deoxynucleotide transferase-mediated dUTP nick-end labeling assay revealed that hypothyroidism by itself did not enhance apoptosis at this stage, but rather increased cell survival, possibly through regulation of these newly identified genes.     

Glutaminase in the postnatally developing rat cerebellum: Comparison of staining and immunocytochemistry activity

Distribution patterns and developmental profiles of phosphate activated glutaminase (PAG) in the cerebellar cortex of the rat were demonstrated by enzyme activity staining (tetrazolium salt technique) and immunolabeling. Histochemical evaluation of enzyme activity stained sections revealed in the molecular and granular layer (i.e. premigratory zone and external germinal zone in neonate rats) an increase from postnatal day 2 to day 50 by 350 and 400%, respectively. The smallest elevation was found in Purkinje cell bodies (140%). Maximum rise of PAG-activity was observed for all of the areas examined between day 12 and 15. The immunocytochemical visualisation of PAG-like immunoreactivity resulted in spatial and developmental patterns which differed from those of PAG-activity staining and displayed, to some extent, dependency on the way of tissue preparation, especially the fixation procedure.     

Neurochemical profile of the developing mouse cortex determined by in vivo 1H NMR spectroscopy at 14.1 T and the effect of recurrent anaesthesia

The neurochemical profile of the cortex develops in a region and time specific manner, which can be distorted by psychiatric and other neurological pathologies. Pre-clinical studies often involve experimental mouse models. In this study, we determined the neurochemical profile of C57BL/6 mice in a longitudinal study design to provide a reference frame for the normal developing mouse cortex. Using in vivo proton NMR spectroscopy at 14 T, we measured the concentrations of 18 metabolites in the anterior and posterior cortex on postnatal days (P) 10, 20, 30, 60 and 90. Cortical development was marked by alterations of highly concentrated metabolites, such as N-acetylaspartate, glutamate, taurine and creatine. Regional specificity was represented by early variations in the concentration of glutamine, aspartate and choline. In adult animals, regional concentration differences were found for N-acetylaspartate, creatine and myo-inositol. In this study, animals were exposed to recurrent isoflurane anaesthesia. Additional experiments showed that the latter was devoid of major effects on behaviour or cortical neurochemical profile. In conclusion, the high sensitivity and reproducibility of the measurements achieved at 14 T allowed us to identify developmental variations of cortical areas within the mouse cortex.     

The number of active Purkinje cells in the microelectrod track as an indicator of maturity of the cerebellum motor function in mature- and immature-born animals

In experiments on 5 age groups of anesthetized guinea pigs (from newborns to 4 weeks of postnatal ontogenesis), activity of cerebellum Purkinje cells (PC) (IV–VII lobules of cerebellar vermis) was studied in the single track of microelectrode passing through cell layers. It has been shown that as early as several hours after birth, in the superficial layer of cerebellar cortex, there are recorded occasional background-active, but functionally mature PC in the form of simple and complex spikes and accordingly reflecting synaptic PC activation by afferent inputs of mossy and climbing fibers. The functional manifestation of the guinea pig motor behavior at this period of ontogenesis is act of their standing. At this period of ontogenesis, in the newborn and one-day old guinea pigs, on average, from 1 to rarely 11 active PC are recorded in the single microelectrode track. At the one-week age, the highest number of active PC in the track somewhat increases, predominantly at the expense of the mean from the total number of cells in the track. In the 2-week old guinea pigs the mean number of active PC in the track somewhat falls, while in the 4-week old and adult animals it again exceeds, although slightly, the maximal number of PC in the track of newborn animals. The relatively high number of active PC at the very initial period of postnatal ontogenesis can indicate importance of motor function in the congenital food-procuring reflex.     

Bimodal septal and cortical triggering and complex propagation patterns of spontaneous waves of activity in the developing mouse cerebral cortex

Spontaneous waves of activity that propagate across large structures during specific developmental stages play central roles in CNS development. To understand the genesis and functions of these waves, it is critical to understand the spatial and temporal patterns of their propagation. We recently reported that spontaneous waves in the neonatal cerebral cortex originate from a ventrolateral pacemaker region. We have now analyzed a large number of spontaneous waves using calcium imaging over the entire area of coronal slices from E18‐P1 mouse brains. In all waves, the first cortical region active is this ventrolateral pacemaker. In half of the waves, however, the cortical pacemaker activity is itself triggered by preceding activity in the septal nuclei. Most waves are restricted to the septum and/or ventral cortex, with only some invading the dorsal cortex or the contralateral hemisphere. Waves fail to propagate at very stereotyped locations at the boundary between ventral and dorsal cortex and at the dorsal midline. Waves that cross these boundaries pause at these same locations. Waves at these stages are blocked by both picrotoxin and CNQX, indicating that both GABA_A and AMPA receptors are involved in spontaneous activity. © 2010 Wiley Periodicals, Inc. Develop Neurobiol 70: 679–692, 2010