Developmental expression of the tenascin-C is altered by hypothyroidism in the rat brain

Tenascin-C is an extracellular matrix glycoprotein involved in cell adhesion and migration, and neurite outgrowth. Since these processes have been found to be under thyroid control in the developing rat brain, we have investigated the effect of congenital hypothyroidism on tenascin-C expression. At birth, in situ hybridization studies in hypothyroid rats show an abnormal up-regulation of tenascin-C in some areas (caudate-putamen, geniculate nuclei, ependymal epithelium of the lateral ventricles, hippocampus) and down-regulation in others (occipital and retrosplenial cortex, subiculum). With subsequent development, hypothyroid animals show higher tenascin-C expression also in the upper layers of the cerebral cortex and subplate, and the Bergmann glia of the cerebellum. Significantly, thyroxine treatment of hypothyroid rats led to normalization of tenascin-C levels in most areas. In agreement with the messenger RNA data, hypothyroid rats contain an uniformly higher level of immunoreactive tenascin-C protein throughout the brain, particularly in the cerebellum. Suggesting a direct cellular effect, thyroid hormone also decreases tenascin-C expression in two glial cell lines (C6, B3.1) expressing thyroid receptors. Our results show that congenital hypothyroidism causes specific alterations in the pattern of tenascin-C expression in the rat brain which may at least partially be responsible for some of the developmental disturbances observed in this syndrome.     

Neuroblastic migration: embryological aspects and mechanisms

INTRODUCTION: The migration of immature neurons of the cerebrum is genetically programmed from the primitive neuroepithelium before the end of the final mitotic cycle. The orientation of the mitotic spindle determines when a neuroepithelial cell is ready to start migration and the proportion of major genetic material it is destined to receive. DEVELOPMENT: The gene LIS1, defective in lissencephaly type 1 of Miller and Dieker, is expressed in the neuroepithelial cells, in the ependyma and the Cajal-Retzius neurons. These transitory fetal cells are the first neurons of the cerebral cortex. Most of the neurons of the cortical plate arrive by means of glial radial cells which guide them towards their destination. Cell adhesion molecules from the neuroblasts themselves, the glial radial cell, the extracellular matrix and perhaps the ependymal cells are important in adhering the neuroblasts to the glial radial cells. Genetic deficiency of these molecules results in defective migration. The mechanism of cellular movement is still not fully understood. Disorders of migration may also be induced by non-genetic factors, such as infarcts or other lesions which damage or destroy the glial radial fibres during the fetal period.     

The nuclear receptor PPARgamma - bigger than fat

Several recent findings have advanced our understanding of the composition and function of the brain extracellular matrix (ECM). BEHAB/brevican, a recently identified CNS-specific proteoglycan, is a component of the brain ECM and is upregulated during glial cell motility. It is expressed at high levels during development, in response to injury, and in primary brain tumors. Cleavage of the BEHAB/brevican protein may increase invasion of tumor cells.     

Astroglia-microvessel relationship in the developing human telencephalon

The telencephalon of 12 and 18 week-old human foetuses was examined for evidence of astroglia-microvessel relationship. Immature astroglia cells (radial glia and astroblasts) and astrocytes were immunostained using antibodies to the cytoskeletal proteins vimentin (VIM) and glial fibrillary acidic protein (GFAP). The microvessels were detected using an antibody to the blood-brain barrier (BBB)-specific glucose transporter GLUT1. Two extracellular matrix (ECM) glycoproteins, laminin (LM), an endothelial-derived molecule, and tenascin-C (TN-C), a glia-derived molecule, were also analyzed. In the two stages examined, VIM- and GFAP-positive fibers of the radial glia establish close relationships with the radial and periventricular microvessels, which are GLUT1-positive and lined by an LM-positive basal lamina-like matrix. At the 18th week, also radial glia transitional forms and immature astrocytes exhibit extensive contacts with the microvasculature. A TN-C-rich ECM is revealed around the vascular plexus of ventricular zones at the 12th week, and around the newly growing radial microvessels and the microvessel branching sites at the 18th week. The observations taken as a whole, suggest that during the telencephalon morphogenesis the immature astroglia cells play a role in the early establishment of the distribution pattern of the neural microvessels and in their growth and maturation.     

Glial cell interactions with tenascin-C: adhesion and repulsion to different tenascin-C domains is cell type related

The multimodular glycoprotein tenascin-C is transiently expressed, predominantly by glial cells, during the development of the central and peripheral nervous systems. This extracellular matrix glycoprotein is involved in the control of cell adhesion, neuron migration and neurite outgrowth. Distinct functional properties for neuronal cell types have been attributed to separate tenascin-C domains using antibody perturbation studies and in vitro experiments on tenascin-C fragments. In order to study potential roles of tenascin-C for glial cell biology, a library of recombinant tenascin-C domains was used in a bioassay in vitro. Embryonic day 14 astrocytes, various astroglial-derived cell lines (C6, A7 and Neu7) and oligodendroglial-derived cell types (Oli-neu and G26-20) were examined in an adhesion assay and compared to the neuroblastoma cell line N2A. A binding site for most cell types, except for A7 and N2A, could be assigned to the first three fibronectin type III domains. Repulsive properties could be mapped to three different sites the epidermal growth factor-like repeats, fibronectin type III repeats 4 and 5 and to the alternatively spliced region of the molecule. The responses to these repulsive sites varied according to the cell type. These data are consistent with the interpretation that different cell types express distinct sets of tenascin-C receptors which might regulate cellular responses via distinct second messenger pathways.     

T lymphocyte migration: the influence of interactions via adhesion molecules, the T cell receptor, and cytokines

Although lymphocytes have been studied extensively with respect to a number of motile aspects the understanding of directed lymphocyte motility and its regulation has increased relatively slowly. T lymphocyte migration/translocation in vivo and in vitro are critically dependent on the avidity of adhesive lymphocyte receptors for endothelial cell ligands and extracellular matrix (ECM) components and on the capacity of the lymphocytes to undergo a motile response. Lymphocytes are rendered motile by adhesion to endothelial cells and ECM components. Thus, T lymphocytes exhibit chemotactic and haptotactic migration to the ECM components fibronectin, laminin, and collagen type IV. This directed migration is mediated by beta 1-integrins and separate T-lymphocyte lines have a functional specialization using either alpha 4 beta 1 or alpha 5 beta 1 during chemo- and haptotaxis to ECM components, although the same cell line may use both integrins for adhesion. Noteworthy, signals triggering T cell migration to ECM components seem to be delivered preferentially via alpha 4 beta 1 or alpha L beta 2. The T cell antigen receptor cannot by itself trigger T lymphocyte migration to fibronectin, laminin, or collagen type IV but does so in collaboration with signals via alpha 4 beta 1. It follows that the migration-triggering signals can be separated from the integrin interactions with matrix components that mediate the chemo- and haptotactic migration per se. This suggests that T cell recruitment to inflammatory sites is induced by antigen receptor signals and beta 1- and beta 2-integrin signals in synergy. Cytokines with chemokinetic properties may collaborate with lymphocyte counterreceptors on endothelial cells and with ECM components in control of the lymphocyte migratory pathways and specifically attract lymphocyte subsets to different compartments. T lymphocytes are endowed with multiple enzymes, classified as serine proteinases or metalloproteinases, which can degrade extracellular matrix components. These enzymes may play an important role for the capacity of T cells to migrate and infiltrate tissues.     

Extracellular matrix. 2: Role of extracellular matrix molecules and their receptors in the nervous system

Extracellular matrix molecules help regulate many aspects of neural development, including survival, migration, axon growth, and synapse formation by neurons. These same molecules have been shown to modulate regeneration of neurons after injuries. They also regulate the development and differentiation of other neural cells, such as astroglia and Schwann cells. Significant progress has been made recently in characterizing both ECM constituents and their receptors in the nervous system. Extracellular matrix molecules promote cell adhesion, activate intracellular signaling pathways, and modulate the activities of several growth factors and proteins. Our current understanding of the extracellular matrix, its receptors, and its functions in the nervous system are discussed.     

The origin and differentiation of microglial cells during development

Some authors claim that microglia originate from the neuroepithelium, although most now believe that microglial cells are of mesodermal origin, and probably belong to the monocyte/macrophage cell line. These cells must enter the developing central nervous system (CNS) from the blood stream, the ventricular space or the meninges. Afterward microglial cells are distributed more or less homogeneously through the entire nervous parenchyma. Stereotyped patterns of migration have been recognized during development, in which long-distance tangential migration precedes radial migration of individual cells. Microglial cells moving through the nervous parenchyma are ameboid microglia, which apparently differentiate into ramified microglia after reaching their definitive location. This is supported by the presence of cells showing intermediate features between those of ameboid and ramified microglia. The factors that control the invasion of the nervous parenchyma, migration within the developing CNS and differentiation of microglial cells are not well known. These phenomena apparently depend on environmental factors such as soluble or cell-surface bound molecules and components of the extracellular matrix. Microglial cells within the developing CNS are involved in clearing cell debris and withdrawing misdirected or transitory axons, and presumably support cell survival and neurite growth.     

Ontogeny of vesicular monoamine transporter mRNAs VMAT1 and VMAT2. I. The developing rat central nervous system

We used in situ hybridization histochemistry to study the expression of the mRNA of the two vesicular monoamine transporters (VMAT1 and VMAT2) during embryonic and postnatal development of the central nervous system (CNS) in the rat. In the adult rat, VMAT2 mRNA is present exclusively in monoaminergic cell groups of the CNS and VMAT1 mRNA was reported to be present in the adrenal medulla and certain intestinal epithelial cells. In contrast to the above, the expression of VMAT1 mRNA has previously never been detected in the central nervous system. This study shows the first evidence that both transporter molecules are expressed in CNS during ontogenesis. We here demonstrate four main expression patterns detected during development: 1. VMAT2 mRNA expression in monoaminergic neurons of the brainstem beginning as early as embryonic day E13. 2. Expression of VMAT2 mRNA in all major sensory relay nuclei of central nervous system. 3. Co-expression of VMAT1 and VMAT2 mRNA in most limbic structures, basal ganglia, as well as in some hypothalamic nuclei. 4. Exclusive expression of VMAT1 mRNA in the neocortical subventricular zone, in the amygdala at early (E15-18) and late (P1-P28) timepoints, the granular cell layer of cerebellum, and in several brainstem motor nuclei. Based on their distribution during development we suggest that monoamines, released in a controlled fashion, might affect wiring of sensory and also motor circuits. VMAT1 mRNA expression may reflect a specific effect of monoamines in glial differentiation and cerebellar granule cell migration and/or differentiation.     

A key function for alphav containing integrins in mesodermal cell migration during Pleurodeles waltl gastrulation

During cleavage of Pleurodeles waltl amphibian embryos, inner cells of the blastocoel roof (presumptive ectodermal and mesodermal cells) organize a fibrillar extracellular matrix (ECM) containing fibronectin on their basal surface by a beta1-integrin-dependent process. This matrix is used as a migratory substrate by mesodermal cells during gastrulation. While alpha5beta1 integrin is expressed on both ectodermal and mesodermal cell surface, we have shown previously that alphav containing integrins are essentially restricted to the surface of mesodermal cells (Alfandari, D., Whittaker, C. A., DeSimone, D. W., and Darribère, T., Dev. Biol. 170, 249-261, 1995). To investigate the function of alphav integrins during gastrulation, we have generated a function blocking antibody directed against the extracellular domain of the Pleurodeles integrin alphav subunit. The antibody did not prevent fibronectin fibril formation, whereas an antibody against the alpha5beta1 integrin did. When injected into the blastocoel, the antibody against integrin alphav subunit perturbed gastrulation and further development in a stage-dependent manner. Developmental defects were correlated to an abnormal positioning of the mesoderm layer. In vitro, the antibody blocked spreading of mesodermal cell to fibronectin or blastocoel roof ECM but not their attachment. In contrast, the antibody directed against the alpha5beta1 integrin inhibited both cell attachment and spreading to the same substrates. We propose that the alpha5beta1 integrin is required for fibronectin assembly into fibrils and mesodermal cell attachment to the blastocoel roof ECM, while the alphav containing integrins are necessary for cell spreading, and possibly migration, on this complex network.     

The role of the extracellular matrix in neoplastic glial invasion of the nervous system

Intrinsic tumours of the central nervous system (CNS) are generally derived from the glial cells: the astrocytes, oligodendrocytes and ependymal cells. Although such tumours rarely metastasize to distant organs, they show a marked propensity for local invasion of the surrounding nervous tissue. Sub-populations of neoplastic glia may migrate several millimetres away from main tumour mass into the contiguous CNS parenchyma, resulting in poor demarcation of the tumour. These migratory, so-called "guerrilla" cells give rise to recurrent tumours following surgical debulking and adjuvant radio- and chemo-therapeutic intervention. As in other organs, tumour cell invasion is, in part, facilitated by interaction with the extracellular matrix (ECM); however, apart from the vascular basal lamina and the glia limitans externa, the CNS lacks a well-defined ECM. Invading neoplastic cells must, therefore, provide their own ECM, a process which may be stimulated by such agents as gangliosides or growth factors. Glioma cell-derived laminin and hyaluronic acid may provide the most important substrates for invasion, cell adhesion to these substrates being achieved largely through integrin receptors (the function of which may be determined by interaction with cell surface gangliosides) and CD44, respectively. Modulation of these ECM components is facilitated by a variety of proteinases including the matrix metalloproteinases and hyaluronidase, the activity of which is also thought to stimulate angiogenesis. Interference with the mechanisms which promote glioma cell adhesive properties may provide suitable targets for novel anti-invasive therapies. These might include ECM components, growth factors, gangliosides, integrin receptors and proteases and their inhibitors.     

SPARC/osteonectin induces matrix metalloproteinase 2 activation in human breast cancer cell lines

Activation of the matrix metalloproteinase 2 (MMP-2) has been shown to play a major role in the proteolysis of extracellular matrix (ECM) associated with tumor invasion. Although the precise mechanism of this activation remains elusive, levels of the membrane type 1-MMP (MT1-MMP) at the cell surface and of the tissue inhibitor of MMP-2 (TIMP-2) appear to be two important determinants. Induction of MMP-2 activation in cells cultivated on collagen type I gels indicated that the ECM is important in the regulation of this process. In this study, we show that SPARC/osteonectin, a small ECM-associated matricellular glycoprotein, can induce MMP-2 activation in two invasive breast cancer cell lines (MDA-MB-231 and BT549) but not in a noninvasive counterpart (MCF-7), which lacks MT1-MMP. Using a set of peptides from different regions of SPARC, we found that peptide 1.1 (corresponding to the NH2-terminal region of the protein) contained the activity that induced MMP-2 activation. Despite the requirement for MT1-MMP, seen in MCF-7 cells transfected with MT1-MMP, the activation of MMP-2 by SPARC peptide 1.1 was not associated with increased steady-state levels of MT1-MMP mRNA or protein in either MT1-MMP-transfected MCF-7 cells or constitutively expressing MDA-MB-231 and BT549 cells. We did, however, detect decreased levels of TIMP-2 protein in the media of cells incubated with peptide 1.1 or recombinant SPARC; thus, the induction of MMP-2 activation by SPARC might be due in part to a diminution of TIMP-2 protein. We conclude that SPARC, and specifically its NH2-terminal domain, regulates the activation of MMP-2 at the cell surface and is therefore likely to contribute to the proteolytic pathways associated with tumor invasion.     

Tissue plasminogen activator mRNA expression in granule neurons coincides with their migration in the developing cerebellum

Tissue plasminogen activator activity in the developing cerebellum, as quantified by zymography of cerebellar homogenates from embryonic day (E) 17 to adult mice, shows a peak of activity at postnatal day (P) 7, followed by a steady 75% decrease into adulthood. Northern blot analysis reveals a similar pattern for tissue plasminogen activator mRNA levels, which are low at E17 but increase dramatically, reaching their highest levels of specific mRNA/micrograms RNA in P1-P7 mice and declining about threefold in the adult mouse. In situ hybridization of whole mouse brain sections with a tissue plasminogen activator antisense cRNA probe shows pronounce reactivity in the cerebellum. Although some binding is associated with the cerebellar meninges, the external granule layer is devoid of tissue plasminogen activator mRNA at all ages. However, highly labeled elongated cells, which also bind antibody to neuronal nuclear antigen and are adjacent to Bergmann glial fibers (i.e., migrating granule neurons), are readily visible throughout the molecular and Purkinje layers at P7 and P14. In the adult mouse cerebellum, tissue plasminogen activator mRNA labeling is restricted to cells in the Purkinje/internal granule layers. Thus, tissue plasminogen activator gene expression is induced as granule neurons leave the external granule layer and begin their inward migration.     

Can gonadal steroids influence cell position in the developing brain?

The preoptic area/anterior hypothalamus (POA/AH) is a site where hormones dramatically influence development. The POA/AH is comprised of multiple subgroups, but little is known about the derivation of these subgroups during development. Results from several laboratories suggest that some cells in the POA/AH originate from progenitor cells in other regions of the developing nervous system. We are exploring pathways for migration in the developing POA/AH in two ways. First, we are examining the distribution of radial glial processes as potential migratory guides using immunocytochemistry. We have identified a transient pattern of radial glial processes from the lateral ventricles to the pial surface at the base of the POA/AH. Additionally, the expression of a molecule in radial glial processes originating in the third ventricle was decreased by prenatal treatment with testosterone. Second, we are utilizing time-lapse video microscopy in vitro to assess the extent and direction of movements of fluorescent dye-labeled cells at different ages in brain slice preparations from the POA/AH of developing rats. Data from these studies indicate that cell migration in the POA/AH includes movements along dorsal-ventral routes and from lateral to medial positions, in addition to the predicted medial to lateral pathway away from the third ventricle. Several researchers have examined effects of gonadal steroids on neurite outgrowth, cell differentiation, cell death, and synaptogenesis. The determination of cell position, however, may be a key event influenced by gonadal steroids earlier in development. The characterization of migratory pathways that contribute to permanent changes in brain structure and ultimately function is essential for unraveling the process of sexual differentiation.     

Experimental jaw-muscle pain does not change heteronymous H-reflexes in the human temporalis muscle

The role of growth factors in controlling the development of glial cells in both the peripheral and central nervous systems has been investigated for a number of years. The recent discovery of a new family of growth factors termed the neuregulins (NRGs) has led to an explosion of information concerning the putative role of these growth factors in the development of Schwann cells (SC), oligodendrocytes (OLG), and astrocytes. Many of these previous studies have focused on the effects of exogenous NRGs on glial cell development and differentiation. We now review the evidence that these glial cells themselves produce NRGs and discuss the major implications of these findings with respect to glial cell development and diseases which affect glial cell function. We also discuss the potential role of endogenous NRGs following neural injury.     

Stem cell factor regulates human melanocyte-matrix interactions

Stem cell factor (SCF) is hypothesized to play a critical role in the migration of melanocytes during embryogenesis because mutations in either the SCF gene, or its ligand, c-kit, result in defects in coat pigmentation in mice and in skin pigmentation in humans. In this report we directly show that SCF alters the adhesion and migration of human melanocytes to extracellular matrix (ECM) ligands and regulates integrin expression at the protein level. SCF decreased adhesion of neonatal and fetal cells to collagen IV, and increased attachment of fetal cells to laminin. Attachment of fetal cells to fibronectin was decreased, but was unchanged in neonatal cells. Flow cytometry analysis of neonatal melanocytes showed that SCF down-regulated the expression of the alpha 2 receptor, and up-regulated the expression of the alpha 3, alpha 5 and beta 1 integrin receptors. SCF down-regulated expression of alpha 2, alpha 5 and beta 1 integrins by fetal melanocytes, and up-regulated expression of the alpha v and alpha 3 integrin receptors. Analysis of melanocyte migration using time-lapse videomicroscopy showed that SCF significantly increased migration of neonatal, but not fetal, melanocytes on fibronectin (FN). We conclude that SCF regulates integrin expression at the protein level and that SCF has pleiotropic effects on melanocyte attachment and migration on ECM ligands. We suggest that this may be one mechanism by which SCF regulates melanocyte migration during development of the skin.     

Calcium binding protein calcyphosine in dog central astrocytes and ependymal cells and in peripheral neurons

Calcyphosine is a calcium binding protein discovered in the dog thyroid in 1979. Calcyphosine mRNA and immunoreactivity were detected using Western and Northern blotting in the cerebral cortex, cerebral white matter and cerebellum. Using immunohistochemistry and in situ hybridization, both are present in ependymal cells, choroid plexus cells and several types of astrocytes of the subependymal cerebral layer, the cerebellar Bergmann layer, the retinal ganglion cell layer, the optic nerve and the posterior pituitary. Both are also present in neurons of nasal olfactory mucosa, enteric Auerbach and Meissner plexuses, orthosympathic and spinal cord ganglia as well as in endocrine cells of neural crest origin in the adrenal medulla. Calcyphosine immunoreactive astrocytes were also present mainly in hemispheric cerebral gray and white matter, hemispheric subcortical structures, brain stem and spinal cord. These results show that calcyphosine is a characteristic calcium binding protein of astrocytes and ependymal cells in the central nervous system and of neurons in the peripheral nervous system. This is of interest in view of the importance of calcium regulation in these cells, and since calcyphosine a calcium binding protein phosphorylated by cAMP dependent process, may be an intermediate between cAMP and inositol phosphate cascades.     

Expression of osteonectin mRNA in human breast tumours is inversely correlated with oestrogen receptor content

Osteonectin is a secreted glycoprotein which is detected in a number of normal and neoplastic human tissues in vivo. It is an extracellular matrix (ECM)-associated protein which is postulated to regulate cell migration, adhesion, proliferation and matrix mineralisation and previous reports suggest that it may be modulated by steroid hormones in target tissues. The aim of this study was to measure osteonectin mRNA and protein expression in breast tumour biopsies and compare these with oestrogen (ER) and progesterone receptor (PR) levels in the same tumours. An inverse correlation was seen between osteonectin mRNA expression and ER level. Samples with low ER protein expression had a mean osteonectin mRNA level which was almost 4-fold greater than the mean level of expression observed in tumours containing high concentrations of ER protein. This inverse correlation was statistically significant. Despite the strong inverse relationship between osteonectin mRNA levels and tumour ER content, no correlation was seen when osteonectin protein concentration was measured in tumour cytosols on immunoblots and compared to ER and PR levels in the same tumours. However, since it is a secreted protein, osteonectin protein expression may not reflect cellular osteonectin levels in breast tumours. In summary, these data suggest that ER-mediated suppression of osteonectin gene expression may contribute to the less aggressive characteristics associated with receptor-positive tumours and that loss of ER expression may lead to over-expression of osteonectin and contribute to a poorer differentiated, more invasive phenotype.