Migration Of Neuronal Precursors Research Articles

α6β1 integrin directs migration of neuronal precursors in adult mouse forebrain

New neuroblasts are constantly generated in the adult mammalian subventricular zone (SVZ) and migrate via the very-restricted rostral migratory stream (RMS) to the olfactory bulb, where they differentiate into functional neurons. Several facilitating and repulsive molecules for this migration have been identified, but little is known about chemoattractive molecules involved in the directed nature of this migration in vivo. Here, we investigated the role of the α6β1 integrin, and its ligand, laminin, in controlling guidance of the migrating neuroblasts in adult mice. Immunostaining for the α6β1 integrin was present in neuroblasts and their processes in the anterior/rostral SVZ and the RMS. Inhibition of the endogenous α6 or β1 subunit with locally injected antibodies disrupted the cohesive nature of the RMS, but did not kill the neuroblasts. Infusion of a 15 a.a. peptide, representing the E8 domain of the laminin α chains that bind α6β1 integrin, into the neostriatum redirected the neuroblasts away from the RMS towards the site of infusion. Injection of a narrow tract of intact laminin also drew the neuroblasts away from the RMS, but in a more restricted localization. These results suggest a critical role for integrins and laminins in adult SVZ-derived neuroblast migration. They also suggest that integrin-based strategies could be used to direct or restrict neuroblasts to CNS regions where they are needed for cell replacement therapies in the nervous system.

MMP-9 deficiency affects axonal outgrowth, migration, and apoptosis in the developing cerebellum

Matrix metalloproteinases (MMPs) are responsible for the extensive extracellular proteolysis that plays a central role in regulating the pericellular environment, contributing to morphogenesis and developmental remodeling. In the CNS, there is increasing in vitro evidence for the involvement of MMPs in neurite elongation and axonal guidance. Here, we show that expression of MMP-9 is spatiotemporally related to cerebellar granule cell migration during postnatal development. Using cerebellar explant cultures, we demonstrated that a specific MMP-9-blocking antibody affects granular cell axonal outgrowth and migration in a dose-dependent manner. In addition, the in vivo analysis of MMP-9-deficient mice revealed abnormal accumulation of granular precursors (GPs) in the external granular layer (EGL) at a time when migration is normally extensive. Furthermore, GP migration was delayed and their programmed cell death was reduced in MMP-9-deficient mice, suggesting that MMP-9 is involved in the control of granule cell migration and apoptosis. These results provide direct evidence for a physiological role of MMP-9 in neuronal precursor migration and apoptosis in the developing cerebellum, and emphasize the importance of MMP-9 in the temporal regulation of the cerebellar microenvironment.

Directed migration of neuronal precursors into the ischemic cerebral cortex and striatum

Pathological processes, including cerebral ischemia, can enhance neurogenesis in the adult brain, but the fate of the newborn neurons that are produced and their role in brain repair are obscure. To determine if ischemia-induced neuronal proliferation is associated with migration of nascent neurons toward ischemic lesions, we mapped the migration of cells labeled by cell proliferation markers and antibodies against neuronal marker proteins, for up to 2 weeks after a 90-min episode of focal cerebral ischemia caused by occlusion of the middle cerebral artery. Doublecortin-immunoreactive cells in the rostral subventricular zone, but not the dentate gyrus, migrated into the ischemic penumbra of the adjacent striatum and, via the rostral migratory stream and lateral cortical stream, into the penumbra of ischemic cortex. These results indicate that after cerebral ischemia, new neurons are directed toward sites of brain injury, where they might be in a position to participate in brain repair and functional recovery.

Temporal profile of stem cell division, migration, and differentiation from subventricular zone to olfactory bulb after transient forebrain ischemia in gerbils.

The stage of neurogenesis can be divided into three steps: proliferation, migration, and differentiation. To elucidate their detailed relations after ischemia, the three steps were comprehensively evaluated, in the subventricular zone (SVZ) through the rostral migratory stream (RMS) to the olfactory bulb (OB), in adult gerbil brain after 5 minutes of transient forebrain ischemia. Bromodeoxyuridine (BrdU), highly polysialylated neural cell adhesion molecule (PSA-NCAM), neuronal nuclear antigen (NeuN), and glial fibrillary acidic protein (GFAP) were used as markers for proliferation, migration, and differentiation, respectively. The number of BrdU-labeled cells that coexpressed PSA-NCAM and the size of PSA-NCAM-positive cell colony increased in the SVZ with a peak at 10 d after transient ischemia. In the RMS, the number of BrdU-labeled cells that coexpressed PSA-NCAM increased, with a delayed peak at 30 d, when the size of RMS itself became larger and the number of surrounding GFAP-positive cells increased. In the OB, BrdU + NeuN double positive cells were detected at 30 and 60 d. NeuN staining and terminal deoxynucleotidyl dUTP nick-end labeling staining showed no neuronal cell loss around the SVZ, and in the RMS and the OB after transient ischemia. These findings indicate that transient forebrain ischemia enhances neural stem cell proliferation in the SVZ without evident neuronal cell loss, and has potential neuronal precursor migration with activation of GFAP-positive cells through the RMS to the OB.

Pax6 regulates regional development and neuronal migration in the cerebral cortex

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.

Movement through Slits: cellular migration via the Slit family.

First isolated in the fly and now characterised in vertebrates, the Slit proteins have emerged as pivotal components controlling the guidance of axonal growth cones and the directional migration of neuronal precursors. As well as extensive expression during development of the central nervous system (CNS), the Slit proteins exhibit a striking array of expression sites in non-neuronal tissues, including the urogenital system, limb primordia and developing eye. Zebrafish Slit has been shown to mediate mesodermal migration during gastrulation, while Drosophila slit guides the migration of mesodermal cells during myogenesis. This suggests that the actions of these secreted molecules are not simply confined to the sphere of CNS development, but rather act in a more general fashion during development and throughout the lifetime of an organism. This review focuses on the non-neuronal activities of Slit proteins, highlighting a common role for the Slit family in cellular migration.

Prolonged seizures increase proliferating neuroblasts in the adult rat subventricular zone-olfactory bulb pathway.

Neuronal precursors in the adult rodent forebrain subventricular zone (SVZ) proliferate, migrate to the olfactory bulb in a restricted pathway known as the rostral migratory stream (RMS), and differentiate into neurons. The effects of injury on this neurogenic region of the mature brain are poorly understood. To determine whether seizure-induced injury modulates SVZ neurogenesis, we induced status epilepticus (SE) in adult rats by systemic chemoconvulsant administration and examined patterns of neuronal precursor proliferation and migration in the SVZ-olfactory bulb pathway. Within 1-2 weeks after pilocarpine-induced SE, bromodeoxyuridine (BrdU) labeling and Nissl staining increased in the rostral forebrain SVZ. These changes were associated with an increase in cells expressing antigenic markers of SVZ neuroblasts 2-3 weeks after prolonged seizures. At these same time points the RMS expanded and contained more proliferating cells and immature neurons. BrdU labeling and stereotactic injections of retroviral reporters into the SVZ showed that prolonged seizures also increased neuroblast migration to the olfactory bulb and induced a portion of the neuronal precursors to exit the RMS prematurely. These findings indicate that SE expands the SVZ neuroblast population and alters neuronal precursor migration in the adult rat forebrain. Identification of the mechanisms underlying the response of neural progenitors to seizure-induced injury may help to advance brain regenerative therapies by using either transplanted or endogenous neural precursor cells.

Prolonged Seizures Increase Proliferating Neuroblasts in the Adult Rat Subventricular Zone–Olfactory Bulb Pathway

Neuronal precursors in the adult rodent forebrain subventricular zone (SVZ) proliferate, migrate to the olfactory bulb in a restricted pathway known as the rostral migratory stream (RMS), and differentiate into neurons. The effects of injury on this neurogenic region of the mature brain are poorly understood. To determine whether seizure-induced injury modulates SVZ neurogenesis, we induced status epilepticus (SE) in adult rats by systemic chemoconvulsant administration and examined patterns of neuronal precursor proliferation and migration in the SVZ–olfactory bulb pathway. Within 1–2 weeks after pilocarpine-induced SE, bromodeoxyuridine (BrdU) labeling and Nissl staining increased in the rostral forebrain SVZ. These changes were associated with an increase in cells expressing antigenic markers of SVZ neuroblasts 2–3 weeks after prolonged seizures. At these same time points the RMS expanded and contained more proliferating cells and immature neurons. BrdU labeling and stereotactic injections of retroviral reporters into the SVZ showed that prolonged seizures also increased neuroblast migration to the olfactory bulb and induced a portion of the neuronal precursors to exit the RMS prematurely. These findings indicate that SE expands the SVZ neuroblast population and alters neuronal precursor migration in the adult rat forebrain. Identification of the mechanisms underlying the response of neural progenitors to seizure-induced injury may help to advance brain regenerative therapies by using either transplanted or endogenous neural precursor cells.

Bag1 is a regulator and marker of neuronal differentiation.

Bag 1 acts as a co-chaperone for Hsp70/Hsc70. We report here that stable over-expression of Bag1 in immortalized neuronal CSM14.1 cells prevents death following serum deprivation. Bag1 over-expression slowed the proliferative rate of CSM14.1 cells, resulted in increased levels of phospo-MAP kinases and accelerated neuronal differentiation. Immunocytochemistry revealed mostly nuclear localization of Bag1 protein in these cells. However, during differentiation in vitro, Bag1 protein shifted from predominantly nuclear to mostly cytosolic in CSM14.1 cells. To explore in vivo parallels of these findings, we investigated Bag1 expression in the developing mouse nervous system using immunohistochemical methods. Early in brain development, Bag1 was found in nuclei of neuronal precursor cells, whereas cytosolic Bag1 staining was observed mainly after completion of neuronal precursor migration and differentiation. Taken together, these findings raise the possibility that the Bag1 protein is expressed early in neurogenesis in vivo and is capable of modulating neuronal cell survival and differentiation at least in part from a nuclear location.

Extracellular Signals That Regulate the Tangential Migration of Olfactory Bulb Neuronal Precursors: Inducers, Inhibitors, and Repellents

Neuronal migration is an essential developmental step in the construction of the vertebrate nervous system, but the extracellular signals involved in initiating and regulating neuronal movement remain unclear. Here we report the identification of a novel astrocyte-derived migration-inducing activity (MIA). Using an in vitro assay, we show that MIA induces the migration of olfactory bulb interneuron precursors, increasing the number of migrating cells and the distance they move. We established quantitative criteria to distinguish between the biological effects of inducers, inhibitors, repellents, and attractants on migrating cells and used them to compare the effects of MIA with those of Slit, a putative repulsive guidance cue. Our analysis demonstrates that, by themselves, MIA induces and Slit inhibits migration from subventricular zone explants. However, when presented together with MIA, Slit acts as a repellent. This study shows that glial cells play a critical role in initiating and modulating the movement of neuronal precursors through the release of a diffusible protein. Moreover, this study provides evidence that the guidance of migrating neuronal precursors is an integrative process, resulting from the cooperation of distinct extracellular factors, and that the function of Slit is context dependent.

Early-generated preplate neurons in the developing telencephalon: inward migration into the developing striatum.

Specialized subsets of early-generated neurons provide the cellular cues that are necessary for the establishment of characteristic cell and fiber interactions in each brain region. During the development of the mammalian cerebral cortex, the early-generated cells line up in the most superficial part of the telencephalic pallium forming the preplate. It has been generally thought that the preplate derivatives are exclusively located in the cortical region and govern the early histogenetic phase of cortical development. However, we here disclose an unexpected evidence that a subset of early-generated neurons of the piriform preplate migrate inward into and disperse within the subcortical structure striatum during the embryonic stage. Their migratory route is unique and its direction is opposite to the ordinary migration of neuronal precursors directed outward from the periventricular germinal zone. After immigrating into the developing striatum, these early-generated cells are closely associated with the intrastriatal fascicules of axons. The majority of these cells are eliminated by apoptotic cell death during the early postnatal stage. Based on these findings, we propose a new concept: the preplate neurons may not only direct cortical histogenesis but also change their location to play a role in the development of subcortical structures.

The 9-O-acetyl GD3 gangliosides are expressed by migrating chains of subventricular zone neurons in vitro.

Neurons from the anterior subventricular zone (SVZ) of the cerebral cortex migrate tangentially to become interneurons in the olfactory bulb during development and in adult rodents. This migration was defined as neuronophilic, independent of a radial glial substrate. The cortical SVZ and the rostral migratory stream to the olfactory bulb were shown to be rich in 9-O-acetyl GD3 gangliosides (9-O-acGD3), which have been previously shown to be implicated in gliophilic migration in the rodent cerebral cortex and cerebellum. In the present study, we performed SVZ explant cultures using rats during their first postnatal week to analyze the expression of these gangliosides in chain migration of neuronal precursors. We characterized migrating chains of these neuroblasts through morphological analysis and immunocytochemistry for the neural cell adhesion molecule. By using the Jones monoclonal antibody which binds specifically to 9-O-acGD3 we showed that migrating chains from the SVZ explants express 9-O-acGD3 which is distributed in a punctate manner in individual cells. 9-O-acGD3 is also present in migrating chains that form in the absence of radial glia, typical of the neuronophilic chain migration of the SVZ. Our data indicate that 9-O-acetylated gangliosides may participate in neuronophilic as well as gliophilic migration.

Altered distribution of dopaminergic neurons in the brain of L1 null mice

Dopaminergic neurons of the mouse mesencephalon originate in the ventricular zone and migrate radially along radial glia then tangentially along nerve fibers that express the neural cell adhesion molecule L1 to form the substantia nigra (A9 group) and ventral tegmental area (VTA) (A10 group). The role of L1 in migration of dopaminergic neuronal precursors was investigated in L1 knockout mice by tyrosine hydroxylase (TH) immunostaining. An altered rostrocaudal distribution of dopaminergic neurons was observed within the substantia nigra and VTA of L1-minus mice. In L1-minus mice at postnatal day 0, TH-positive cells were present abnormally in the dorsomedial mesencephalon, suggesting impaired migration. Axons projecting from the substantia nigra to the caudate putamen also exhibited an abnormal targeting pattern. There was no evidence of dopaminergic cell loss in the mutant SN. Abnormal localization of dopaminergic neurons in L1-minus mice was also evident in the zona incerta of the thalamus (A13 group), and the arcuate (A12) and periventricular nucleus (A14) of the hypothalamus. Cell bodies and axons in the substantia nigra, VTA, and hypothalamus of wild type mouse embryos expressed L1. These results suggested that L1 plays an important developmental role in the organization of dopaminergic neuronal cell groups in the mesencephalon and diencephalon.

Migrating neurons cross a reelin-rich territory to form an organized tissue out of embryonic cortical slices.

In this study we show that the radial migration of neuronal precursors out of cerebral cortex of embryonic brain slices cultured for 4-7 days gives rise to an organized tissue that forms de novo off developing slices. In our in vitro preparations, migrating neuronal precursors overshot the marginal zone, as did the elongation of radial glial processes out of the slices. These cells detached from radial glia at a distance from the cortex and differentiated into pyramidal and nonpyramidal profiles that expressed different neuronal markers. Glial precursors were shown to proliferate in the slice and in the neotissue, and to differentiate into astrocytes. We show that cells expressing reelin in the marginal zone of embryonic cortical slices persist after a week in culture, which implies that neuronal migration is not necessarily hindered by the presumed stop signals provided by reelin in the marginal zone. Furthermore, our results provide a new model for in vitro studies of migration and differentiation during cortical development.

Analysis of cerebrocortical neuronal migration in three-dimensional fetal mouse cerebral explants: comparison with in vivo

Organotypic cultures of cerebral mantle offer a useful method for in vitro investigation of mechanisms involved in the migration of neuronal precursors to the neocortex. Studies reported to-date have indicated that, while neurons continue to migrate in cerebral explant cultures for a few days, they eventually stop migrating and fail to reach their pre-programmed destinations and thus distinct layers fail to develop within the cortical plate. Hypothesizing that this migrational arrest stemmed, in part, from severe distortion and flattening of explants in the roller-tube culture method used in previous studies by others, we studied neocortical neuronal migration using a recently developed three-dimensional explant culture system. Explants 300–350 μm thick were derived from E16 fetal mouse cerebrum 24 h following administration of 5-bromodeoxyuridine (BrdU), encased in collagen, placed on porous membranes, and alternately exposed to medium and air on a rocking platform. Cultures were maintained for 7, 11 and 14 days, then analyzed for the location of BrdU-labeled cells. Newborn mice labeled at E15 and analyzed at P1 and P9 served as controls. In the explants, labeled cells continued to migrate into the cortical plate until 11 days in culture, at which point the ventricular zone had disappeared and two distinct cortical cell layers sometimes developed. BrdU-labeled cells were present throughout the cortical plate, particularly the mid portion. In P9 controls, five distinct cortical layers were present with labeled cells mostly in the outer half (layers II, III). Thus, preservation of three-dimensional tissue integrity in culture was accompanied by a modest improvement in migration of neurons to the neocortex in comparison with previous reports. The fact that normal layer formation failed to develop in thick explants suggests that factors other than tissue distortion contribute to this outcome.

Impaired cerebral cortex development and blood pressure regulation in FGF-2-deficient mice.

Fibroblast growth factor-2 (FGF-2) has been implicated in various signaling processes which control embryonic growth and differentiation, adult physiology and pathology. To analyze the in vivo functions of this signaling molecule, the FGF-2 gene was inactivated by homologous recombination in mouse embryonic stem cells. FGF-2-deficient mice are viable, but display cerebral cortex defects at birth. Bromodeoxyuridine pulse labeling of embryos showed that proliferation of neuronal progenitors is normal, whereas a fraction of them fail to colonize their target layers in the cerebral cortex. A corresponding reduction in parvalbumin-positive neurons is observed in adult cortical layers. Neuronal defects are not limited to the cerebral cortex, as ectopic parvalbumin-positive neurons are present in the hippocampal commissure and neuronal deficiencies are observed in the cervical spinal cord. Physiological studies showed that FGF-2-deficient adult mice are hypotensive. They respond normally to angiotensin II-induced hypertension, whereas neural regulation of blood pressure by the baroreceptor reflex is impaired. The present genetic study establishes that FGF-2 participates in controlling fates, migration and differentiation of neuronal cells, whereas it is not essential for their proliferation. The observed autonomic dysfunction in FGF-2-deficient adult mice uncovers more general roles in neural development and function.

Determination of the migratory capacity of embryonic cortical cells lacking the transcription factor Pax-6

The cerebral cortex forms by the orderly migration and subsequent differentiation of neuronal precursors generated in the proliferative ventricular zone. We studied the role of the transcription factor Pax-6, which is expressed in the ventricular zone, in cortical development. Embryos homozygous for a mutation of Pax-6 (Small eye; Sey) had abnormalities suggesting defective migration of late-born cortical precursors. When late-born Sey/Sey precursors were transplanted into wild-type embryonic rat cortex, they showed similar integrative, migrational and differentiative abilities to those of transplanted wild-type mouse precursors. These results suggest that postmitotic cortical cells do not need Pax-6 to acquire the capacity to migrate and differentiate, but that Pax-6 generates a cortical environment that permits later-born precursors to express their full developmental potential.

Focal Ventricular Origin and Migration of Oligodendrocyte Precursors into the Chick Optic Nerve

During central nervous system (CNS) development, oligodendrocyte precursors originate in specific locations and subsequently migrate to all regions of the CNS. Here, we demonstrate that the chick optic nerve is populated by oligodendrocyte precursors, which initially appeared in a focal region at the ventral midline of the third ventricle at stage 26–27. Oligodendrocyte precursors migrated into the chiasmal end of the nerve by stage 29 and became uniformly distributed by stage 35. Migrating precursors were restricted to the anterior region of the nerve, closely apposed to axons, and had a bipolar morphology. In contrast to the polysialic acid (PSA)–dependent cooperative streaming migration of olfactory neuronal precursors, the migration of oligodendrocyte precursors along the optic nerve appeared axophilic and unaffected by removal of neural cell adhesion molecule (N-CAM)–associated PSA. These data indicate that during development, defined domains of the ventricular zone give rise to distinct cell types that utilize discrete mechanisms to navigate specific migrational pathways.

Neuregulin and erbB Receptors Play a Critical Role in Neuronal Migration

The migration of neuronal precursors along radial glial fibers is a critical step in the formation of the nervous system. In this report, we show that neuregulin–erbB receptor signaling plays a crucial role in the migration of cerebellar granule cells along radial glial fibers. Granule cells express neuregulin (NRG), and radial glia cells express erbB4 in the developing cerebellum and in vitro. When the glial erbB receptors are blocked, neurons fail to induce radial glia formation, and their migration along radial glial fibers is impaired. Moreover, soluble NRG is as effective as neuron–glia contact in the induction of radial glia formation. These results suggest that the activation of glial erbB4 by NRG is an early critical step in the neuronal migration program.

Motility and cytoskeletal organization of migrating cerebellar granule neurons

To characterize CNS neuronal precursor migration along astroglial fibers, we examined the motility of the migratory leading process and cytoskeletal-based mechanisms of locomotion of early postnatal mouse cerebellar granule neurons in vitro. To visualize the surface motility of the leading process, granule neurons were labeled with the fluorescent lipophilic dye, PKH-26, and imaged by time lapse fluorescence microscopy. The motile behavior and cytoskeletal organization of the migrating neuron had several distinctive features. As the migrating neuron moved along the glial fiber, the leading process rapidly extended, projecting up to 40 microns, and retracted, withdrawing towards the cell soma. Broad lamellipodia were common along the entire length of the leading process, giving it a ruffled appearance. Within the cell soma, a cage-like distribution of microtubules encircled the nucleus and actin filaments formed a subcortical rim underneath the plasma membrane. Disruption of actin filaments with cytochalasin B inhibited migration, suggesting involvement of actin subunit assembly in neuronal migration. Both microtubules and actin filaments were heavily concentrated in the leading process; the leading process did not show the development of a distinct actin-rich domain at its tip.