Differential mRNA expression of the related extracellular matrix glycoproteins SC1 and SPARC in the rat embryonic nervous system and skeletal structure

SPARC is a multifunctional extracellular matrix glycoprotein that shares partial sequence homology with SC1. These extracellular matrix molecules are thought to play important roles in modulating cellular interactions. In vitro, SPARC has been shown to exhibit anti-adhesive activity. In the present investigation, in situ hybridization is used to compare the expression patterns of SC1 and SPARC mRNA in the rat embryo. Results show that SC1 and SPARC expression is spatially and temporally regulated. SC1 mRNA is strongly expressed in the embryonic brain and spinal cord, whereas SPARC mRNA is enriched in craniofacial cartilage and skeletal structures. This differential expression pattern in the rat embryo suggests that SC1 plays an important role in the developing nervous system, whereas SPARC participates primarily in events associated with skeletal development. However at embryonic day 17, SC1 and SPARC mRNA show parallel expression patterns in areas of the cerebellum undergoing cell migratory events.     

SC1, a brain extracellular matrix glycoprotein related to SPARC and follistatin, is expressed by rat cerebellar astrocytes following injury and during development

In the nervous system, extracellular matrix components are believed to influence cell shape, proliferation and migration during development and following injury. SC1 is a secreted glycoprotein expressed during neural development and in the adult brain. The molecule shows partial sequence homology to the anti-adhesive extracellular matrix molecule SPARC/osteonectin and to follistatin. We have made a surgical lesion in the adult rat cerebellum and examined changes in SC1 expression at 1 to 14 days after injury. Dual in situ hybridization/immunohistochemistry demonstrated that SC1 mRNA was induced in astrocytes surrounding the wound, reaching maximal levels at 10 days post-lesion. Immunohistochemistry revealed changes in the deposition of SC1 protein in radial fibres of Bergmann glia. SC1 protein was also detected at the border of the lesion, suggesting an association with the glial scar. Double immunohistochemistry with the astrocytic marker GFAP demonstrated that astrocytes also express SC1 during postnatal development.     

Conditional gene expression and lineage tracing of tuba1a expressing cells during zebrafish development and retina regeneration

The tuba1a gene encodes a neural-specific α-tubulin isoform whose expression is restricted to the developing and regenerating nervous system. By using zebrafish as a model system for studying CNS regeneration, we recently showed that retinal injury induces tuba1a gene expression in Müller glia that reentered the cell cycle. However, because of the transient nature of tuba1a gene expression during development and regeneration, it was not possible to trace the lineage of the tuba1a-expressing cells with a reporter directly under the control of the tuba1a promoter. To overcome this limitation, we generated tuba1a:CreER(T2) and β-actin2:loxP-mCherrry-loxP-GFP double transgenic fish that allowed us to label tuba1a-expressing cells conditionally and permanently via ligand-induced recombination. During development, recombination revealed transient tuba1a expression in not only neural progenitors but also cells that contribute to skeletal muscle, heart, and intestine. In the adult, recombination revealed tuba1a expression in brain, olfactory neurons, and sensory cells of the lateral line, but not in the retina. After retinal injury, recombination showed tuba1a expression in Müller glia that had reentered the cell cycle, and lineage tracing indicated that these cells are responsible for regenerating retinal neurons and glia. These results suggest that tuba1a-expressing progenitors contribute to multiple cell lineages during development and that tuba1a-expressing Müller glia are retinal progenitors in the adult.     

SPARC, an extracellular matrix glycoprotein containing the follistatin module, is expressed by astrocytes in synaptic enriched regions of the adult brain

Although extracellular matrix (ECM) glycoproteins play important roles in neural development, their levels are generally believed to decrease in the adult brain. Immunohistochemical analysis indicates that the anti-adhesive ECM glycoprotein SPARC/osteonectin, which contains a follistatin ‘module’, is expressed in the adult rabbit nervous system. In the cerebellum, SPARC is present in Bergmann glia, with a strong signal along their radial fibres. SPARC, while enriched in membrane fractions, is not a transmembrane protein. In the hippocampus, colocalization of SPARC is observed in cells which express the astrocytic marker GFAP. The expression of SPARC by a subset of astrocytes, particularly in synaptic enriched areas, suggests a continuing role for the ECM in the adult brain.     

Effect of hyperthermia on the transport of mRNA encoding the extracellular matrix glycoprotein SC1 into Bergmann glial cell processes

SC1 is an extracellular matrix glycoprotein that is related to the multifunctional protein SPARC. These matricellular members play regulatory roles in modulating cellular interactions. SC1 expression is enriched in the central nervous system during embryonic and postnatal development as well as in the adult brain. In the rat cerebellum, SC1 is expressed at high levels in Bergmann glial cells and their radial fibers which project into the synaptic-rich molecular layer. At specific stages of development and in the adult, SC1 mRNA is selectively transported into cellular processes of these cells. In the present study, we have examined the effect of whole-body hyperthermia on the transport of SC1 mRNA in Bergmann glial cells of the rat cerebellum. Our results show that SC1 mRNA transport is diminished at 10 and 15 h post-hyperthermia, but returns to control levels by 24 h after heat shock. One of the characteristics of a heat shock on cells grown in tissue culture is a collapse of the cytoskeletal network. Intact components of the cytoskeleton are necessary for the transport of mRNA into peripheral processes of cells. However, in vivo hyperthermia does not appear to affect the morphology of the intermediate filament proteins GFAP, vimentin, or the beta-tubulin component of microtubules in Bergmann glial cell processes. During the hyperthermic time course, levels of vimentin protein increase, which is reflected by immunoreactivity of activated astrocytes and microvasculature in cerebellar white matter.     

Selective transport of SC1 mRNA, encoding a putative extracellular matrix glycoprotein, during postnatal development of the rat cerebellum and retina

The selective transport of mRNA species into peripheral processes of cells is an important aspect of gene expression in the nervous system. In this study, we report the transport of SC1 mRNA into the distal processes of Bergmann glial (BG) cells at particular stages of development. SC1 is a putative anti-adhesive extracellular matrix (ECM) glycoprotein that is expressed not only in the developing central nervous system (CNS) but also in the adult brain. The intracellular distribution of SC1 mRNA was examined in two highly laminated neural structures, the cerebellum and retina, during postnatal development and in the adult rat. Our results indicate that SC1 mRNA expression is both spatially and temporally regulated. SC1 message was localized to BG cell bodies at postnatal day 5 (P5) and P10. However, by P15 through to the adult, SC1 mRNA was transported to distal processes of BG cells in the synapse-rich molecular layer (ML) of the cerebellum. In the developing rat retina, SC1 mRNA was expressed in specific neuronal populations by P10, however, transport of SC1 message to the dendrites of these retinal neurons was not detected during development or in the adult. These results indicate neural mechanisms which control the timing and cell type in which selective transport of SC1 mRNA is observed. The localization of SC1 mRNA to the distal processes of BG cells in the synapse-rich ML of the cerebellum could facilitate local control of SC1 protein synthesis which may play roles in synapse formation during development and in synaptic plasticity in the adult.     

Cytokines regulate expression of the type 1 interleukin-1 receptor in rat hippocampal neurons and glia

Interleukin-1 beta is a key mediator of inflammation and stress in the central nervous system (CNS). This cytokine induces CNS glial cells to produce numerous additional cytokines and growth factors under inflammatory conditions. We have investigated regulation of the signal transducing type 1 interleukin-1 receptor (IL-1R1) in the CNS. In vivo, IL-1R1 was not detected in glial cells under basal conditions but was strongly induced after a stab lesion. Cultured astrocytes were used to identify specific signals that regulate expression of the receptor. IL-1R1 mRNA and protein were induced by inflammatory stimuli including tumor necrosis factor (TNF alpha) and IL-1 beta itself. Although expression of the receptor was not detected in glia under basal conditions in vivo, pyramidal neurons in the hippocampus expressed the IL-1 receptor in the normal, unlesioned brain. Cultured embryonic hippocampal neurons were used to investigate specific stimuli that regulate IL-1R1 in neurons. As in astrocytes, IL-1 and TNF alpha induced expression of IL-1R1. The expression of IL-1R1 in hippocampal neurons suggests a possible role for IL-1 in regulating neuronal function, and indicates that these neurons may be directly influenced by cytokines.     

Role of stimulator of interferon genes (STING) in the enteric nervous system in health and disease

Stimulator of Interferon Genes (STING) is a crucial protein that controls the immune system's reaction to bacterial and viral infections. As a pattern-recognition receptor, STING is found in immune cells as well as in neurons and glia in the enteric nervous system (ENS). Recent studies have linked STING to the pathogenesis of several neurological disorders like multiple sclerosis (MS), Alzheimer's disease (AD), and gastrointestinal disorders, including irritable bowel syndrome (IBS) and inflammatory bowel disease (IBD), which are characterized by chronic inflammation and dysregulation of the enteric nervous system (ENS) in the digestive tract. STING plays a crucial role in the pathway that induces the production of interferon in response to viral infection in the central nervous system (CNS). A new study by Dharshika et al. in the current issue of Neurogastroenterology and Motility has demonstrated distinct roles for STING in enteric neurons and glia, namely activation of STING leads to IFN-β production in enteric neurons but not in glia and reducing STING activation in enteric glia does not modulate the severity of Dextran sulfate sodium (DSS) colitis or subsequent loss of enteric neurons. Rather, the role of STING in enteric glia is related to enhancing autophagy. STING can influence gastrointestinal motility and barrier function and therefore be involved in the pathophysiology of IBS and IBD. This mini review highlights the current knowledge of STING in the pathophysiology of CNS and gastrointestinal diseases as well as these newly uncovered roles STING in enteric neurons and glia.     

Expression of the cell adhesion proteins BEN/SC1/DM-GRASP and TAG-1 defines early steps of axonogenesis in the human spinal cord

We have studied the expression pattern of two cell adhesion proteins of the immunoglobin (Ig) superfamily, BEN/SC1/DM-GRASP (BEN) and the transient axonal glycoprotein TAG-1, during the development of the human nervous system. This study was performed by immunocytochemistry on sections of human embryos ranging from 4 to 13 weeks postconception. The overall distribution of the two proteins during development is very similar to that reported in other vertebrate species, but several important differences have been observed. Both proteins exhibit a transient expression on selected neuronal populations, which include the motor and the sensory neurons. In addition, BEN was also detected on virtually all neurons derived from the neural crest as well as in nonneuronal tissues. A major difference of expression with the chick embryo is that, in the motor neurons, BEN expression was not observed at early stages of development, thus arguing against a role of this molecule in pathfinding and fasciculation. BEN was observed to be restricted to subsets of motor neurons, such as the medial column at the upper limb level. Expression was also detected in a laterodorsal population of the ventral horn cells, which are likely to correspond to migrating preganglionic neurons that originate from the motor pool at the thoracic level. TAG-1 was found on commissural neurons and weakly on the sympathetic neurons; it was also detected on restricted nonneuronal populations. In addition, we observed TAG-1 expression in fibers that could correspond either to subsets of dorsal root ganglia (DRGs) central afferences (including the Ia fibers) or to the axons of association interneurons and in scattered motoneurons likely to correspond either to preganglionic neurons, to γ-motoneurons, or to late-born motoneurons. Therefore, our results indicate that the molecular strategies used to establish the axonal scaffolding of the nervous system in humans are extremely conserved among the different vertebrates. J. Comp. Neurol. 379:415–427, 1997. © 1997 Wiley-Liss, Inc.     

Expression of PTHrP and PTH/PTHrP receptor 1 in the superior cervical ganglia of rats

PTHrP and its receptor PTHR1 are found in the CNS and peripheral nervous system. The presence of PTHrP mRNA has been detected in the superior cervical ganglion (SCG), but there are no data on the cellular distribution of PTHrP and PTHR1 in the SCG. Although it is known that ovarian activity and reproductive status influence sympathetic activity, and the PTHrP/PTHR1 system is influenced by estrogens in different tissues, it is not known whether these factors have a similar effect on expression of PTHrP and PTHR1 in the nervous system. Hence, we investigated the presence and distribution of PTHrP and PTHR1 in neurons and glia of the SCG of rats, as well as the influence of ovariectomy on their expression, by using immunohistochemistry. PTHrP and PTHR1 immunoreactivity was observed in cytoplasm as well as in nuclei of almost all neurons in the SCG. In male rats, intensity of PTHrP fluorescence was significantly higher in cytoplasm of NPY−, in comparison to NPY+ neurons (p < 0.05). In female rats, 2 months post-ovariectomy, significantly lower intensity of PTHrP fluorescence in cytoplasm of the SCG neurons was observed in comparison to sham operated animals (p < 0.05). In addition to neurons, PTHrP and PTHR1 immunoreactivity was observed in most of the glia and was not influenced by ovariectomy. Results show the expression of PTHrP and its receptor, PTHR1, in the majority of neurons and glial cells in the SCG of rats. Expression of PTHrP, but not PTHR1 in the cytoplasm of SCG neurons is influenced by ovarian activity.     

Expression and localization of the fibronectin receptor in the mouse nervous system

Cell surface receptors for extracellular matrix components have recently been characterized as integral membrane complexes with common features in their structural and functional properties. We have investigated the expression of the mammalian fibronectin receptor in the mouse nervous system using immunocytological and immunochemical methods. The fibronectin receptor was detectable on immature oligodendrocytes and immature and mature astrocytes in culture, while central nervous system neurons did not reveal detectable levels of fibronectin receptor at the developmental stages studied. In the peripheral nervous system both glia and neurons were found to express the fibronectin receptor. The receptor complex in both peripheral and central nervous system has an apparent molecular weight of approximately 140 kD under reducing conditions and resolves into two or three distinct protein bands under nonreducing conditions. The fibronectin receptor expresses the L2/HNK-1 epitope that is characteristic of several adhesion molecules, including L1, N-CAM, the myelin-associated glycoprotein, and J1 and thus is another member of the L2/HNK-1 family of adhesion molecules. The L2/HNK-1 carbohydrate epitope is expressed differently and independently of the fibronectin receptor protein backbone in that it is detectable in neonatal brain but not in adult brain. Our observations attribute a functional role to the fibronectin receptor and its L2/HNK-1 carbohydrate epitope during development and maintenance of cell interactions in the central and peripheral nervous systems.     

Neuronal dependency of the glycine transporter GLYT1 expression in glial cells

Two membrane-localized transporter proteins (GLYT1 and GLYT2) are responsible for removal of extracellular glycine in the mammalian CNS. Whereas GLYT1 seems to be expressed mainly in glial cells, GLYT2 is neuronal. The highest concentrations of both transporters are found in glycinergic areas of the nervous system. The expression of these proteins may be under regulatory control. We demonstrate here that GLYT1 is not expressed in pure glial cultures, but it is expressed by diverse types of glial cells in mixed neuronal/glial cultures. In these mixed cultures, the glial expression of GLYT1 is down-regulated after selective elimination of the neurons. The absence of expression in pure glial cultures and the observed reduction in the GLYT1 expression after neuronal loss support the existence of a regulatory cross-talk between neurons and glia to initiate and sustain the glial expression of GLYT1. GLIA 20:155–162, 1997. © 1997 Wiley-Liss Inc.     

SC1: a marker for astrocytes in the adult rodent brain is upregulated during reactive astrocytosis

Astrocytes are the most abundant cell type in the mammalian central nervous system (CNS), and are involved in many processes critical for normal CNS maintenance and function. We have used double-label immunocytochemistry and in situ analysis to show that the SPARC (secreted protein acidic and rich in cysteine)-related protein SC1, co-localizes with the astrocyte marker glial fibrillary acidic protein (GFAP) in the adult rodent brain. Thus, SC1 is an astrocyte marker that may be used to investigate astrocyte heterogeneity and analyze glial cell lineages during neural development. Consistent with the presence of SC1 and GFAP in astrocytes, both proteins were markedly upregulated following reactive astrocytosis induced by focal mechanical trauma. Therefore, SC1 may play an important role in reactive astrocytosis subsequent to a wide variety of neural trauma, including neurodegenerative diseases and acute neural damage.     

Glycosyltransferase encoding gene EXTL3 is differentially expressed in the developing and adult mouse cerebral cortex

Exostosin tumor-like 3 (EXTL3) is a glycosyltransferase involved in heparan sulfate (HS) biosynthesis. HS proteoglycans are critically involved in different steps during brain development. The present in situ hybridization in mice revealed wide EXTL3 expression at different grades in the central and peripheral nervous system components including the neural retina and neural crest-derived structures at embryonic days (E) 11.5, E12.5, E14.5, and E16.5. In the neopallial cortex, an intense EXTL3 expression was observed in the neuroepithelial cells lining the ventricular zone at E11.5 and E12.5. The signal decreased at E14.5 and was further downregulated at E16.5 in the ventricular zone. The pioneer neurons of the preplate at E12.5 differentially expressed the gene. Heavily stained among weakly or negatively stained neurons were observed. At E14.5, the cortical plate cells were moderately and homogeneously stained. In contrast, at E16.5, an upregulated and differential expression pattern was detected. The labeling pattern at E16.5 subdivided the cortical plate cells into a large number of heavily, a moderate number of less intensely, and some negatively stained cell populations. Interestingly, the distinct expression pattern displayed by the three main cell types of the adult cerebral cortex was similar to that of the late corticogenesis stage (E16.5). In the adult, the strongest expression was observed in the pyramidal neurons. The granule-type neurons showed less intense staining while the glia cells were devoid of signals. Our data revealed that EXTL3 expression is developmentally regulated in the mouse nervous system and suggested that it differentially contributes to brain development and corticogenesis.     

Radial glial cells as neuronal precursors: a new perspective on the correlation of morphology and lineage restriction in the developing cerebral cortex of mice

Radial glia is a ubiquitous cell type in the developing central nervous system (CNS) of vertebrates, characterized by radial processes extending through the wall of the neural tube which serve as guiding cables for migrating neurons. Radial glial cells were considered as glial precursor cells due to their astroglial traits and later transformation into astrocytes in the mammalian CNS. Accordingly, a hypothetical morphologically distinct type of precursor was attributed the role of neurogenesis. Recent evidence obtained in vitro and in vivo, however, revealed that a large subset of radial glia generates neurons. We further demonstrate here that the progeny of radial glial cells does not differ from the progeny of precursors labeled from the ventricular surface, implying that there is no obvious relation between precursor morphology and neuron-glia lineage decisions in the developing cerebral cortex of mice. Moreover, we show that many radial glial cells seem to maintain their process during cell division and discuss the implications of this observation for the orientation of cell division. These new data are then related to radial glial cells in other non-mammalian vertebrates persisting into adulthood and suggest that radial glia are not only neurogenic during development, but also in adulthood.     

R- and B-cadherin expression defines subpopulations of glial cells involved in axonal guidance in the optic nerve head of the chicken

Glial cells play a crucial role in the organization and function of the nervous system. Cell-cell adhesion receptors of the cadherin family have been shown to participate in distinct morphogenetic processes throughout the development of the CNS, but little is known about glial expression of cadherins. Applying immunofluorescence and confocal laser scanning microscopy, we investigated R- and B-cadherin expression in relation to the glial cell differentiation in the optic nerve head and pecten oculi of developing chicken. Throughout embryonic development, R- and B-cadherin were expressed in distinct cell populations, which differentiated into distinct subtypes of glial cells. R-cadherin was located in the glia limitans perivascularis et superficialis of the optic nerve and in cells bordering the optic nerve head, where it comes in contact with the retina. B-cadherin was located in the glia limitans perivascularis et superficialis of the pecten oculi and in a subset of cells at the retinal border. R-cadherin-expressing cells differentiated unequivocally into a glial fibrillary acidic protein (GFAP)-positive but glutamine synthetase (GS)-negative phenotype, whereas B-cadherin-expressing glia developed into a GS-positive but GFAP-negative phenotype. In addition, the B-cadherin-positive population developed into a highly pigmented cell type, which was consistently associated with pecten-type capillaries. By contrast, the R-cadherin-positive glia remained unpigmented and surrounded normal brain-phenotype capillaries. These data suggest that glial cells, like neurons, may use the expression of different cadherins to segregate and differentiate into distinct subtypes, which goes hand in hand with their involvement in special functions and morphogenetic processes. To address this issue, we selectively lysed both glial subtypes in developing embryos by microinjection of R- and B-cadherin antibodies with complement. First evidence is presented for R-cadherin-positive glial cells as crucial to the organization of the optic nerve and axonal guidance at its lateral margin. B-cadherin-positive cells are involved in the axonal guidance at the pecteneal margin, avoiding the ingrowth of axons into the pecten.