Highly segregated localization of the functionally related vps10p receptors sortilin and SorCS2 during neurodevelopment.

Nervous system development is a precisely orchestrated series of events requiring a multitude of intrinsic and extrinsic cues. Sortilin and SorCS2 are members of the Vps10p receptor family with complementary influence on some of these cues including the neurotrophins (NTs). However, the developmental time points where sortilin and SorCS2 exert their activities in conjunction or independently still remain unclear. In this study we present the characterization of the spatiotemporal expression pattern of sortilin and SorCS2 in the developing murine nervous system. Sortilin is highly expressed in the fetal nervous system with expression localized to distinct cell populations. Expression was high in neurons of the cortical plate and developing allocortex, as well as subpallial structures. Furthermore, the neuroepithelium lining the ventricles and the choroid plexus showed high expression of sortilin, together with the developing retina, spinal ganglia, and sympathetic ganglia. In contrast, SorCS2 was confined in a marked degree to the thalamus and, at E13.5, the floor plate from midbrain rostrally to spinal cord caudally. SorCS2 was also found in the ventricular zones of the ventral hippocampus and nucleus accumbens areas, in the meninges and in Schwann cells. Hence, sortilin and SorCS2 are extensively present in several distinct anatomical areas in the developing nervous system and are rarely co-expressed. Possible functions of sortilin and SorCS2 pertain to NT signaling, axon guidance and beyond. The present data will form the basis for hypotheses and study designs for unravelling the functions of sortilin and SorCS2 during the establishment of neuronal structures and connections.

Mural lymphatic endothelial cells regulate meningeal angiogenesis in the zebrafish.

Mural cells of the vertebrate brain maintain vascular integrity and function, play roles in stroke and are involved in maintenance of neural stem cells. However, the origins, diversity and roles of mural cells remain to be fully understood. Using transgenic zebrafish, we identified a population of isolated mural lymphatic endothelial cells surrounding meningeal blood vessels. These meningeal mural lymphatic endothelial cells (muLECs) express lymphatic endothelial cell markers and form by sprouting from blood vessels. In larvae, muLECs develop from a lymphatic endothelial loop in the midbrain into a dispersed, nonlumenized mural lineage. muLEC development requires normal signaling through the Vegfc-Vegfd-Ccbe1-Vegfr3 pathway. Mature muLECs produce vascular growth factors and accumulate low-density lipoproteins from the bloodstream. We find that muLECs are essential for normal meningeal vascularization. Together, these data identify an unexpected lymphatic lineage and developmental mechanism necessary for establishing normal meningeal blood vasculature.

The Cerebellum and Its Wrapping Meninge: Developmental Interplay between Two Major Structures.

Meninges have long been considered as a protective and supportive tissue for the central nervous system. Nevertheless, new developmental roles are now attributed to them. The meninges that surround the cerebellum come from the cephalic mesoderm. They are essential for the cerebellum to develop normally. They induce and maintain the basal lamina and glia limitans. In the absence of these structures, the external granular cells of the cerebellum migrate aberrantly and penetrate the subarachnoid space. The molecules involved in the recognition between the cerebellar primordium and the basal lamina belong to two groups in humans: dystroglycan and laminin on the one hand, and GPR56 and collagen III on the other. Finally, molecules secreted by the meninges and acting on the cerebellum begin to be demonstrated; such is the case of SDF1 secreted under the action of FOXC1.

Segmental and restricted localization pattern of Fras1 in the developing meningeal basement membrane in mouse.

The Fras1/Frem family of extracellular matrix proteins consists of Fras1 and its structurally related proteins, Frem1 (Fras1-related extracellular matrix protein 1), Frem2 and Frem3. These are co-localized in embryonic epithelial basement membranes (BMs), where they contribute to epithelial-mesenchymal adhesion. Although Fras1 localization pattern in epithelial BMs has been well defined, it has not yet been comprehensively studied in the central nervous system. Here, we demonstrate the immunohistochemical profile of Fras1 in the developing mouse brain and reveal an exclusively meningeal BM protein deposition. Interestingly, Fras1 displays a segmental localization pattern, which is restricted to certain regions of the meningeal BM. Frem2 protein displays a similar localization pattern, while Frem3 is rather uniformly distributed throughout the meningeal BM. Fras1 and Frem2 proteins are detected in regions of the BM that underlie organizing centers, such as the roof plate (RP) of diencephalon, midbrain and hindbrain, and the RP-derived structures of telencephalon (choroid plexus and hem). Organizing centers exert their activity via the production of bioactive molecules, which are potential Fras1 ligands. The restricted pattern of Fras1 and Frem2 proteins indicates a molecular compartmentalization of the meningeal BM that could reflect, yet unspecified, functional and structural differences.

Laminin α1 is essential for mouse cerebellar development.

Laminin α1 (Lama1), which is a subunit of laminin-1 (laminin-111), a heterotrimeric ECM protein, is essential for embryonic development and promotes neurite outgrowth in culture. Because the deletion of Lama1 causes lethality at early embryonic stages in mice, the in vivo role of Lama1 in neural development and functions has not yet been possible to determine. In this study, we generated conditional Lama1 knockout (Lama1(CKO)) mice in the epiblast lineage using Sox2-Cre mice. These Lama1(CKO) mice survived, but displayed behavioral disorders and impaired formation of the cerebellum. Deficiency of Lama1 in the pial basement membrane of the meninges resulted in defects in the conformation of the meninges. During cerebellar development, Lama1 deficiency also caused a decrease in the proliferation and migration of granule cell precursors, disorganization of Bergmann glial fibers and endfeet, and a transient reduction in the activity of Akt. A marked reduction in numbers of dendritic processes in Purkinje cells was observed in Lama1(CKO) mice. Together, these results indicate that Lama1 is required for cerebellar development and functions.

Visualizing myeloarchitecture with magnetic resonance imaging in primates.

The pattern of myelination over the cerebral cortex, termed myeloarchitecture, is an established and often-used feature to visualize cortical organization with histology in a variety of primate species. In this paper, we use in vivo magnetic resonance imaging (MRI) and advanced image processing using surface rendering to visualize and characterize myeloarchitecture in a small nonhuman primate, the common marmoset (Callithrix jacchus). Through images made in four female adult marmosets, we produce a representative 3D map of marmoset myeloarchitecture and flatten and annotate this map to show the location and extent of a variety of major areas of the cortex, including the primary visual, auditory, and somatosensory areas. By treating our MRI data as a surface, we can measure the surface area of cortical areas, and we present these measurements here to summarize cortical organization in the marmoset.

Developmental changes of mast cell populations in the cerebral meninges of the rat.

It is known that both the dura and the pia mater attract and support the differentiation of mast cells. The present study shows that unevenly distributed mast cells in the cerebral meninges of the rat can be found in perivascular sites and vessel ramification points, but can also be unrelated to the meningeal vasculature. It also documents changes in the number, localization and staining preferences of the mast cells in the two meninges of the developing and mature rat brain. Quantitative examination of all types of histochemically differentiated meningeal mast cells reveals no major (although some exist) differences between right and left side subpopulations, but strongly suggests a different origin and fate of the dural and the pial mast cells. The number of dural mast cells, already high from postnatal day 0, although declining from postnatal day 21 onwards, remains conspicuous up to postnatal day 180. In contrast, pial mast cells are comparatively very few in the first day of the postnatal life, and despite a transient significant increase in the following two weeks, they reach almost zero levels from postnatal day 21.

Integrin-linked kinase regulates Bergmann glial differentiation during cerebellar development.

We demonstrate here that integrin-linked kinase (ILK), a serine/threonine kinase that binds to the beta1 integrin cytoplasmic domain, regulates cerebellar development. Mice with a CNS-restricted knock-out of the Ilk gene show perturbations in the laminar structure of the cerebellar cortex that are associated with defects in Bergmann glial fibers and the formation of meningeal basement membranes. Similar defects have been observed in mice lacking beta1 integrins in the CNS. ILK and beta1 integrins are coexpressed in Bergmann glial cells, and studies with primary cells in culture demonstrate that ILK and CDC42 are required for beta1-integrin-dependent glial process outgrowth. Consistent with these findings, the amount of GTP-bound CDC42 is impaired in the cerebellum of Ilk-deficient mice. We conclude that beta1 integrin, ILK and CDC42 are components of the signaling machinery that regulates glial process outgrowth in the cerebellum. We also show that granule cell precursor proliferation is affected in ILK-deficient mice, but our findings provide strong evidence that proliferative defects are a secondary consequence of ILK function in glia.

Phenotypic integration of neurocranium and brain.

Evolutionary history of Mammalia provides strong evidence that the morphology of skull and brain change jointly in evolution. Formation and development of brain and skull co-occur and are dependent upon a series of morphogenetic and patterning processes driven by genes and their regulatory programs. Our current concept of skull and brain as separate tissues results in distinct analyses of these tissues by most researchers. In this study, we use 3D computed tomography and magnetic resonance images of pediatric individuals diagnosed with premature closure of cranial sutures (craniosynostosis) to investigate phenotypic relationships between the brain and skull. It has been demonstrated previously that the skull and brain acquire characteristic dysmorphologies in isolated craniosynostosis, but relatively little is known of the developmental interactions that produce these anomalies. Our comparative analysis of phenotypic integration of brain and skull in premature closure of the sagittal and the right coronal sutures demonstrates that brain and skull are strongly integrated and that the significant differences in patterns of association do not occur local to the prematurely closed suture. We posit that the current focus on the suture as the basis for this condition may identify a proximate, but not the ultimate cause for these conditions. Given that premature suture closure reduces the number of cranial bones, and that a persistent loss of skull bones is demonstrated over the approximately 150 million years of synapsid evolution, craniosynostosis may serve as an informative model for evolution of the mammalian skull.

Directional neurite outgrowth is enhanced by engineered meningeal cell-coated substrates.

After injury to the CNS, the anatomical organization of the tissue is disrupted, posing a barrier to the regeneration of axons. Meningeal cells, a central participant in the CNS tissue response to injury, migrate into the core of the wound site in an unorganized fashion and deposit a disorganized extracellular matrix (ECM) that produces a nonpermissive environment. Previous work in our laboratory has shown that the presentation of nanometer-scale topographic cues to these cells influences their morphological, cytoskeletal, and secreted ECM alignment. In the present study, we provided similar environmental cues to meningeal cells and examined the ability of the composite construct to influence dorsal root ganglion regeneration in vitro. When grown on control surfaces of meningeal cells lacking underlying topographic cues, there was no bias in neurite outgrowth. In contrast, when grown on monolayers of meningeal cells with underlying nanometer-scale topography, neurite outgrowth length was greater and was directed parallel to the underlying surface topography even though there exists an intervening meningeal cell layer. The observed outgrowth was significantly longer than on laminin-coated surfaces, which are considered to be the optimal substrata for promoting outgrowth of dorsal root ganglion neurons in culture. These results suggest that the nanometer-level surface finish of an implanted biomaterial may be used to organize the encapsulation tissue that accompanies the implantation of materials into the CNS. It furthermore suggests a simple approach for improving bridging materials for repair of nerve tracts or for affecting cellular organization at a device-tissue interface.

The structure and development of avian lumbosacral specializations of the vertebral canal and the spinal cord with special reference to a possible function as a sense organ of equilibrium.

The avian lumbosacral vertebral column and spinal cord show a number of specializations which have recently been interpreted as a sense organ of equilibrium. This sense organ is thought to support balanced walking on the ground. Although most of the peculiar structures have been described previously, there was a need to reevaluate the specializations with regard to the possible function as a sense organ. Specializations were studied in detail in the adult pigeon. The development of the system was studied both in the pigeon (semiprecocial at hatching) and in the chicken (precocial). Specializations in the vertebral canal consist of a considerable enlargement, which is not due to an increase in the size of the spinal nervous tissue, but to a large glycogen body embedded in a dorsal rhomboid sinus. The dorsal wall of the vertebral canal shows segmented bilateral dorsal grooves, which are covered by the meninges towards the lumen of the vertebral canal leaving openings in the midline and laterally. This results in a system of lumbosacral canals which look and may function similar to the semicircular canals in the inner ear. Laterally these canals open above ventrolateral protrusions or accessory lobes of the spinal cord which contain neurons. There are large subarachnoidal cerebrospinal fluid spaces, lateral and ventral to the accessory lobes. Movement of this fluid is thought to stimulate the lobes mechanically. As to the development of avian lumbosacral specializations, main attention was given to the organization of the lobes and the adjacent fluid spaces including the dorsal canals. In the pigeon the system is far from being adult-like at hatching but maturates rapidly after hatching. In the chicken the system looks already adult-like at hatching. The implications derived from the structural findings are discussed with regard to a possible function of the lumbosacral specializations as a sense organ of equilibrium. The adult-like organization in the newly hatched chickens, which walk around immediately after hatching, supports the assumed function as a sense organ involved in the control of locomotion on the ground.

Role of presenilin-1 in cortical lamination and survival of Cajal-Retzius neurons.

Presenilin-1 (PS1), the major causative gene of familial Alzheimer disease, regulates neuronal differentiation and Notch signaling during early neural development. To investigate the role of PS1 in neuronal migration and cortical lamination of the postnatal brain, we circumvented the perinatal lethality of PS1-null mice by generating a conditional knockout (cKO) mouse in which PS1 inactivation is restricted to neural progenitor cells (NPCs) and NPC-derived neurons and glia. BrdU birthdating analysis revealed that many late-born neurons fail to migrate beyond the early-born neurons to arrive at their appropriate positions in the superficial layer, while the migration of the early-born neurons is largely normal. The migration defect of late-born neurons coincides with the progressive reduction of radial glia in PS1 cKO mice. In contrast to the premature loss of Cajal-Retzius (CR) neurons in PS1-null mice, generation and survival of CR neurons are unaffected in PS1 cKO mice. Furthermore, the number of proliferating meningeal cells, which have been shown to be important for the survival of CR neurons, is increased in PS1-null mice but not in PS1 cKO mice. These findings show a cell-autonomous role for PS1 in cortical lamination and radial glial development, and a non-cell-autonomous role for PS1 in CR neuron survival.

Meningeal cell-derived semaphorin 3A inhibits neurite outgrowth.

The neural scar that forms after injury to the mammalian central nervous system is a barrier to sprouting and regenerating axons. In addition to reactive astrocytes that are present throughout the lesion site, leptomeningeal fibroblasts invade the lesion core. When isolated in vitro, these cells form a very poor substrate for growing neurites, even more so than reactive astrocytes. Nevertheless the molecular mechanisms involved in this growth inhibition are not well understood. Semaphorins have been reported to be upregulated in meningeal cells (MCs) on mechanical injury to the brain and spinal cord. In the present study, we show that Sema3A mRNA and active protein are produced by cultured meningeal cells. A protein extract from these cells induces the collapse of embryonic dorsal root ganglion (DRG) growth cones. This collapsing activity is partially blocked by neuropilin-1 antibodies and is absent in meningeal cells derived from Sema3A-knockout mice. In addition to growth cone collapse, recombinant Sema3A but not Sema3C inhibits neurite outgrowth of embryonic DRGs. Consistent with this result we find that the inhibitory effect of meningeal cells on neurite outgrowth is partially overcome on Sema3A-deficient MCs. Furthermore we show that the inhibitory effect of MC-derived Sema3A on neurite outgrowth is modulated by nerve growth factor. Our results show that Sema3A, a chemorepellent during nervous system development, is a major neurite growth-inhibitory molecule in meningeal fibroblasts and is therefore likely to contribute to the inhibitory properties of the neural scar.

Expression of a membrane-bound form of the ferroxidase ceruloplasmin by leptomeningeal cells.

Ceruloplasmin is a key enzyme involved in detoxifying ferrous iron, which can generate free radicals. The secreted form of ceruloplasmin is produced by the liver and is abundant in serum. We have previously identified a membrane-bound glycosylphosphatidylinositol (GPI)-anchored form of ceruloplasmin (GPI-Cp) that is expressed by astrocytes in the central nervous system (CNS) (Patel and David. 1997. J Biol Chem 272:20185-20190). We now provide direct evidence that rat leptomeningeal cells, which cover the surface of the brain, also express GPI-Cp. The expression of GPI-Cp on the surface of these cells increases with postnatal development and is regulated in vitro by cell density, time in culture, and various extracellular matrix molecules. The expression of GPI-Cp also appears to be regulated differently in astrocytes and leptomeningeal cells in vitro. The abundant expression of GPI-Cp on the surface of leptomeningeal cells suggests that these cells play a role in antioxidant defense along the surface of the postnatal CNS possibly by detoxifying the cerebrospinal fluid.

Remodeling of the leptomeningeal microvascular plexus in neonatal rats.

In order to better understand the mechanisms of cerebrovascular development and differentiation, the leptomeningeal microcirculation was analyzed using in vivo fluorescence video-microscopy in neonatal (1, 9, and 12 day old) Sprague-Dawley rats. The pattern of flow distribution was reconstructed, the location of radially emerging intracortical veins was identified and the minimum distance between these vessels was measured. We found no AV connections in the leptomeningeal vasculature of the neonatal rat in all examined animals. The LVP in the neonatal period of life is a venous system and probably does not serve as a major source of oxygen supply to cerebral tissue. After birth, the number of radial vessels does not increase, suggesting that intracortical vascularization continues by the branching of existing radial vessels.

The morphological picture of developing meningo-encephalitis in central nervous system.

In this paper we are summarizing our observations collected during several years and concerning the inflammatory changes in maturing human brains. Studies of inflammatory reactions in developing central nervous system (CNS) revealed that their morphological picture changes along with the maturation of the brain and formation of fetal immune response. We have analyzed various types of inflammatory processes (meningeal infiltrations, meningoencephalitis with perivascular infiltrates and glial interstitial changes, inflammatory-necrotic lesions) occurring in different stages of brain development. In all these processes gradual maturation of cell lines belonging to the immune system was seen. Evident correlation between tissue inflammatory reaction and necrosis was confirmed. The necrotic changes mainly within hemispheric white matter may result in the cortical malformations. The morphologic manifestation of inflammation in the developing CNS is dependent on the stage of maturation of the brain structure and immunological response.

The tight junctions of the leptomeningeal blood-cerebrospinal fluid barrier during development.

The outer blood-cerebrospinal fluid barrier is formed by leptomeningeal cells of the arachnoidea. The structures underlying this barrier are tight junctions. In contrast to the tight junctions of endo- and epithelial cells, which have been investigated by means of ultrastructural as well as by molecular methodology, equivalent studies on meningeal cells are lacking. In the present study, therefore, the ultrathin section and freeze-fracture morphology of cranial leptomeningeal cells of carp, frog, chicken and rat was investigated by quantitative morphometry. In addition, the developmental features of the meningeal barrier in chicken and rat were compared. The parameters determined were the complexity of the tight junctions, the density of strands and branchpoints and the degree of association of tight junction particles with the protoplasmatic and exoplasmatic leaflets of cellular membranes. The complexity of tight junctions was highest in chicken and lowest in frog meningeal cells, whereas intermediate values were reached in the carp and the rat. E- and P-face associations were similar in carp and frog, whereas in chicken, the P-face association and in rat the E-face association dominated. During development, tight junction complexity continuously increased up to adult stages in the chicken, whereas in the rat, the adult value was already reached at postnatal day 2. At early embryonic stages, particle insertion into tight junctions was incomplete but occurred equally into both membranous leaflets; if completed at E19 in the chicken, a redistribution of particles toward a higher P-face association was observed. In the rat, tight junction particles were redistributed toward a higher E-face association. These results are discussed in the context of blood-brain barrier induction, maintenance and regulation.

Postnatal remodeling of the leptomeningeal vascular network as assessed by intravital fluorescence video-microscopy in the rat.

An intriguing characteristic of the ontogenic development of the cerebral vasculature is the rapid differentiation of the neonatal leptomeningeal vascular plexus into the mature, adult network form. The physiological and cellular mechanisms of this cerebrovascular remodeling process are unclear. The objective of this work was to determine and correlate changes in vascular density, network pattern and flow velocity in leptomeningeal microvessels of the rat during postnatal development in vivo. To this end, microvascular diameter, segment length, and vascular density of reconstructed leptomeningeal networks were measured from video-recordings of the microcirculation visualized through a cranial window in 0-15-day-old Sprague-Dawley rats. The velocity of erythrocytes in the microvessels was measured by frame to frame tracking of fluorescently labeled red blood cells. We found that surface vascular density (total vessel length per area), node density and segment density (object per area) decreased significantly by the second week after birth. Anastomosing vascular polygons, characteristic to newborn networks, became less numerous and larger in diameter during the postnatal 2-week period, indicating progressive rarefaction of the networks. Vessel diameter and red cell velocity showed transient increases at 1.5 weeks. The velocity/diameter ratio (V/D), an index of wall shear rate, increased by the age of 1.5 weeks and remained unchanged afterwards. There was a negative correlation between V/D and diameter at 1 week; this relationship was reversed to a positive correlation at 2 weeks. We conclude that postnatal remodeling of the leptomeningeal vascular network is associated with rarefaction and an adaptation of vessel caliber to wall shear rate. These changes may contribute to arterio-venous differentiation and redistribution of blood flow from the superficial to the intracortical vasculature in the developing brain.

Cytokeratin expression in human spinal meninges and ependymal cells.

The expression of intermediate filament protein in human spinal cord arachnoid cells and ependyma was studied by immunohistochemistry and immunoblotting. Monoclonal antibodies specific for individual cytokeratin polypeptides indicated a developmental change in the presence of cytokeratin 8 and 18 in spinal leptomeninx and tanycytes of the spinal cord ependyma. While in fetal material cytokeratin 8 and 18 were abundant in arachnoid cells, in adults immunoreactivity was restricted to a few cells. Immunoblots prepared from adult as well as fetal arachnoid membranes showed significant amounts of cytokeratin 8. These findings indicate that although cytokeratin is represented in both fetal and adult arachnoid cells there is development regulation of its specific localization.

Development of the rat meninx: experimental study using bromodeoxyuridine.

The development of the rat meninx from the viewpoint of cell proliferation was studied microscopically and immunohistochemically using bromodeoxyuridine (BUdR). A compact cell layer around the neural tube, the meninx primitiva, was observed in 12- and 13-day fetuses. A reticular structure resembling the subarachnoid space appeared in the 14-day fetus. The ectomeninx, consisting of a collagen fiber layer, part of which became the dura mater, appeared in 15-day fetuses, allowing discrimination of the endomeninx, the arachnoid cell layer. The primordium of the choroid plexus also appeared in the lateral ventricle on the same day. Bone appeared in the primitive dura mater, and stratification of the meninx was almost complete in 21-day fetuses. BUdR-positive cells were confirmed in the meninx from day 12 of gestation to day 15 postpartum. The number of BUdR-positive cells was greatest in fetuses aged about 12 or 13 days, reaching nearly 50%, but decreased gradually toward the neonatal period. The findings of this study suggest that, after the migration of neural crest cells, marked cell proliferation in the meninx begins. Differentiation into various layers then follows and is almost complete before birth, whereas the proliferation of arachnoid cells continues even in the early neonatal period.