Expression pattern of LINGO-1 in the developing nervous system of the chick embryo

We isolated a chick homologue of LINGO-1 (cLINGO-1), a novel component of the Nogo-66 receptor (NgR)/p75 neurotrophin receptor (NTR) signaling complex, and examined the expression of cLINGO-1 in the developing brain and spinal cord of the chick embryo by in situ hybridization and immunohistochemistry. cLINGO-1 was expressed broadly in the spinal cord, including the ventral portion of the ventricular zone, and motor neurons. cLINGO-1 was also expressed in the dorsal root ganglion and boundary cap cells at dorsal and ventral roots. In the early embryonic brain, cLINGO-1 was first expressed in the prosencephalon and the ventral mesencephalon, and later in the telencephalon, the rostral part of the mesencephalon and some parts of the hindbrain. cLINGO-1 was also expressed in the ventral part of the neural retina and trigeminal and facial nerves. We also found that cLINGO-1, cNgR1 and p75NTR were expressed in overlapped patterns in the spinal cord and the dorsal root ganglion, but that these genes were expressed in distinct patterns in the early embryonic brain.     

Quantitative Proteome Analysis of Brain Subregions and Spinal Cord from Experimental Autoimmune Encephalomyelitis Mice by TMT‐Based Mass Spectrometry

Multiple sclerosis (MS) is an autoimmune disease of the central nervous system (CNS); its cause is unknown. To understand the pathogenesis of MS, researchers often use the experimental autoimmune encephalomyelitis (EAE) mouse model. Here, the aim is to build a proteome map of the biological changes that occur during MS at the major onset sites—the brain and the spinal cord. Quantitative proteome profiling is performed in five specific brain regions and the spinal cord of EAE and healthy mice with high‐resolution mass spectrometry based on tandem mass tags. On average, 7400 proteins per region are quantified, with the most differentially expressed proteins in the spinal cord (1691), hippocampus (104), frontal cortex (83), cerebellum (63), brainstem (50), and caudate nucleus (41). Moreover, region‐specific and commonly expressed proteins in each region are identified and bioinformatics analysis is performed. Pathway analysis reveals that protein clusters resemble their functions in disease pathogenesis (i.e., by inducing inflammatory responses, immune activation, and cell–cell adhesion). In conclusion, the study provides an understanding of the pathogenesis of MS in the EAE animal model. It is expected that the comprehensive proteome map of the brain and spinal cord can be used to identify biomarkers for the pathogenesis of MS.     

Expression pattern of BM88 in the developing nervous system of the chick and mouse embryo

We isolated a chick homologue of BM88 (cBM88), a cell-intrinsic nervous system-specific protein and examined the expression of BM88 mRNA and protein in the developing brain, spinal cord and peripheral nervous system of the chick embryo by in situ hybridization and immunohistochemistry. cBM88 is widely expressed in the developing central nervous system, both in the ventricular and mantle zones where precursor and differentiated cells lie, respectively. In the spinal cord, particularly strong cBM88 expression is detected ventrally in the motor neuron area. cBM88 is also expressed in the dorsal root ganglia and sympathetic ganglia. In the early neural tube, cBM88 is first detected at HH stage 15 and its expression increases with embryonic age. At early stages, cBM88 expression is weaker in the ventricular zone (VZ) and higher in the mantle zone. At later stages, when gliogenesis persists instead of neurogenesis, BM88 expression is abolished in the VZ and cBM88 is restricted in the neuron-containing mantle zone of the neural tube. Association of cBM88 expression with cells of the neuronal lineage in the chick spinal cord was demonstrated using a combination of markers characteristic of neuronal or glial precursors, as well as markers of differentiated neuronal, oligodendroglial and astroglial cells. In addition to the spinal cord, cBM88 is expressed in the HH stage 45 (embryonic day 19) brain, including the telencephalon, diencephalon, mesencephalon, optic tectum and cerebellum. BM88 is also widely expressed in the mouse embryonic CNS and PNS, in both nestin-positive neuroepithelial cells and post-mitotic betaIII-tubulin positive neurons.     

GFAP transgenic zebrafish

We have generated transgenic zebrafish that express green fluorescent protein (GFP) in glial cells driven by the zebrafish glial fibrillary acidic protein (GFAP) regulatory elements. Transgenic lines Tg(gfap:GFP) were generated from three founders; the results presented here are from the mi2001 line. GFP expression was first visible in the living embryo at the tail bud-stage, then in the developing brain by the 5-somite-stage ( approximately 12 h post-fertilization, hpf) and then spreading posteriorly along the developing spinal cord by the 12-somite stage (approximately 15 hpf). At 24 hpf GFP-expressing cells were in the retina and lens. By 72 hpf GFP expression levels were strong and localized to the glia of the brain, neural retina, spinal cord, and ventral spinal nerves, with moderate expression in the enteric nervous system and weaker levels in the olfactory sensory placode and otic capsule. GFP expression in glia co-localized with anti-GFAP antibodies, but did not co-localize with the neuronal antibodies HuC/D or calretinin in mature neurons.     

Substance P Receptor Expression and Cellular Responses to Substance P in Prenatal Rat Spinal Cord Cells

Abstract

Substance P receptors (SPRs) are expressed by prenatal rat spinal cord neurons and glial cells early in their differentiation, and SPRs may mediate developmental influences in the developing spinal cord. In order to understand better early SPR expression, we quantified SPR mRNA in the rat spinal cord during prenatal development using a cDNA probe for the rat SPR in nuclease protection assays. SPR mRNA was present in the rat spinal cord at E14, the earliest stage examined, and the presence of specific binding sites for radiolabeled SP suggested that SPRs were expressed at the protein level as well. Comparisons of samples from rats at different prenatal ages showed that the relative abundance of SPR mRNA declined by about 75% from E14 through the remainder of prenatal development. Assays of the hydrolysis of phosphatidyl inositol performed on prenatal spinal cord cells in culture revealed that SP caused a small but significant stimulation. These results show that expression of SPRs is an early molecular event in the development of the rat spinal cord in vivo and that SPRs on young spinal cord cells can mediate functional responses at early developmental stages.     

Fetal Calf Spinal Cords as Enriched Source of mRNA Encoding Glial Fibrillary Acidic Protein

Abstract: Total poly(A)⁺ RNA was isolated from fetal calf spinal cord, adult rat spinal cord, and young rat brain, and was translated using the rabbit reticulocyte lysate system. The amount of glial fibrillary acidic protein in the translation products was measured by immunoprecipitation with antiserum against glial fibrillary acidic protein. RNA from fetal calf spinal cord could direct glial fibrillary acidic protein synthesis such that this protein comprised approximately 1.4% of the total products. RNAs from adult rat spinal cord and brain could direct glial fibrillary acidic protein synthesis much less efficiently, with this protein comprising <0.3% of the total products. These results suggest that the gene for glial fibrillary acidic protein is strongly expressed in fetal calf spinal cord and that this tissue is an enriched source of mRNA encoding glial fibrillary acidic protein.     

Keratin 8 of simple epithelia is expressed in glia of the goldfish nervous system

The intermediate filament protein composition in glial cells of goldfish optic nerve differs from that found in glial cells of the goldfish spinal cord and brain. Brain and spinal cord glial cells contain glial fibrillary acidic protein (GFAP), whereas glial cells in the optic nerve contain ON3. The ON3 protein of the goldfish optic nerve was recently identified as the goldfish equivalent to the mammalian type II keratin 8 protein. In addition to the ON3 protein, the goldfish optic nerve also contains a 48-kDa protein. Immunoblotting experiments suggest that this protein is equivalent to the mammalian type I keratin 18 protein, which typically pairs with keratin 8 to form filaments. We show that these proteins are not specific to the optic nerve. The ON3 and 48-kDa proteins of the goldfish optic nerve share common antigenic properties with the predominant keratin pair expressed in the goldfish liver. These proteins are also expressed at low levels in the goldfish brain and spinal cord. In addition RNase protection assays and Northern blots indicate that the mRNA for the ON3 protein in optic nerve is identical to the message found in other goldfish tissues. The expression of ON3 was also examined in cultured glial cells from goldfish spinal cord and optic nerve and cultured fibroblast cells. Analysis of intermediate filament protein expression in cultured glial cells taken from goldfish spinal cord demonstrated the absence of GFAP in these cells and the expression of ON3. This protein was also the predominant intermediate filament protein of cultured optic nerve glial cells and fibroblasts. The differences in the expression of intermediate filament proteins in mammals and lower vertebrates are discussed. In addition, we discuss how the expression of a simple epithelial keratin pair in glial cells of the goldfish optic nerve may be associated with this system's capacity for continuous growth and regeneration.     

Region-specific and stage-dependent regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 homeodomain transcription factor

During early neural development, the Nkx6.1 homeodomain neural progenitor gene is specifically expressed in the ventral neural tube, and its activity is required for motoneuron generation in the spinal cord. We report that Nkx6.1 also controls oligodendrocyte development in the developing spinal cord, possibly by regulating Olig gene expression in the ventral neuroepithelium. In Nkx6.1 mutant spinal cords, expression of Olig2 in the motoneuron progenitor domain is diminished, and the generation and differentiation of oligodendrocytes are significantly delayed and reduced. The regulation of Olig gene expression by Nkx6.1 is stage dependent, as ectopic expression of Nkx6.1 in embryonic chicken spinal cord results in an induction of Olig2 expression at early stages, but an inhibition at later stages. Moreover, the regulation of Olig gene expression and oligodendrogenesis by Nkx6.1 also appears to be region specific. In the hindbrain, unlike in the spinal cord, Olig1 and Olig2 can be expressed both inside and outside the Nkx6.1-expressing domains and oligodendrogenesis in this region is not dependent on Nkx6.1 activity.     

Slit2-Mediated chemorepulsion and collapse of developing forebrain axons

Diffusible chemorepellents play a major role in guiding developing axons toward their correct targets by preventing them from entering or steering them away from certain regions. Genetic studies in Drosophila revealed a novel repulsive guidance system that prevents inappropriate axons from crossing the CNS midline; this repulsive system is mediated by the Roundabout (Robo) receptor and its secreted ligand Slit. In rodents, Robo and Slit are expressed in the spinal cord and Slit can repel spinal motor axons in vitro. Here, we extend these findings into higher brain centers by showing that Robo1 and Robo2, as well as Slit1 and Slit2, are often expressed in complementary patterns in the developing forebrain. Furthermore, we show that human Slit2 can repel olfactory and hippocampal axons and collapse their growth cones.     

Disproportional Expression of Proteolipid Protein and DM-20 in the X-Linked, Dysmyelinating Shaking Pup Mutant

The shaking pup is an X-linked canine mutant with a severe hypomyelination of the CNS. Proteolipid protein (PLP) and the related DM-20 protein were examined in this mutant by densitometric scanning of Western blots stained with PLP antiserum. In the spinal cord of 4-week-old mutants, PLP was reduced to less than 1% of the control level, which is a greater deficiency than was found for other myelin proteins. On Western blots of control spinal cord, PLP stained much more intensely than DM-20. However, on Western blots of the mutant spinal cord, a component with the electrophoretic mobility of DM-20 stained slightly more intensely with PLP antiserum than PLP itself. This component was shown to be DM-20 by its lack of reactivity with an antiserum raised to a synthetic peptide corresponding to part of the PLP sequence that is missing in DM-20. Thus PLP and DM-20 are expressed in approximately equal and greatly reduced amounts in the mutant spinal cord. Although PLP or DM-20 could not be detected in brain from the 4-week-old mutant, similar disproportional expression of these two proteins was demonstrated in both spinal cord and brain from a 10-week-old mutant pup. Immunostaining of tissue sections showed that the small amounts of PLP and/or DM-20 synthesized in the mutant are present in the thin myelin sheaths. The results suggest that the shaking pup could have a primary defect in the PLP gene leading to a severe deficiency of PLP and DM-20 as well as disproportional expression of these two proteins.     

A gene trap knockout of the Tiam-1 protein results in malformation of the early embryonic brain

Tiam-1 has been implicated in the development of the central nervous system. However, the in vivo function of Tiam-1 has not been fully determined in the developing mouse brain. In this study, we generated Tiam-1 knockout mice using a Tiam-1 gene-trapped embryonic stem cell line. Insertion of a gene trap vector into a genomic site downstream of exon 5 resulted in a mutant allele encoding a truncated protein fused with the β-geo LacZ gene. Primary mouse embryonic fibroblasts lacking Tiam-1 revealed a significant decrease in Rac activity and cell proliferation. In addition, whole-mount embryonic LacZ expression analysis demonstrated that Tiam-1 is specifically expressed in regions of the developing brain, such as the caudal telencephalon and rostral diencephalon. More importantly, mouse embryos deficient in Tiam-1 gene expression displayed a severe defect in embryonic brain development, including neural tube closure defects or a dramatic decrease in brain size. These findings suggest that embryonic Tiam-1 expression plays a critical role during early brain development in mice.     

Expression of Plxdc2/TEM7R in the developing nervous system of the mouse

Plexin-domain containing 2 (Plxdc2) is a relatively uncharacterised transmembrane protein with an area of nidogen homology and a plexin repeat (PSI domain) in its extracellular region. Here, we describe Plxdc2 expression in the embryonic mouse, with particular emphasis on the developing central nervous system. Using light microscopy and optical projection tomography (OPT), we analyse RNA in situ hybridization patterns and expression of two reporter genes, beta-geo (a fusion of beta-galactosidase to neomycin phosphotransferase) and placental alkaline phosphatase (PLAP) in a Plxdc2 gene trap mouse line (KST37; [Leighton, P.A., Mitchell, K.J., Goodrich, L.V., Lu, X., Pinson, K., Scherz, P., Skarnes, W.C., Tessier-Lavigne, M., 2001. Defining brain wiring patterns and mechanisms through gene trapping in mice. Nature 410, 174-179]). At mid-embryonic stages (E9.5-E11.5) Plxdc2-betageo expression is prominent in a number of patterning centres of the brain, including the cortical hem, midbrain-hindbrain boundary and the midbrain floorplate. Plxdc2 is expressed in other tissues, most notably the limbs, lung buds and developing heart, as well as the spinal cord and dorsal root ganglia. At E15.5, expression is apparent in a large number of discrete nuclei and structures throughout the brain, including the glial wedge and derivatives of the cortical hem. Plxdc2-betageo expression is particularly strong in the developing Purkinje cell layer, especially in the posterior half of the cerebellum. The PLAP marker is expressed in a number of axonal tracts, including the posterior commissure, mammillotegmental tract and cerebellar peduncle. We compare Plxdc2-betageo expression in the embryonic brain with the much more restricted expression of the related gene Plxdc1 and with members of the Wnt family (Wnt3a, Wnt5a and Wnt8b) that show a striking overlap with Plxdc2 expression in certain areas.     

Complementary expression of transmembrane ephrins and their receptors in the mouse spinal cord: a possible role in constraining the orientation of longitudinally projecting axons

In the developing spinal cord, axons project in both the transverse plane, perpendicular to the floor plate, and in the longitudinal plane, parallel to the floor plate. For many axons, the floor plate is a source of long- and short-range guidance cues that govern growth along both dimensions. We show here that B-class transmembrane ephrins and their receptors are reciprocally expressed on floor plate cells and longitudinally projecting axons in the mouse spinal cord. During the period of commissural axon pathfinding, B-class ephrin protein is expressed at the lateral floor plate boundaries, at the interface between the floor plate and the ventral funiculus. In contrast, B-class Eph receptors are expressed on decussated commissural axon segments projecting within the ventral funiculus, and on ipsilaterally projecting axons constituting the lateral funiculus. Soluble forms of all three B-class ephrins bind to, and induce the collapse of, commissural growth cones in vitro. The collapse-inducing activity associated with B-class ephrins is likely to be mediated by EphB1. Taken together, these data support a possible role for repulsive B-class Eph receptor/ligand interactions in constraining the orientation of longitudinal axon projections at the ventral midline.