Developing Rat Nervous System Research Articles

Expression of the gene for the neuronal intermediate filament protein alpha-internexin coincides with the onset of neuronal differentiation in the developing rat nervous system.

While neurofilaments have long been considered early markers of neuronal differentiation, they cannot be detected in most newly postmitotic neurons of the developing central nervous system (CNS). Here we show that these neurons already express the neuronal intermediate filament protein alpha-internexin at high levels. alpha-internexin is expressed by most, if not all, neurons as they begin differentiation and shows no overlap with vimentin, whose expression in the CNS is restricted to mitotic neuronal precursors. In the adult, alpha-internexin is the only intermediate filament gene expressed by the cerebellar granule cells, the source of the thin-caliber parallel fibers; conversely, neurofilament proteins are highly expressed in large neurons, which express alpha-internexin at low levels. These data suggest that neuronal intermediate filaments may regulate axonal stability and/or diameter through changes not only in their number, but also in their subunit composition.

The expression of a neuronal nuclear antigen (Ri) recognized by the human anti-Ri autoantibody in the developing rat nervous system

Anti-Ri is a human autoantibody that recognizes a neuronal nuclear antigen (Ri) of unknown function. To ascertain the possible role of the Ri antigen in neuronal development, we analysed the pattern of the anti-Ri immunoreactivity in the developing rat nervous system. Neurons of the retina, except the photoreceptors, and central but not peripheral nervous system were anti-Ri-positive. The intensity of the immunoreactivity followed the sequence of neuronal maturation. Germinal cells of the periventricular layer and migrating neuroblasts in the intermediate zone were Ri-negative. These data suggest Ri antigen may play a role in the postmigratory maturation of neurons of the CNS.

A phosphorylation epitope on MAP 1B that is transiently expressed in growing axons in the developing rat nervous system.

We have isolated a monoclonal antibody (150) that recognizes a phosphorylation epitope on the microtubule-associated protein (MAP) 1B. Immunoblot analysis of the developing rat central nervous system shows that monoclonal antibody 150 is directed against a protein of approximately 325 kDa (MAP 1B) that copolymerizes with microtubules through successive cycles of temperature-dependent assembly and disassembly. Furthermore, immunoprecipitated MAP 1B contains the epitope recognized by monoclonal antibody 150. Removal of phosphate from blotted proteins using alkaline phosphatase abolishes the binding of monoclonal antibody 150 to MAP 1B, indicating that the epitope is phosphorylated. In the developing rat nervous system, immunohistochemistry with monoclonal antibody 150 shows that the phosphorylation epitope on MAP 1B is transiently expressed in growing axons but not in dendrites. For instance, in the neonatal rat cerebellum, the parallel fibres of granule cells are stained only during elongation and not after synaptogenesis. The monoclonal antibody 150 epitope is also transiently expressed in radial glial fibres and in certain cell nuclei. All immunostaining of sections with monoclonal antibody 150 was completely abolished by alkaline phosphatase treatment. These observations and previous ones made by us in cell culture (Mansfield et al., J. Neurocytol., 20, 654-666, 1991) suggest that the phosphorylation epitope on MAP 1B recognized by monoclonal antibody 150, which has not been previously detected in vivo, may be important in axonogenesis.

Expression of the F84.1 glycoprotein in the spinal cord and cranial nerves of the developing rat

The monoclonal antibody designated as F84.1 was used for an immunohistochemical study of the developing rat nervous system. The most prominent neural components recognized by F84.1 are motor and sensory components of the spinal cord and cranial nerves. F84.1 is first detected in the dorsal root ganglia of embryonic day 11 spinal cord. The expression in the dorsal roots persists in the adult. In contrast, a more transient expression of F84.1 is found in the spinal motor system. F84.1 labels primary neurons of cranial nerves V, VIII, IX and X. F84.1 is also expressed by the non-neuronal cells of the notochord and the floor plate. Immunoprecipitation experiments from several types of cells in culture show that the F84.1 antigen is a cell-surface glycoprotein with a molecular weight of 90–105 kDa. An analysis of the amino terminal sequence demonstrates that the F84.1 antigen is similar to the chick cell adhesion molecule SC1/DM-GRASP, a member of the immunoglobulin superfamily. The pattern of expression of F84.1 in the rat differs in several aspects from that of the chick molecules, leaving a possibility that F84.1 may be a variant of SC1/DM-GRASP.

Spatial and temporal expression of alpha- and beta-thyroid hormone receptor mRNAs, including the beta 2-subtype, in the developing mammalian nervous system

Thyroid hormone exerts profound effects on the developing mammalian brain, and its deficiency can lead to severe mental retardation and motor abnormalities. To identify specific anatomic targets of thyroid hormone action in the developing mammalian nervous system, we examined thyroid hormone receptor gene expression by hybridization histochemistry on serial adjacent sections from 12 stages of the developing rat nervous system. 35S-labeled cRNA probes were generated from divergent sequences of rat alpha 1-, alpha 2-, beta 1-, and beta 2-thyroid hormone receptor and related cDNAs. We found that alpha- and beta-thyroid hormone receptor genes have distinct patterns of spatiotemporal expression in the embryonic and postnatal rat nervous system. alpha 1- and alpha 2-mRNAs were widely expressed in similar patterns; highest levels were found in the fetal neocortical plate, site of cortical neuronal differentiation. In contrast, beta 1-transcripts were restricted in distribution, with prominent expression in zones of neuroblast proliferation such as the germinal trigone and the cortical ventricular layer. Surprisingly, the "pituitary-specific" beta 2-transcript was detected in the developing hippocampus and striatum. Our results suggest that alpha- and beta-thyroid hormone receptors may play distinct functional roles during development of the mammalian nervous system.

NT-3, BDNF, and NGF in the developing rat nervous system: Parallel as well as reciprocal patterns of expression

To obtain insight into the site and stage specificity of nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF), and neurotrophin-3 (NT-3) action in vivo, we compared the expression patterns of the genes for these three related neurotrophic factors as well as for the NGF receptor in developing and adult rats. Initial embryonic expression of these related neurotrophic factors approximately coincides with the onset of neurogenesis. However, the levels at which the three factors are expressed at this time and throughout the developing nervous system are dramatically different. NT-3 is by far the most highly expressed in immature regions of the CNS in which proliferation, migration, and differentiation of neuronal precursors is ongoing. NT-3 expression dramatically decreases with maturation of these regions. By contrast, BDNF expression is low in developing regions of the CNS and increases as these regions mature. NGF expression varies during the development of discrete CNS regions, but not in any consistent manner compared with NT-3 and BDNF Despite the dramatic variations, NT-3, BDNF, and NGF do share one striking similarity-high level expression in the adult hippocampus. Our observations are consistent with the idea that NT-3, BDNF, and NGF have parallel as well as reciprocal roles in vivo.

Granule cell induction of 9- O-acetyl gangliosides on cerebellar glia in microcultures

In previous studies we have shown that the expression of acetylated gangliosides recognized by the JONES monoclonal antibody is correlated with regions of cell migration in the developing rat nervous system. In this study we have investigated the expression of these gangliosides in two different types of cultures prepared from dissociated postnatal rat cerebella. In the first type, cells are plated after dissociation under conditions where most of the glial cells develop a stellate morphology that anchors neurons but does not support their migration. In the second type of culture, cells are plated in a ratio of four neurons to one glial cell and under these conditions the predominant form of astroglia is an elongate form that supports the migration of granule neurons. Granule neurons express JONES antigens in dissociated cell suspensions and in cultures in which cells are plated either after dissociation or in a 4:1 neuron:glia ratio. On the other hand, glial cells grown in the absence of neurons are JONES negative. In addition, the expression of JONES gangliosides by glial cells is different in the two types of culture. In cultures where the astroglial cells display the stellate morphology only a small proportion show JONES staining. Cultures in which the glial cells assume the elongate morphology have a significantly higher number of JONES-positive astroglia.

Spatial and temporal expression of sialic acid-containing portions of glycoprotein in developing rat nervous system and muscle

Spatial and temporal expression of sialic acid-containing portions of glycoprotein in developing rat nervous system and muscle

Spatial and temporal expression of sialic acid-containing portions of glycoprotein in developing rat nervous system and muscle : Tatsunori Seki, Yasumasa Arai, Department of Anatomy, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyou-ku, Tokyo 113, Japan

Spatial and temporal expression of sialic acid-containing portions of glycoprotein in developing rat nervous system and muscle : Tatsunori Seki, Yasumasa Arai, Department of Anatomy, Juntendo University, School of Medicine, 2-1-1 Hongo, Bunkyou-ku, Tokyo 113, Japan

Simultaneous up‐regulation of neurofilament proteins during the postnatal development of the rat nervous system

The expression of neurofilament (NF) proteins was examined during postnatal development of the rat nervous system in order to elucidate the nature of NF expression during the period of transition from the embryonic (immature) to the adult (mature) stages of NF expression. mRNAs to the light (NF-L), mid-sized (NF-M), and heavy (NF-H) NF proteins were compared by Northern blots and by in situ hybridizations, in NF-rich (i.e., DRG, spinal cord and brainstem) and in NF-poor (i.e., cerebellum and cerebral cortex) regions of the developing rat nervous system. NF proteins were analyzed by gel electrophoresis and by immunoblots. In each tissue, the expression of NF-H was delayed compared to that of NF-L and NF-M, as previously reported. The present study now shows that the delayed expression of NF-H is accompanied by parallel up-regulations in the expressions of NF-L and NF-M, both at the levels of mRNA and protein. Similar rates of increase of all three NF mRNAs occur between postnatal days 5 (P5) and 24 (P24) in rat spinal cord and DRG. Furthermore, the postnatal up-regulation of NF expression is characterized by a progressive accumulation of all three NF proteins in the tissues. The findings indicate that the adult pattern of NF expression (with high levels of expression of all three NF proteins) becomes established during the postnatal period of development, raising questions as to the nature of factors that coregulate the expression of NF subunits.

Homology between the cDNAs Encoding Phosphoprotein p19 and SCG10 Reveals a Novel Mammalian Gene Family Preferentially Expressed in Developing Brain

We have isolated and sequenced a rat testis cDNA encoding p19, a 19-kD cytosolic phosphoprotein that is abundant in immature brain, testis, and neuroendocrine tumor cells. The cDNA was identified using bovine brain p19 peptide sequences, which indicate that the gene encoding p19 has been highly conserved during mammalian evolution. Using Northern blot analysis on rat tissues, p19 mRNA was readily detected in brain and testis and showed a 15-fold greater abundance in newborn than in adult brain. Low levels of p19 mRNA were observed in spleen, kidney, and heart, but not in liver. Thus, the expression of the gene encoding p19 shows a strong tissue preference and is developmentally regulated. The predicted amino acid sequence of p19 is highly homologous to that of SCG10, another protein expressed in the developing rat nervous system, suggesting that the two proteins serve similar functions. Based on a comparison of the two cDNAs, we conclude that p19 and SCG10 are encoded by distinct but related genes constituting a novel gene family.

Rapid induction of the major embryonic alpha-tubulin mRNA, T alpha 1, during nerve regeneration in adult rats

The mRNAs for 2 isotypes of alpha-tubulin, termed T alpha 1 and T26, are differentially regulated in the developing rat nervous system. T alpha 1 alpha-tubulin mRNA is expressed at high levels when neurons extend processes whereas T26 mRNA is expressed constitutively (Miller et al., 1987b). We have examined the expression of these 2 alpha-tubulin mRNAs in regenerating facial and sciatic motor neurons of the rat using Northern blot and in situ hybridization analyses. T alpha 1 alpha-tubulin mRNA is rapidly induced in axotomized motor neurons of the facial nerve: increased levels of mRNA are detectable 4 hr after a lesion is made 1.5 cm distal to the neuronal cell bodies. T alpha 1 mRNA levels are highest from 3-7 d postcrush and decline slowly to control levels following functional reinnervation of facial muscles. In contrast, T26 mRNA levels remain constant throughout the regeneration process. Total alpha-tubulin mRNA levels do not change until 1 d postaxotomy; otherwise the changes in expression are similar to T alpha 1 mRNA, although the relative increase is not as great. Enhanced T alpha 1 alpha-tubulin mRNA expression also occurs in motor neurons of crushed or tied sciatic nerve. Ligature or crush of the sciatic nerve leads to approximately the same peak in the expression of T alpha 1 mRNA at 7-15 d postaxotomy. Following the facial nerve transection, under conditions in which reinnervation is prevented, T alpha 1 alpha-tubulin mRNA levels remain elevated significantly longer than when the nerve is crushed. Taken together, the data indicate that T alpha 1 alpha-tubulin mRNA is rapidly induced following neuronal axotomy, remains elevated during the period of axonal regrowth, and is subsequently down-regulated at the approximate time of target contact. These results are reminiscent of changes in T alpha 1 mRNA that occur during neuronal development. This growth-associated pattern of T alpha 1 gene expression can be modified by inhibiting appropriate regeneration of the damaged nerve.

A developmentally regulated antigen associated with neural cell and process migration

The distribution of an epitope recognized by the monoclonal antibody JONES has been studied immunohistochemically in the developing nervous system of the rat. In the present report, we survey selected regions of the fetal, postnatal, and adult rat nervous system to test the hypothesis that JONES binding is invariably associated with neural cell migration and axon growth in the developing rat. A series of selected developmental stages extending from embryonic day (E) 9 to adult were used in this investigation. The distribution of JONES binding was examined using indirect immunofluorescence, as well as the immunogold procedure. Particular attention was paid to regions where the positions and timing of cell and axon migrations have been well described for the rat. JONES immunoreactivity first appears at E11-12, when it is localized to the lamina terminalis, the telencephalic-diencephalic junction, the midbrain, and the rhombic lip regions of the cytologically undifferentiated neural tube. In all the regions studied, during embryonic and early postnatal life, the labeling is very intense in the ventricular zone and shows a radial array in the adjacent intermediate and marginal zones. The expression of JONES epitope correlates particularly with times of cell migration in the retina, superior colliculus, cerebellum, and telencephalon and in regions undergoing neurite extension, such as the developing optic tract, the white matter of the cerebellum, the dorsal roots, the trigeminal system, and olfactory nerve. JONES binding becomes progressively restricted in the postnatal period. In the adult brain, immunoreactivity is present only in the retina and cerebellum. In the retina, JONES labeling is present in the outer plexiform layer and optic fiber layer. The labeling in the optic fiber layer extends to the optic nerve head and stops abruptly outside the orbit. In the cerebellum, JONES shows a radially oriented pattern throughout the molecular layer and delineates the cell bodies in the Purkinje cell layer. The only non-neural regions that show JONES immunoreactivity are the adrenal medulla and the kidney glomeruli. We conclude that the antigens recognized by the JONES monoclonal antibody are associated with the migration of subsets of cells and axons within the developing rat nervous system and, consequently, may play a role in conveying selectivity to these processes.

Changes in Axonally Transported Proteins in the Mature and Developing Rat Nervous System During Early Stages of Methyl Mercury Exposure

It was established by means of SDS polyacrylamide gel electrophoresis that direct injections of methyl mercury (10 micrograms Hg) into the mature rat vitreous body of the eye decrease protein synthesis in the retina and optic nerve at 4 hours after injection. Although the global spectrum of polypeptides did not change, a specific decrease in the volume of polypeptides of 20-23 K daltons molecular weight was evident. Conversely, systemic exposure to methyl mercury resulted in increased protein synthesis of polypeptides of 20-23 K molecular weight both in adult (8 mgHg/kg/day for 8 days) and neonatal rats (2 mgHg/kg/day for 10 days). In addition, specific changes in the volume of polypeptides 75-90 K molecular weight were noted in sciatic nerves of neonatal rats. These data are consistent with a bimodal response in protein synthesis following MeHg treatment. Local presence of MeHg following direct injection into the eye causes a reduction in protein synthesis, while chronic systemic exposure results in increased synthesis and transport of proteins in both mature and developing optic nerves and neonatal sciatic nerves. Thus, these systems possess the capacity to attempt regenerative processes through induction of a small subset of proteins known as GAPs (Growth-Associated Proteins) during the early stages of systemic methyl mercury exposure. These wide spread and system-specific changes are consistent with growth-specific functions during the early stages of methyl mercury exposure.

Cell surface-directed monoclonal antibodies for analysis of cellular transformation risk and tumor cell surface phenotypes in oncogenesis by ethylnitrosourea in the developing rat nervous system

Cell surface-directed monoclonal antibodies for analysis of cellular transformation risk and tumor cell surface phenotypes in oncogenesis by ethylnitrosourea in the developing rat nervous system

Antibody against nerve growth factor-inducible large external (NILE) glycoprotein labels nerve fiber tracts in the developing rat nervous system

The NILE (nerve growth factor-inducible large external) glycoprotein is a 230,000-dalton molecule found on the surface of PC12 cells. Immunologically cross-reactive glycoproteins in the molecular weight range of 215,000 to 230,000 have been found on many types of neurons in culture. Using immunohistochemical methods, we have shown that NILE-related glycoproteins are present in neuronal fiber tracts of the developing rat brain. Antibody against the NILE glycoprotein specifically labels processes that appear identical to those recognized by antibodies against the neurofilament triplet of proteins. These processes are clearly distinct from the radial glial fibers recognized by antibody against the intermediate filament protein vimentin. NILE glycoprotein is not distributed uniformly over the entire neuronal surface but is concentrated on neurites and is much less abundant on cell bodies. NILE-positive fiber tracts are first seen in the spinal cord and rhombencephalon on embryonic day 11 and over the next 2 days appear in the mesencephalon and diencephalon. Staining in the telencephalon is not seen until embryonic day 15. The appearance of NILE immunoreactivity in these various regions closely parallels the appearance of neurofilament polypeptides, suggesting that NILE-related glycoproteins are present during the early phases of fiber tract formation. This idea is supported by the finding that the NILE glycoprotein can be found postnatally in parts of the nervous-system such as the cerebellar cortex and olfactory bulb which undergo major histogenesis during the postnatal period. In the cerebellum the appearance of NILE immunoreactivity in the two major fiber zones, the molecular layer and the white matter, parallels the development of the fiber structure of these layers. These findings support tissue culture studies which suggest a role for the NILE glycoprotein in mediating nerve fiber fasciculation.

Comparative localization of Wolfgram W1 and myelin basic proteins in the rat brain during ontogenesis.

Antisera raised in rabbits against myelin basic proteins (MBP) and Wolfgram W1 protein isolated from rat myelin were used to study the maturation of oligodendrocytes in the developing rat nervous system. Both proteins were localized immunohistochemically at the light and electron microscopical levels in rat brain from the time of their first appearance to the adult stage. Oligodendrocytes were first detected by their positive staining with W1 antiserum two days after birth and at 1-3 days later with MBP antiserum. At 8-10 days, the number of oligodendrocytes labelled with both sera increases and the myelinated fibre pathways were clearly visible. Labelling with W1 antiserum was observed in oligodendrocytes at all stages from 2 days after birth to adulthood and in myelin fibers when they were present. In contrast, staining of oligodendroglial cells with MBP declined during the period of rapid myelination (20-25 days after birth) and finally disappeared, whereas myelin staining was still apparent. The electron microscopical study revealed that the synthesis of Wolfgram proteins occurred mostly at the peripheral cytoplasmic ribosomes of the cells, from where they were probably transported to processes engaged in myelination. The electron micrographs also showed that the sites of MBP synthesis seemed to be more uniformly distributed over the entire cytoplasm.

Physiology of nerve growth factor.

Physiology of nerve growth factor.

Myelin-associated glycoprotein demonstrated immunocytochemically in myelin and myelin-forming cells of developing rat.

The unlabeled peroxidase-antiperoxidase method has been used with antiserum against "myelin-associated glycoprotein" to establish the presence of the glycoprotein in myelin and myelin-forming cells of the developing rat nervous system. Myelin-associated glycoprotein is found in oligodendroglial cytoplasm before the beginning of myelination. Staining intensity of oligodendroglia increases during early development and slowly declines during the period of rapid myelination. Myelin staining is confined to the periaxonal region of the myelin sheath and does not increase as large, compact sheaths are formed. Antiserum to central nervous system myelin-associated glycoprotein also stains Schwann cells in developing trigeminal ganglia and the periaxonal region of peripheral myelin sheaths.