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.     

Prenatal ontogeny of the epidermal growth factor receptor and its ligand,transforming growth factor alpha,in the rat brain

Transforming growth factor alpha (TGFα) interacts with the epidermal growth factor receptor (EGF-R) to produce its biological effects. TGFα induces the proliferation and differentiation of central nervous system (CNS) astrocytes and pluripotent stem cells, as well as the survival and differentiation of postmitotic CNS neurons. Both TGFα and EGF-R have been localized to the postnatal CNS. As the majority of CNS neuronal proliferation and migration occurs antenatally, we have examined the ontogeny of TGFα and EGF-R in the embryonic rat brain by in situ hybridization. EGF-R mRNA was expressed in the brain as early as embryonic day 11 (E11; the earliest age examined). It was initially detected in the midbrain, with subsequent expression first in multiple germinal zones, followed by expression in numerous cells throughout the brain. In many brain areas, EGF-R mRNA appeared in germinal centers during the later stages of neurogenesis and the early stages of gliogenesis. In the midbrain, the distribution of EGF-R mRNA overlapped extensively with that of tyrosine hydroxylase mRNA, suggesting that fetal dopaminergic neurons express EGF-R. Immunocytochemistry was used to demonstrate the presence of EGF-R-immunoreactive protein in brain areas that expressed EGF-R mRNA on E15 and E20. The expression of TGFα in many brain structures preceded that of EGF-R mRNA. TGFα mRNA was distributed throughout many non-germinal centers of the brain on E12 and later. Some brain areas, such as the external granule cell layer of the cerebellum, expressed EGF-R, but not TGFα mRNA. Northern blot analysis demonstrated that mRNA species for both TGFα and EGF-R were similar in embryos and adults. These data indicate that TGFα and EGF-R are positioned to have a role in the genesis, differentiation, migration, or survival of numerous cell populations in the embryonic brain. J. Comp. Neurol. 380:243–261, 1997. © 1997 Wiley-Liss, Inc.     

Localization and ontogeny of the orphan receptor OR-1 in the rat brain

This study describes the expression of the OR-1 orphan receptor in embryonic, postnatal, and adult brain tissue studied byin situ hybridization. This newly characterized member of the nuclear receptor superfamily functions as a modulator of retinoic acid and thyroid hormone signalling by influencing gene activation by these hormones from a distinct promoter region. In the fetal brain OR-1 mRNA was observed from E13–E16 in the developing pons, tegmentum, pontine flexure, medulla, inferior and superior colliculi, cerebellum, hippocampus, thalamus, striatum, and cortical plate. At E18, OR-1 was expressed in the hippocampus, cerebellum, ventricular layer of the developing cortex and cortical plate, striatum, and olfactory bulb. In the E21 to early postnatal brain the highest expression of OR-1 mRNA was seen in the hippocampus, cerebellum, striatum, and olfactory bulb. The expression of OR-1 in the cerebellum increased during postnatal development and by d P21 OR-1 mRNA had reached the levels present in the adult in the cerebellar cortex. In the adult brain the highest expression of OR-1 mRNA was observed in the Ca1 area of the hippocampus and the cerebellar cortex. We conclude that OR-1 is widely expressed in the fetal brain, whereas in the postnatal and adult brains OR-1 mRNA is more discretely localized, and that the amount of OR-1 mRNA increases in the cerebellum during postnatal development. The results of this study suggest that, in the fetal brain, OR-1 has a spatially widespread role in modulating gene activation by retinoids and thyroid hormone, whereas in the adult brain this modulation occurs only in distinct neuronal populations.     

In situ hybridization analysis of PrP mRNA in human CNS tissues

Expression of the prion protein gene (Prnp) and production of the PrP protein are essential requirements for acquisition and spread of transmissible spongiform encephalopathies such as Creutzfeldt-Jakob disease (CJD) in humans. Here we have developed an in situ hybridization method for use on human post-mortem central nervous system (CNS) tissues in order to determine those cell which are transcribing the Prnp gene and thus expressing PrP mRNA. Tissues from 11 adult individuals (age range 21-79 years) were analysed. Similar to previous studies in other animal systems, it was shown that PrP production occurs primarily in neuronal populations throughout the human brain. Neurones of the hippocampus, cortex, thalamus, cerebellum and medulla all synthesize PrP mRNA at readily detectable levels. No age-related differences were observed between the cases studied. It was also found that the ependymal cells produced PrP mRNA; these were the only non-neuronal cell type expressing the Prnp gene in the CNS. It is hoped that the information produced here will be helpful in understanding the pathology associated with CJD and other prion diseases in humans.     

Localization and developmental changes of the expression of two inward rectifying K-channel proteins in the rat brain

We have raised affinity-purified polyclonal antibodies specific for the inward rectifying K⁺ channel (IRK1/Kir2.1) and the G protein-activated inward rectifying K⁺ channel (GIRK1/Kir3.1) examined their distributions in the rat brain immunohistochemically. The regional expression pattern of the IRK1 and GIRK1 proteins were similar to those of mRNA of the previous in situ hybridization study. The subcellular distribution was studied in the cerebellum, cerebral cortex and hippocampus. In the cerebellum, the IRK1 protein was clearly detected in the somata and proximal dendrites of Purkinje cells, while the GIRK1 protein was present in the somata and clustered dendrites of granule cells. In the cerebral cortex and hippocampus, both IRK1- and GIRK1-immunoreactivities were detected in the somata and apical dendrites of the pyramidal cells. The presence of IRK1 or GIRK1 proteins in the axons could not proved by the present study. The developmental changes of the expression pattern of the GIRK1 protein were also investigated in the hippocampus and in the cerebellum of postnatal day (P) 7 to P17 rats. The GIRK1 protein was detected neither in the subgranular zone of the dentate gyrus nor in the proliferative zone of the external granule cell layer of the cerebellum, in which granule cell precursors are reported to proliferate, while it was clearly detected in the adjacent layer in which postmitotic but immature cells exist. These results imply that the expression of the GIRK1 protein starts just after the neuronal precursors finished the last mitotic cell division. ©1997 Elsevier Science B.V. All rights reserved.     

Identification of the novel developmentally regulated gene, Bdm2, which is highly expressed in fetal rat brain

Most of the neurogenesis take place during the embryonic stage; the genes expressed predominantly in this stage may play important roles in the control of development of the central nervous system. Using a differential display method, we identified the novel rat gene, brain development-related molecule 2 (Bdm2), that is expressed more abundantly in the embryonic brain than in the adult brain. Full-length Bdm2 cDNA consists of 1842 base pairs (bp) and contains an open reading frame of 1260 bp. Northern blot analysis demonstrated that Bdm2 was strongly expressed in the late embryonic brain and was still detected at lower levels in an early postnatal period; in adults, Bdm2 mRNA was decreased to an undetectable level in brain, though the expression of this mRNA was revealed in other tissues. Level of Bdm2 mRNA was maintained during neuronal differentiation of mouse embryonal carcinoma cell P19, but decreased during the differentiation to glial and unidentified non-neuronal cells. In situ hybridization study demonstrated the wide distribution of Bdm2 mRNA in the embryonic brain; in the adult brain, the hybridization signals became more restricted to the hippocampus, olfactory bulb, cerebellum, and neocortex, almost coinciding with the regions where nascent and immature neurons are present. Thus, it appears likely that Bdm2 encodes a protein that is involved in both the regulation of growth of undifferentiated neural cells and the terminal differentiation of neuronal cells.     

Neurogranin: immunocytochemical localization of a brain-specific protein kinase C substrate

The developmental expression and the cellular localization of neurogranin (formerly designated p17), a brain-specific protein kinase C (PKC) substrate, were investigated. The developmental expression of neurogranin was studied by immunoblotting of rat brain and neuronal cell-culture extracts using neurogranin polyclonal antibodies. Neurogranin synthesis was found to be developmentally regulated, with no expression in the embryonic and neonatal period and an abrupt increase between 2 and 3 weeks of age. By immunohistochemistry, neurogranin was found essentially in the adult rat telencephalon, specifically located in the cell bodies and dendritic processes of neurons of the cerebral cortex, hippocampus, striatum, and a few other discreet areas. Neurogranin immunoreactivity was nearly absent in the thalamus, cerebellum, and brain stem. The late developmental expression and the dendritic localization of neurogranin in neurons are 2 features that also characterize the type I PKC isozyme. The specific localization of the protein in integrative areas of the rat brain suggests a highly specialized function of neurogranin in the CNS. A possible role for neurogranin in the transduction of the PKC activation signals at the postsynaptic level is suggested.     

Expression of Trio, a member of the Dbl family of Rho GEFs in the developing rat brain

Trio, a member of the Dbl family of guanine nucleotide exchange factors (GEFs), has a series of spectrin repeats, two GEF domains, protein interaction domains, and a putative kinase domain, potentially important in neuronal axon guidance and cell migration. Most knowledge about Trio is based on studies of Caenorhabditis elegans and Drosophila, while the function of Trio in vertebrates is unclear. The aim of these experiments was to establish the patterns of Trio expression in the postnatal rat brain. During postnatal (P) development, high levels of Trio mRNA are found in the cerebral cortex, hippocampus, thalamus, caudate/putamen, and olfactory bulb, with lower levels in the septal nucleus, nucleus accumbens, amygdala, and hypothalamus. Except for the cerebellum, Trio mRNA in major brain areas is highest at P1, decreasing gradually during development, with low but detectable levels at P30. In P14 cerebral cortex, pyramidal neurons with strongly staining soma and dendrites are observed primarily in layer 5. In hippocampus, strong staining is observed in pyramidal neurons, granule cells, and isolated interneurons. Cerebellar Purkinje neurons exhibit intense staining in the soma and in extensive dendritic arbors at P7 and P14. Levels of Trio mRNA and the intensity of Trio immunostaining in cerebellar Purkinje cells increase from P1 to P30. Consistent with the in situ hybridization pattern, Western blot analyses show that Trio levels in the hippocampus and cortex are high at P1, decreasing until P30. The data suggest that Trio plays an important role during neuronal development.     

Na/HCO3 cotransporters in rat brain: expression in glia, neurons, and choroid plexus.

We studied the expression and distribution of Na/HCO(3) cotransporters in rat brain using polynucleotide probes and polyclonal antibodies derived from the electrogenic rat kidney Na/HCO(3) cotransporter (rkNBC). In whole brain, we observed a single mRNA ( approximately 7.5 kb) by Northern hybridization and a major approximately 130 kDa protein by immunoblotting with a polyclonal antiserum directed against the C terminus of rkNBC. NBC mRNA and protein were present in cortex, brainstem-diencephalon, and cerebellum. In situ hybridization revealed NBC mRNA expression throughout the CNS, with particularly high levels in olfactory bulb, hippocampal dentate gyrus, and cerebellum. NBC mRNA was present in glial cells (e.g., Bergmann glia of cerebellum and hippocampal astrocytes) and neurons (e.g., granule cells of dentate gyrus and neurons of cortex or striatum). Double hybridization of mRNA encoding NBC and glutamate transporter 1 (glial marker) confirmed that both glia and neurons express NBC. Indirect immunofluorescence microscopy demonstrated NBC protein throughout the CNS, particularly in hippocampus and cerebellum. Although NBC mRNA was restricted to cell bodies, NBC protein was distributed diffusely, compatible with a localization in cell processes and perhaps cell bodies. Double labeling with glial fibrillary acidic protein (astrocytic marker), microtubule-associated protein 2 (neuronal marker), or 2',3'-cyclic mononucleotide 3'-phosphodiesterase (oligodendrocytic marker) demonstrated expression of NBC protein in specific subpopulations of both glia and neurons. Moreover, NBC protein was present in both cultured hippocampal astrocytes and cortical neurons. NBC mRNA and protein were also present in epithelial cells of choroid plexus, ependyma, and meninges. Our results are thus consistent with multiple novel roles for Na/HCO(3) cotransport in CNS physiology.     

Neurokinin 1 receptor and relative abundance of the short and long isoforms in the human brain

Substance P exerts its various biochemical effects mainly via interactions through neurokinin-1 receptors (NK1). Recently, the NK1 receptor has attracted considerable interest for its possible role in a variety of psychiatric disorders including depression and anxiety. However, little is known regarding the anatomical distribution of NK1 in the human central nervous system (CNS). Riboprobe in situ hybridization, quantitative PCR and in vitro autoradiography were performed. Highest NK1 mRNA levels were localized in the locus coeruleus and ventral striatum, while moderate hybridization signals were observed in the cerebral cortex (most abundant in the visual cortex), hippocampus and different amygdaloid nuclei. Very low levels of the NK1 mRNA were detected in the cerebellum and thalamus. In view of the existence of a long and short isoform of the NK1 receptor, it was of interest to assess whether there was a differential distribution of the two splice variants in the human CNS and peripheral tissues. A quantitative TaqMan PCR analysis showed that the long NK1 isoform was the most prevalent throughout the human brain, while in peripheral tissues the truncated form was the most represented. 3H-Substance P autoradiography revealed a good correlation between receptor binding sites and NK1 mRNA expression throughout the brain, with the highest levels of binding in the locus coeruleus. These results provide the anatomical evidence that the NK1 receptors have a strong association with neuronal systems relevant to mood regulation and stress in the human brain, but do not suggest a region-specific role of the two isoforms in the CNS.     

Monoclonal antibody Br4 recognizes specific neuronal cell types.

A monoclonal antibody termed as Br4 was prepared by a fusion of the myeloma P3 x 63-AG8-653 with spleen cells from BALB/c mice immunized with chick embryonic brain cells. Immunocytochemically, it reacts strongly with certain neurons in cerebral cortex, hippocampus, substantia nigra, and granular layer of cerebellum from rat brain but does not react with the white matter. A monoclonal antibody Br4 also reacts with primary cultured chick neurons, but not with cultured astrocytes. Western blots show that Br4 recognizes three proteins of 145,000, 108,000, and 97,000 molecular weight from the rat brain. The protein with molecular weight 145,000 (p145) and the protein with 97,000 molecular weight (p97) are essentially soluble; p145 is especially enriched in the synaptosomal soluble fraction. The protein with 108,000 molecular weight (p108) is found in both membrane-bound and soluble forms. Western blots show that among the tissues examined the three proteins are found most abundant in brain and especially enriched in the cerebral cortex and hippocampus. The Br4 antigens may be useful in identifying neuronal cell subpopulations in the central nervous system.     

Identification of PSF, the polypyrimidine tract-binding protein-associated splicing factor, as a developmentally regulated neuronal protein.

The polypyrimidine tract-binding protein-associated splicing factor (PSF), which plays an essential role in mammalian spliceosomes, has been found to be expressed by differentiating neurons in developing mouse brain. The sequence of a fragment of mouse PSF was found to be remarkably similar to that of human PSF. Both the expression of PSF mRNA in cortex and cerebellum and PSF immunoreactivity in all brain areas were high during embryonic and early postnatal life and almost disappeared in adult tissue, except in the hippocampus and olfactory bulb where various neuronal populations remained PSF-immunopositive. Double-labeling experiments with anti-PSF antibody and anti-neurofilaments or anti-glial fibrillary acidic protein antibodies on sections of cortex, hippocampus, and cerebellum indicate that PSF is expressed by differentiating neurons but not by astrocytic cells. In vitro, mouse PSF was found to be expressed by differentiating cortical and cerebellar neurons. Radial glia or astrocyte nuclei were not immunopositive; however, oligodendrocytes differentiating in vitro were found to express PSF. The restricted expression of PSF suggests that this splicing factor could be involved in the control of neuronal-specific splicing events occurring at particular stages of neuronal differentiation and maturation.     

Dscam is associated with axonal and dendritic features of neuronal cells.

Dscam, a novel cell-adhesion molecule belonging to the Ig-superfamily mediates homophilic intercellular adhesion and is expressed abundantly in the nervous system during development. To gain better understanding on the role of Dscam in neuronal differentiation, we raised an antibody and characterized its protein product. Anti-Dscam antibody detected an approximately 200-kDa protein band in human and mouse brain lysates. Immunohistochemical studies showed that during embryonic development of mice, mouse Dscam is expressed throughout the neuronal tissues and also in nonneuronal tissues such as lung, liver, and limb buds. In adult brain Dscam expression is predominant in the cerebellum, hippocampus, and olfactory bulb. Immunofluorescence double labeling of hippocampal and cerebellar primary cultures revealed that Dscam is associated with axonal and dendritic processes. In view of its cellular localization and spatiotemporal expression pattern, we suggest that Dscam is involved in cell-cell interactions during axonal-dendritic development, and maintenance of functional neuronal networks.     

Widespread and Developmentally Regulated Expression of Neurotrophin-4 mRNA in Rat Brain and Peripheral Tissues

The neurotrophin gene family includes four structurally related proteins with neurotrophic activities. Two of them, nerve growth factor and brain-derived neurotrophic factor (BDNF), have been studied in detail and information has recently emerged on the expression and function of the third member, neurotrophin-3. In contrast, little information is available on neurotrophin-4 (NT-4), the most recently isolated member of this family. In this report we have used a sensitive RNAase protection assay to analyse the developmental expression of NT-4 mRNA in the rat brain and in 12 different rat peripheral organs. In heart, liver and muscle plus skin NT-4 mRNA levels were maximal at embryonic day (E) E13 (the earliest time point tested), with reduced levels at later times of development. In lung, kidney and thymus similar levels were seen from E13 to postnatal day (P) 1, with reduced levels in the adult. In testis, ovary and salivary gland NT-4 mRNA was detected at E16 with a peak shortly after birth. During brain development, NT-4 mRNA was maximal at E13 followed by a decrease around birth, after which the level increased. The postnatal increase of NT-4 mRNA was also seen in cerebral cortex and brain stem analysed separately, while in the hippocampus similar levels were found from P1 to adulthood. NT-4 mRNA was detected in all ten adult rat brain regions analysed with only small regional variations, being highest in pons–medulla, hypothalamus, thalamus and cerebellum. Adult rat thymus, thyroid, muscle, lung and ovary contained higher levels of NT-4 mRNA than brain, while all other adult tissues showed similar or lower levels than in the brain. The highest level of NT-4 mRNA overall was found in P1 testis. For comparison, BDNF mRNA was analysed in the same tissues. The expression of BDNF mRNA was in many cases different from that of NT-4 mRNA. The peak of NT-4 mRNA expression in several of the peripheral tissues coincided with the peak of naturally occurring neuronal cell death in peripheral ganglia. This is consistent with the possibility that NT-4 acts as a target-derived trophic factor in vivo. The widespread and increased expression of NT-4 mRNA during postnatal brain development could reflect a trophic role of NT-4 for central nervous system neurons. However, non-neuronal functions of NT-4 are also possible, particularly in male and female reproductive tissues, where the NT-4 protein could function as a growth factor for immature germ cells.