Identification of neuronal cell lineage-specific molecules in the neuronal differentiation of P19 EC cells and mouse central nervous system

P19 embryonic carcinoma (EC) cells are one of the simplest systems for analyzing the neuronal differentiation. To identify the membrane-associated molecules on the neuronal cells involved in the early neuronal differentiation in mice, we generated two monoclonal antibodies, SKY-1 and SKY-2, by immunizing rats with a membrane fraction of the neuronally committed P19 EC cells as an antigen. SKY-1 and SKY-2 recognized the carbohydrate moiety of a 90 kDa protein (RANDAM-1) and the polypeptide core of a 40 kDa protein (RANDAM-2), respectively. In the P19 EC cells, the expression of RANDAM-1 was colocalized to a part of Nestin-positive cells, whereas that of RANDAM-2 was observed in most Nestin-positive cells as well as beta-III-tubulin positive neurons. In the embryonic and adult brain of mice, RANDAM-1 was expressed at embryonic day 8.5 (E8.5), and the localization of antigen was restricted on the neuroepithelium and choroid plexus. The RANDAM-2 expression commenced at E6.0, and the antigen was distributed not only on the neuroepithelium of embryonic brain but on the neurons of adult brain. Collectively, it was concluded that RANDAM-1 is a stage specific antigen to express on the neural stem cells, and RANDAM-2 is constitutively expressed on both the neural stem cells and differentiated neuronal cells in mouse central nervous system (CNS).     

Characterization of antibodies against major fish CNS myelin proteins: immunoblot analysis and immunohistochemical localization of 36K and IP2 proteins in trout nerve tissue

Antisera against the trout CNS myelin proteins 36K and IP2 were prepared in rabbits and characterized by immunoblot analysis and immunohistochemistry. The anti-36K antiserum exclusively stained its corresponding antigen from trout CNS myelin but failed to recognize any myelin polypeptide from either trout PNS or mammalian CNS and PNS. Antibodies against the IP2 glycoprotein specifically cross-reacted with related intermediate proteins (IP) of both CNS and PNS myelin from trout but only faintly labeled the PO protein of mouse peripheral nerve. Immunohistochemical localization of both antigens in the CNS of young trout was confined to the myelin sheath, except that anti-36K antiserum also stained oligodendrocytes. Nodes of Ranvier, neuronal cell bodies, and dendrites, as well as other glial elements, were negative. Specificity of the immunofluorescent reaction was established by crossed immunoadsorption experiments. Whereas on adjacent sections through trout brain both antigens exhibited a nearly identical distribution pattern, immunostaining in peripheral nerves was seen only with anti-IP2 antibodies.     

The mouse ortholog of the neuronal ceroid lipofuscinosis CLN5 gene encodes a soluble lysosomal glycoprotein expressed in the developing brain

Neuronal ceroid lipofuscinoses (NCLs) are recessively inherited neurodegenerative lysosomal storage disorders characterized by progressive motor and mental retardation, visual failure, and epileptic seizures. Finnish variant late infantile NCL (vLINCL(Fin)) is caused by mutations in the CLN5 gene. We have isolated the mouse Cln5 gene and analyzed its spatiotemporal expression in the central nervous system (CNS) by in situ hybridization and immunohistochemistry. Cln5 was expressed throughout the embryonic brain already at E15 and the expression steadily increased during development. Prominent expression was observed in cerebellar Purkinje cells, cerebral neurons, hippocampal pyramidal cells, and hippocampal interneurons. The expression pattern correlated with those CNS regions that get degenerated in CLN5 patients. In vitro expression of Cln5 in COS-1, HeLa, and neuronal cells further implied that mouse Cln5 is a soluble lysosomal glycoprotein, closely resembling human CLN5.     

Analysis of JHM central nervous system infections in rats

Intracerebral inoculation of murine coronavirus JHM into 2- to 3-day-old Wistar Furth rats causes an acute encephalomyelitis, while inoculations at 10 days of age usually result in hind leg paralysis. To examine the distribution of viral antigens within this infected central nervous system (CNS) tissue, we used the avidin-biotin-peroxidase method to detect monoclonal and polyclonal antibodies bound to JHM structural proteins; in addition we used the Western blot technique to detect viral proteins. Our study demonstrated the following characteristics: Infected neuronal and glial cells produced viral nucleocapsid and E2 glycoprotein. The synthesis of these viral structural proteins was not restricted to cells in any particular part of the central nervous system. While JHM E2 proteins could be detected in individual cells of JHM-infected CNS tissue, the relative level of detectable E2 protein in the total CNS tissue of infected rats was reduced by more than 13-fold compared with JHM-infected tissue culture cells. Hippocampus neuronal cells provided a sensitive indication of JHM infection. These cells invariably contained antigens in both acutely and chronically infected animals. The distribution of cells containing viral antigens differed markedly for JHM-induced acute encephalitis and chronic demyelinating disease. Acutely infected brains had large lesions containing low levels of viral antigen scattered throughout the brain. One percent to ten percent of histologically normal cells in many parts of the brain contained viral antigens; in addition, more neuronal cells than glial cells were observed to be antigen-positive. The hippocampus appeared normal with hematoxylin-eosin staining; however, a scattered infection of neuronal cells was apparent.(ABSTRACT TRUNCATED AT 250 WORDS)     

Musashi1: an evolutionally conserved marker for CNS progenitor cells including neural stem cells

In situ detection of neural progenitor cells including stem-like cells is essential for studying the basic mechanisms of the generation of cellular diversity in the CNS, upon which therapeutic treatments for CNS injuries, degenerative diseases, and brain tumors may be based. We have generated rat monoclonal antibodies (Mab 14H1 and 14B8) that recognize an RNA-binding protein Musashi1, but not a Musashi1-related protein, Musashi2. The amino acid sequences at the epitope sites of these anti-Musashi1 Mabs are remarkably conserved among the human, mouse, and Xenopus proteins. Spatiotemporal patterns of Musashi1 immunoreactivity in the developing and/or adult CNS tissues of frogs, birds, rodents, and humans indicated that our anti-Musashi1 Mabs reacted with undifferentiated, proliferative cells in the CNS of all the vertebrates tested. Double or triple immunostaining of embryonic mouse brain cells in monolayer cultures demonstrated strong Musashi1 expression in Nestin(+)/RC2(+) cells. The relative number of Musashi1(+)/Nestin(+)/RC2(+) cells increased fivefold when embryonic forebrain cells were cultured to form 'neurospheres' in which stem-like cells are known to be enriched through their self-renewing mode of growth. Nestin(+)/RC2(-) cells, which included Talpha1-GFP(+) neuronal progenitor cells and GLAST(+) astroglial precursor cells, were also Musashi1(+), as were GFAP(+) astrocytes. Young neurons showed a trace of Musashi1 expression. Cells committed to the oligodendroglial lineage were Musashi(-). Musashi1 was localized to the perikarya of CNS stem-like cells and non-oligodendroglial progenitor cells without shifting to cell processes or endfeet, and is therefore advantageous for identifying each cell and counting cells in situ.     

Selective localization of wild type and mutant mouse hepatitis virus (JHM strain) antigens in CNS tissue by fluorescence, light and electron microscopy

Demyelination may be induced by several different pathogenetic mechanisms. We have been utilizing mouse hepatitis virus (MHV) to study virus-induced demyelination in the central nervous system (CNS). To learn whether the different disease phenotypes in 4-week-old mice, caused by wild type (a model for fatal encephalomyelitis) or mutant ts8 (a model for primary demyelination), is due to an altered cellular tropism, we have developed an immunolabeling technique to evaluate critically the localization of MHV antigens in the unique cells of the CNS. Using mouse derived L-cells and primary neuronal cells in vitro, we determined an appropriate fixative (4% paraformaldehyde and 0.5% glutaraldehyde) that both preserved MHV antigenicity and cell structure. These studies in vitro showed the presence of MHV antigens on the surface of cells. Utilizing immunoperoxidase labeling as developed, we studied the localization of MHV antigens in vivo. MHV antigens associated with wild type (wt) virus were localized in neuronal cells as well as oligodendrocytes, which might account for the encephalomyelitis and primary demyelination, respectively. In contrast, MHV antigens associated with ts8 were localized rarely in neurons but commonly in oligodendrocytes. This might account for the uncommon occurrence of fatal encephalomyelitis, but the frequent presence of primary demyelination. Of interest was the finding of viral antigens during MHV infection in the cytoplasmic processes of oligodendrocytes surrounding intact myelin sheaths. We conclude that the different disease phenotypes caused by wt and mutant ts8 reflect differences in the cellular tropism of the two viruses for cells in the CNS.     

Maturing neurons are selectively sensitive to human immunodeficiency virus type 1 exposure in differentiating human neuroepithelial progenitor cell cultures

Human immunodeficiency virus type 1 (HIV-1) infection of the brain is associated with neuronal injury manifested by dendritic pruning, aberrant neurofilament metabolism, and decreased synaptic density. The central nervous system (CNS) responds to neuronal injury by differentiating new neurons and astrocytes from resident populations of multipotent neuroepithelial progenitor cells (NEP) located in regions such as the subventricular zone or hippocampus. In vitro studies have demonstrated that the HIV-1 virion or envelope glycoprotein gp120 can injure differentiated human neurons and astrocytes, suggesting that HIV-1 proteins could similarly injure NEP or NEP-derived glial and neuronal lineage-committed precursor cells. To answer this question, human fetal brain-derived "neurospheres" containing NEP and NEP-derived precursor cells were cultured in low serum differentiation medium containing lymphotropic HIV-1(SF2), macrophage-tropic HIV-1(SF128A), or recombinant gp120SF2 from HIV-1(SF2). These experiments indicate that exposure to HIV-1 does not affect the ability of the NEP to differentiate into cells expressing either astrocyte-specific or neuron-specific cytoskeletal antigens. However prolonged exposure to HIV-1 does selectively decrease expression of neuronal antigens (microtubule beta-III-tubulin and intermediate filament neurofilament-L) but not astrocyte antigens (intermediate filament glial fibrillary acidic protein). The effects of continuous exposure to HIV-1 or gp120 may result from injury to developing neurons and/or impairment of the neuronal developmental process itself. By depressing neuronal microtubule and neurofilament protein expression, HIV-1 and gp120 exposure compromise the potential for postmitotic neuronal dendrite and axon development.     

Distinct functions of human numb isoforms revealed by misexpression in the neural stem cell lineage in the Drosophila larval brain

Mammalian Numb (mNumb) has multiple functions and plays important roles in the regulation of neural development, including maintenance of neural progenitor cells and promotion of neuronal differentiation in the central nervous system (CNS). However, the molecular bases underlying the distinct functions of Numb have not yet been elucidated. mNumb, which has four splicing isoforms, can be divided into two types based on the presence or absence of an amino acid insert in the proline-rich region (PRR) in the C-terminus. It has been proposed that the distinct functions of mNumb may be attributable to these two different types of isoforms. In this study, we used the outer optic anlage (OOA) of the Drosophila larval brain as an assay system to analyze the functions of these two types of isoforms in the neural stem cells, since the proliferation pattern of neuroepithelial (NE) stem cells in the OOA closely resembles that of the vertebrate neural stem/progenitor cells. They divide to expand the progenitor cell pool during early neurogenesis and to produce neural precursors/neurons during late neurogenesis. Clonal analysis in the OOA allows one to discriminate between the NE stem cells, which divide symmetrically to expand the progenitor pool, and the postembryonic neuroblasts (pNBs), which divide asymmetrically to produce neural precursors (ganglion mother cells), each of which divides once to produce two neurons. We found that in the OOA, the human Numb isoform with a long PRR domain (hNumb-PRRL), which is mainly expressed during early neurogenesis in the mouse CNS, promotes proliferation of both NE cells and pNBs without affecting neuronal differentiation, while the other type of hNumb isoform with a short PRR domain (hNumb-PRRS), which is expressed throughout neurogenesis in the mouse embryonic CNS, inhibits proliferation of the stem cells and promotes neuronal differentiation. We also found that hNumb-PRRS, a functional homologue of Drosophila Numb, more strongly decreases the amount of nuclear Notch than hNumb-PRRL, and could antagonize Notch functions probably through endocytic degradation, suggesting that the two distinct types of hNumb isoforms could contribute to different phases of neurogenesis in the mouse embryonic CNS.     

Expression of neurofilament subunits in neurons of the central and peripheral nervous system: an immunohistochemical study with monoclonal antibodies

The extent to which all neurofilament (NF) subunits (NF68, NF150, NF200) are expressed by different populations of mature CNS and PNS neurons is controversial. We addressed this issue in immunohistochemical studies of mature bovine tissues using monoclonal antibodies specific for each bovine NF subunit. All three NF subunits were detected in the perikarya and neurites of both CNS and PNS neurons; they were seen in nearly all PNS neuronal perikarya, and in all identifiable CNS and PNS axons. Most, but not all, CNS neuronal perikarya contained each of these NF antigens. CNS neurons devoid of immunodetectable NF antigens were generally small. The presence of low levels of NF antigens in neurons with scant perikaryal cytoplasm may account for the apparent absence of NF immunoreactivity in some classes of neurons, although other explanations, such as microheterogeneity among NF proteins, could account for this finding. NF antigens were also seen in some cells of the diffuse neuroendocrine system (adrenal chromaffin cells and cells of the pars distalis and pars intermedia), but not in other cell types. We suggest that the expression of all three NF subunits is a common feature of CNS and PNS neurons and their processes, and of some cells of the diffuse neuroendocrine system. These findings have implications for hypotheses concerning the structure and function of the intermediate filaments of neurons, and for hypotheses concerning neurodegenerative diseases involving NF proteins.     

GFAP-Positive Progenitor Cell Production is Concentrated in Specific Encephalic Regions in Young Adult Mice

Previous genetic fate-mapping studies have indicated that embryonic glial fibrillary acidic protein-positive (GFAP⁺) cells are multifunctional progenitor/neural stem cells that can produce astrocytes as well as neurons and oligodendrocytes throughout the adult mouse central nervous system (CNS). However, emerging evidence from recent studies indicates that GFAP⁺ cells adopt different cell fates and generate different cell types in different regions. Moreover, the fate of GFAP⁺ cells in the young adult mouse CNS is not well understood. In the present study, hGFAP-Cre/R26R transgenic mice were used to investigate the lineage of embryonic GFAP⁺ cells in the young adult mouse CNS. At postnatal day 21, we found that GFAP⁺ cells mainly generated NeuN⁺ neurons in the cerebral cortex (both ventral and dorsal), hippocampus, and cerebellum. Strangely, these cells were negative for the Purkinje cell marker calbindin in the cerebellum and the neuronal marker NeuN in the thalamus. Thus, contrary to previous studies, our genetic fate-mapping revealed that the cell fate of embryonic GFAP⁺ cells at the young adult stage is significantly different from that at the adult stage.     

Developmental expression of voltage-gated potassium channel beta subunits.

Expression of potassium channel beta subunits (Kvbeta) was determined in the developing mouse CNS using an antiserum against an amino acid sequence present in the C-terminus of Kvbeta1, Kvbeta2, and Kvbeta3. Using the anti-Kvbeta antiserum, we determined that Kvbeta expression is restricted to the spinal cord and dorsal root ganglia in the embryonic CNS. At birth, Kvbeta expression is detected in brainstem and midbrain nuclei, but was not detected in the hippocampus, cerebellum or cerebral cortex. During the first postnatal week, Kvbeta expression is present in hippocampal and cortical pyramidal cells and in cerebellar Purkinje cells. Expression of Kvbeta subunits reaches adult levels by the third postnatal week in all of the brain regions examined. A rabbit antiserum directed against a unique peptide sequence in the N-terminus of the Kvbeta1 protein demonstrates that this subunit displays a novel expression pattern in the developing mouse brain. Kvbeta1 expression is high at birth in all brain regions examined and decreases with age. In contrast, Kvbeta2 expression is low at birth and increases with age to reach adult levels by the third postnatal week. These findings support the notion that the differential regulation of distinct potassium channel beta subunits, in the developing mouse nervous system, may confer the functional diversity required to mediate both neuronal survival and maturation.     

Defective neuronogenesis in the absence of Dlx5

Dlx genes play an important role in the control of the development of the central nervous system (CNS). Single or compound inactivation of Dlx1, Dlx2, or Dlx5 in the mouse causes defects of neuronal migration and differentiation. Dlx5, in particular, is essential for the correct development of the olfactory system. Targeted inactivation of Dlx1 and Dlx2 in the mouse results in abnormal neuronal differentiation in the embryonic subcortical forebrain and is associated to the loss of Dlx5 and Dlx6 expression. So far, however, it has been impossible to investigate the role of Dlx genes on late neurogenesis, as their inactivation leads to perinatal death. We have now generated cultures of neural stem cells (NSCs) derived from embryonic and newborn Dlx5-null mice, and we have compared their capacity to differentiate in vitro to that of equivalent cells derived from normal littermates. We show here that in the absence of Dlx5, NSCs derived from newborn animals have a severely reduced capacity to generate neurons. This is not the case for cells derived from E12.5 embryos. Forced expression of Dlx5 in cultures of newborn mutant NSCs fully restores their neuronogenic potential. Our data suggest that Dlx5 is essential for secondary (postnatal) neuronogenesis.     

Domain-restricted expression of two glutamic acid decarboxylase genes in midgestation mouse embryos

Glutamic acid decarboxylase (GAD) is the biosynthetic enzyme for gamma-aminobutyric acid (GABA), the major inhibitory neurotransmitter in the central nervous system (CNS) of vertebrates. In addition to the adult CNS, GABA and GAD also have been detected in embryos, although their precise localization and specific functions in embryonic development have not been elucidated. In this paper, the authors studied the cellular distribution of two GAD isoforms, GAD65 and GAD67, in midgestation mouse embryos by in situ hybridization histochemistry. With few exceptions, it was found that GAD65 and GAD67 mRNAs are localized in overlapping cellular domains of the embryonic CNS that later develop into regions with a strong GABAergic contribution. The GAD-expressing cells are situated in the differentiating zone of the embryonic day 10.5 (E10.5) through E11.5 CNS and in the subventricular zone and the mantle zone of the E12.5 CNS, which suggests that they are committed neuronal precursors. By using a specific serum for GABA, a similar pattern of distribution was obtained, indicating that GAD mRNAs are translated efficiently into enzymatically active GAD, which produces embryonic GABA. The expression domains of GAD overlap with those of genes that are known to be involved in the patterning of the embryonic CNS. The two GAD mRNAs also are detected outside of the embryonic CNS in various cell types, mainly those of placodal and neural crest origin. This pattern of expression is consistent with the notion that GAD and its product, GABA, play a signaling role during development.     

Expression of peroxiredoxins and thioredoxins in the mouse spinal cord during embryonic development

Reactive oxygen and nitrogen species (ROS/RNS) are natural byproducts of cellular metabolism. Although these molecules are deleterious at high concentrations, moderate levels of ROS/RNS are essential for normal cell function and take part in numerous cellular processes. The regulation of ROS/RNS is largely attended by peroxiredoxins (Prdxs) and their main reductants, thioredoxins (Trxs). Through their oxidoreductase activities, the members of the Trx/Prdx system can also affect certain cellular processes, notably many implicated in central nervous system (CNS) development. Although several studies have investigated the expression of Prdxs and Trxs in mouse, rat, and human adult CNS, few data are available concerning embryonic stages. In this work, we use immunofluorescence analyses to study the distribution of these enzymes during prenatal mouse spinal cord development. Our results highlight several patterns that contrast with available data for the adult. Indeed, Prdx1, Prdx4, and Prdx6, which are expressed in glial cells in the adult CNS, present clear neuronal localization in mouse spinal cord during embryonic development. Additionally, Prdx1, Prdx2, and to a lesser extent Prdx4, Prdx6, and Trx1 are localized mainly in the nucleus of neural cells. Finally, we identified a consistent, intense expression of all Prdxs and Trxs in groups of cells located in ventral regions of the spinal cord that express motor neuronal markers. These striking expression patterns suggest novel functions of these enzymes at these stages and offer clues to the role of the Trx/Prdx system during embryonic development of the spinal cord. J. Comp. Neurol. 523:2599–2617, 2015. © 2015 Wiley Periodicals, Inc.     

Alpha-internexin, a novel neuronal intermediate filament protein, precedes the low molecular weight neurofilament protein (NF-L) in the developing rat brain

alpha-Internexin is a 66 kDa protein that copurifies with intermediate filaments (IF) from rat spinal cord and optic nerve. This protein is axonally transported in rat optic nerve along with the neurofilament triplet proteins in slow component a. Polymerization in vitro and distribution in vivo confirm that alpha-internexin is a neuronal IF. We raised 2 highly specific monoclonal antibodies to alpha-internexin which were applied to frozen rat brain sections and Western blots of cytoskeletal extracts. These results indicate that alpha-internexin is primarily an axonal protein found in most, if not all, neurons of the CNS. Immunoreactive proteins of similar molecular weight were found in cytoskeletal extracts of CNS tissue from several additional species, including mouse and cow. While the distribution of alpha-internexin as given by immunocytochemical methods is similar to that of low molecular weight neurofilament protein (NF-L) in the adult, its distribution in the embryo is far more extensive. At embryonic day 16, when the expression of NF-L is still limited to a relatively small number of cells and levels of expression are low, alpha-internexin is already found at much higher levels and in cells not yet expressing NF-L in detectable quantities. Similar results are found at embryonic day 12. These data suggest that neuronal IF in the developing nervous system contain a higher proportion of alpha-internexin than their adult counterparts, and that expression of alpha-internexin precedes that of NF-L in many or most neurons of the developing brain.     

Localization of a neurectoderm-associated cell surface antigen in the developing and adult rat

The distribution of a neurectoderm-associated carbohydrate antigen (termed D1.1) in tissues of the developing and adult rat was determined using indirect immunofluorescent techniques. The antigen was detected as early as embryonic days 8 and 9 when it was localized to cells within the developing neural plate and neural tube. As the central nervous system (CNS) developed, the anti-D1.1 antibody labeled neuroepithelial cells but not terminally differentiated neurons or glial cells. In addition, the notochord and somatic mesoderm were labeled transiently with the antibody. Outside of the CNS, the antibody labeled dorsal root ganglia neurons, adrenal chromaffin cells and cells of the kidney glomerulus. These tissues were labeled at embryonic day 14 and the labeling persisted in the adult. We used a sensitive immunoautoradiography assay to identify antigenic gangliosides present in extracts of these tissues. The anti-D1.1 antibody recognized a ganglioside of kidney and adrenal glands that has a chromatographic mobility identical to that of the D1.1 antigen previously identified from cell lines and developing cerebellum. However, the antibody bound to a separate and distinct set of gangliosides present in extracts of adult dorsal root ganglia. Thus, the carbohydrate sequence recognized by the antibody can be associated with more than one molecular species of ganglioside. These results demonstrate that within the context of the developing CNS, the D1.1 antigen is a stage-specific embryonic antigen, but, as is the case with other cell surface carbohydrate antigens, is also found on a limited but developmentally unrelated set of tissues in the adult.     

Xenografted fetal dorsal root ganglion, embryonic stem cell and adult neural stem cell survival following implantation into the adult vestibulocochlear nerve

Sensorineural hearing loss is a disabling condition. In the post-embryonic and adult mammalian inner ear, the regeneration of auditory hair cells, spiral ganglion neurons or their axons does not occur naturally. This decrease in excitable neurons limits the success of auditory rehabilitation. Allografts and xenografts have shown promise in the treatment of a variety of neurological diseases. Fetal dorsal root ganglion (DRG) neurons can extend functional connections in the rat spinal cord. Embryonic stem cells (ES cells) and adult neural stem cells (ANSC) have the potential to differentiate into neurons. We have implanted embryonic days (E) 13-16 fetal mouse DRGs from transgenic mouse lines that express Enhanced Green Fluorescent Protein (EGFP) or lacZ reporter genes, EGFP-expressing ES cells or lacZ-expressing ANSC into the injured vestibulocochlear nerve of adult rats and guinea pigs. Survival of the implants was assessed 2 to 4 weeks postoperatively. For further evaluation of the differentiation of the implanted ES-cells, we double labeled with the mouse-specific neuronal antibody Thy 1.2. The rats implanted with EGFP- or lacZ-expressing DRGs showed labeled DRGs after sacrifice. In addition, EGFP-positive nerve fibers were seen growing within the proximal nerve. The results from the EGFP ES cells and lacZ ANSC revealed reporter-expressing cells at the site of injection in the vestibulocochlear nerve of the host rats and guinea pigs but also within the brain stem. Thy 1.2 profiles were seen among the EGFP ES cells within the 8th cranial nerve. The findings of this study indicate that the vestibulocochlear nerve of adult rats and guinea pigs will support xenotransplants of embryonic DRG, ES cells and ANSC. This may have future clinical applicability in recreating a neuronal conduit following neuronal injury between the inner ear and the central nervous system (CNS).     

Molecular cloning and characterization of a novel developmentally regulated gene, Bdm1, showing predominant expression in postnatal rat brain.

Postnatal development, such as synapse refinement, is necessary for the establishment of a mature and functional central nervous system (CNS). Using differential display analysis, we identified a novel gene, termed Bdm1, that is more abundantly expressed in the adult brain than in the embryonic brain. The full-length Bdm1 cDNA is 2718 base pairs long and contains an open reading frame of 1059 base pairs encoding a 38-kDa protein. Northern blot analysis revealed that expression of Bdm1 mRNA in the brain was weak on embryonic days and increased in the early postnatal period. Bdm1 mRNA was significantly expressed in the brain and heart, but there was no or little expression in other tissues. During the differentiation of mouse carcinoma cells P19 to neuron-like cells by retinoic acid, Bdm1 mRNA was up-regulated almost parallel to neurofilament mRNA. Expression of Bdm1 mRNA was observed appreciably in PC12 cells after neuronal differentiation but not in the nonneural cell lines examined. In situ hybridization demonstrated that Bdm1 was expressed widely in the olfactory bulb, cerebral cortex, hippocampus, cerebellum, thalamus, and medulla oblongata. Taken together, these data suggest that Bdm1 gene plays a role in the early postnatal development and function of neuronal cells.     

Imprinting as a rapid technique for assessing the morphology of the central nervous system by immunofluorescence

This paper describes a technique that has been developed to assess the in vivo morphology of central nervous system (CNS) tissue by immunofluorescence. This technique permits the study of tissue that is mainly just a monolayer of cells. Unlike routine cryosections that are much thicker (10–15 μm), imprinting does not section the cells, but can result in the detachment of whole cells onto a glass surface for subsequent staining. The imprinting technique is simple and rapid and does not require prior fixation or embedding of the tissue. It has been used to evaluate antigens expressed at the cell surface, in myelin and in the cytoskeleton in the studies of normal and myelin mutant mice. Using the imprinting/immunofluorescence technique one can now assay the genotype of mouse strains that differ in their expression of cell surface antigens within 2 h.     

An adenovirus vector encoding tyrosine hydroxylase activity may enter human CNS cells in primary dissociated cultures

An adenovirus encoding tyrosine hydroxylase (TH) activity was inserted in neuronal and glial cultured cells obtained from human fetal central nervous system (CNS) tissue. Using a double fluorescence immunostaining, we characterized inoculated CNS cells, with a TH antiserum and one of the following antibodies: microtubule-associated protein (MAP₂) and GABA for neuronal cells, vimentin (Vim) for glial cells and glial fibrillary acidic protein (GFAP) for astrocytes. The characterization of inoculated neuronal cells was established by the detection of TH-MAP₂-stained neurons in cultures obtained from the thoracic and lumbar parts of the spinal cord where no intrinsic TH cells are described. Inoculated glial cells were characterized by the detection of TH-Vim and TH-GFAP-stained CNS cultured cells. We also observed GABA neurons expressing TH immunoreactivity which could be considered as inoculated neurons expressing the GABA phenotype. Whatever the time of inoculation, transfection was observed in both neuronal and glial cells, after up to 4 months of culture. Although no precise quantitation was performed, the percentage of inoculation was found on microscopic inspection to be greater in glia than in neurons, as previously reported. We concluded that a gene coding for a key neuronal enzyme can be incorporated in embryonic human glial and neuronal cells through the use of a recombinant adenovirus.