Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations.

Glial progenitors colonize the CNS widely in the perinatal period, but the pathways and mechanisms of migration are not well understood. We investigated the migration of progenitors from the neonatal rat forebrain subventricular zone (SVZ) by labeling them in vivo with a retrovirus encoding green fluorescent protein and visualizing movements by time lapse microscopy in slices. Cells within the dorsolateral SVZ moved in an undirected fashion but migrated radially and tangentially after emigration into white matter, cortex, and striatum. Cells in the striatal SVZ migrated parallel to the ventricular surface. During migration, elongation of the leading process and nuclear translocation were independent or linked. Orthogonal turning involved either cessation of cell body movement and formation of a new leading process or continuous cell body movement and bending of the leading process.     

Unique neuronal tracers show migration and differentiation of SVZ progenitors in organotypic slices.

Continual neurogenesis in the subventricular zone (SVZ) of postnatal and adult mammalian forebrain has been well documented, but the mechanisms underlying cell migration and differentiation in this region are poorly understood. We have developed novel in vivo and in vitro methods to investigate these processes. Using stereotaxic injections of a variety of tracers/tracker [Cholera Toxin beta subunit (CTb-), Fluorogold (FG), and Cell Tracker Green (CTG)], we could efficiently label SVZ cells. Over several days, labeled cells migrate along the rostral migratory stream (RMS) to their final differentiation site in the olfactory bulb (OB). The compatibility of these tracers/trackers with immunohistochemistry allows for cell labeling with multiple dyes (e.g., CTb and CTG) and/or specific cell antigens. To investigate the dynamics of migration we labeled SVZ progenitor cells with small injections of CTG and monitored the movements of individual cells in fresh parasagittal brain slices over several hours using time-lapse confocal microscopy. Our observations suggest that tangential cell migration along the RMS occurs more rapidly than radial cell migration into the OB granule cell layer. To investigate migration over longer time periods, we developed an in vitro organotypic slice in which labeled SVZ progenitors migrate along the RMS and differentiate within the OB. The phenotypic characteristics of these cells in vitro were equivalent to those observed in vivo. Taken together, these methods provide useful tools investigating cell migration and differentiation in a preparation that maintains the anatomical organization of the RMS.     

Unique neuronal tracers show migration and differentiation of SVZ progenitors in organotypic slices

Continual neurogenesis in the subventricular zone (SVZ) of postnatal and adult mammalian forebrain has been well documented, but the mechanisms underlying cell migration and differentiation in this region are poorly understood. We have developed novel in vivo and in vitro methods to investigate these processes. Using stereotaxic injections of a variety of tracers/tracker [Cholera Toxin β subunit (CTb‐), Fluorogold (FG), and Cell Tracker Green (CTG)], we could efficiently label SVZ cells. Over several days, labeled cells migrate along the rostral migratory stream (RMS) to their final differentiation site in the olfactory bulb (OB). The compatibility of these tracers/trackers with immunohistochemistry allows for cell labeling with multiple dyes (e.g., CTb and CTG) and/or specific cell antigens. To investigate the dynamics of migration we labeled SVZ progenitor cells with small injections of CTG and monitored the movements of individual cells in fresh parasagittal brain slices over several hours using time‐lapse confocal microscopy. Our observations suggest that tangential cell migration along the RMS occurs more rapidly than radial cell migration into the OB granule cell layer. To investigate migration over longer time periods, we developed an in vitro organotypic slice in which labeled SVZ progenitors migrate along the RMS and differentiate within the OB. The phenotypic characteristics of these cells in vitro were equivalent to those observed in vivo. Taken together, these methods provide useful tools investigating cell migration and differentiation in a preparation that maintains the anatomical organization of the RMS. © 2001 John Wiley & Sons, Inc. J Neurobiol 49: 326–338, 2001     

Spatiotemporal selectivity of response to Notch1 signals in mammalian forebrain precursors

The olfactory bulb, neocortex and archicortex arise from a common pool of progenitors in the dorsal telencephalon. We studied the consequences of supplying excess Notch1 signal in vivo on the cellular and regional destinies of telencephalic precursors using bicistronic replication defective retroviruses. After ventricular injections mid-neurogenesis (E14.5), activated Notch1 retrovirus markedly inhibited the generation of neurons from telencephalic precursors, delayed the emergence of cells from the subventricular zone (SVZ), and produced an augmentation of glial progeny in the neo- and archicortex. However, activated Notch1 had a distinct effect on the progenitors of the olfactory bulb, markedly reducing the numbers of cells of any type that migrated there. To elucidate the mechanism of the cell fate changes elicited by Notch1 signals in the cortical regions, short- and long-term cultures of E14.5 telencephalic progenitors were examined. These studies reveal that activated Notch1 elicits a cessation of proliferation that coincides with an inhibition of the generation of neurons. Later, during gliogenesis, activated Notch1 triggers a rapid cellular proliferation with a significant increase in the generation of cells expressing GFAP. To examine the generation of cells destined for the olfactory bulb, we used stereotaxic injections into the early postnatal anterior subventricular zone (SVZa). We observed that precursors of the olfactory bulb responded to Notch signals by remaining quiescent and failing to give rise to differentiated progeny of any type, unlike cortical precursor cells, which generated glia instead of neurons. These data show that forebrain precursors vary in their response to Notch signals according to spatial and temporal cues, and that Notch signals influence the composition of forebrain regions by modulating the rate of proliferation of neural precursor cells.     

Glial progenitors of the neonatal subventricular zone differentiate asynchronously, leading to spatial dispersion of glial clones and to the persistence of immature glia in the adult mammalian CNS

The subventricular zone (SVZ) of the developing mammalian forebrain gives rise to astrocytes and oligodendrocytes in the neocortex and white matter, and neurons in the olfactory bulb in perinatal life. We have examined the developmental fates and spatial distributions of the descendants of single SVZ cells by infecting them in vivo at postnatal day 0-1 (P0-1) with a retroviral "library". In most cases, individual SVZ cells gave rise to either oligodendrocytes or astrocytes, but some generated both types of glia. Members of glial clones can disperse widely through the gray and white matter. Progenitors continued to divide after stopping migration, generating clusters of related cells. However, the progeny of a single SVZ cell does not differentiate synchronously: individual clones contained both mature and less mature glia after short or long intervals. For example, progenitors that settled in the white matter generated three types of clonal oligodendrocyte clusters: those composed of only myelinating oligodendrocytes, of both myelinating oligodendrocytes and non-myelinating oligodendrocytes, or of only non-myelinating cells of the oligodendrocyte lineage. Thus, some progenitors do not fully differentiate, but remain immature and may continue to cycle well into adult life.     

The migratory behavior of immature enteric neurons

While they are migrating caudally along the developing gut, around 10%-20% of enteric neural crest-derived cells start to express pan-neuronal markers and tyrosine hydroxylase (TH). We used explants of gut from embryonic TH-green fluorescence protein (GFP) mice and time-lapse microscopy to examine whether these immature enteric neurons migrate and their mode of migration. In the gut of E10.5 and E11.5 TH-GFP mice, around 50% of immature enteric neurons (GFP(+) cells) migrated, with an average speed of around 15 mum/h. This is slower than the speed at which the population of enteric neural crest-derived cells advances along the developing gut, and hence neuronal differentiation seems to slow, but not necessarily halt, the caudal migration of enteric neural crest cells. Most migrating immature enteric neurons migrated caudally by extending a long-leading process followed by translocation of the cell body. This mode of migration is different from that of non-neuronal enteric neural crest-derived cells and neural crest cells in other locations, but resembles that of migrating neurons in many regions of the developing central nervous system (CNS). In migrating immature enteric neurons, a swelling often preceded the movement of the nucleus in the direction of the leading process. However, the centrosomal marker, pericentrin, was not localized to either the leading process or swelling. This seems to be the first detailed report of neuronal migration in the developing mammalian peripheral nervous system.     

Generation and migration of cells in the developing striatum.

The development of the rat striatum was investigated using a combination of two histochemically distinguishable retrovirus vectors. Using this method, it was possible to identify clonal boundaries within the embryonic striatum and thus determine patterns of proliferation, migration, and some lineal relationships. Several novel aspects of striatal histogenesis were discovered. Striatal progenitor cells do not exhibit a stem cell pattern of division between embryonic day 15 (E15) and E19; a progenitor-progeny relationship appears to exist for ventricular zone and subventricular zone (SVZ) cells; striatal progenitors produce a variety of clone types; some SVZ cells migrate radially, and some migrate tangentially within the SVZ; and radial glia and presumptive neurons can occur in the same clone.     

Auto-reverse nuclear migration in bipolar mammalian cells on micropatterned surfaces

A novel assay based on micropatterning and time-lapse microscopy has been developed for the study of nuclear migration dynamics in cultured mammalian cells. When cultured on 10-20-microm wide adhesive stripes, the motility of C6 glioma and primary mouse fibroblast cells is diminished. Nevertheless, nuclei perform an unexpected auto-reverse motion: when a migrating nucleus approaches the leading edge, it decelerates, changes the direction of motion, and accelerates to move toward the other end of the elongated cell. During this process, cells show signs of polarization closely following the direction of nuclear movement. The observed nuclear movement requires a functioning microtubular system, as revealed by experiments disrupting the main cytoskeletal components with specific drugs. On the basis of our results, we argue that auto-reverse nuclear migration is due to forces determined by the interplay of microtubule dynamics and the changing position of the microtubule organizing center as the nucleus reaches the leading edge. Our assay recapitulates specific features of nuclear migration (cell polarization, oscillatory nuclear movement), while it allows the systematic study of a large number of individual cells. In particular, our experiments yielded the first direct evidence of reversive nuclear motion in mammalian cells, induced by attachment constraints.     

Nucleokinesis in neuronal migration

Neuronal migration is a critical phase of nervous system development and can be divided into two distinct phases: extension of the leading process and movement of the cell body and nucleus (nucleokinesis). Nucleokinesis appears to require many of the same cytoskeletal and signaling molecules used in cell mitosis. Converging studies suggest it requires cytoplasmic dynein, cell polarity genes, and microtubule-associated proteins that coordinate microtubule remodeling. These coordinate first the positioning of the centrosome (microtubule organizing center) in the leading process in front of the nucleus and then the movement of the nucleus towards the centrosome. The positioning of the centrosome and the dynamic regulation that couples and uncouples the nucleus underlies directed migration of neurons.     

Intranasal HB-EGF administration favors adult SVZ cell mobilization to demyelinated lesions in mouse corpus callosum

In the adult rodent brain, the subventricular zone (SVZ) represents a special niche for neural stem cells; these cells proliferate and generate neural progenitors. Most of these migrate along the rostral migratory stream to the olfactory bulb, where they differentiate into interneurons. SVZ-derived progenitors can also be recruited spontaneously to damaged brain areas to replace lost cells, including oligodendrocytes in demyelinated lesions. In this study, we searched for factors able to enhance this spontaneous recruitment of endogenous progenitors. Previous studies have suggested that epidermal growth factor (EGF) could stimulate proliferation, migration, and glial differentiation of SVZ progenitors. In the present study we examined EGF influence on endogenous SVZ cell participation to brain repair in the context of demyelinated lesions. We induced a focal demyelinated lesion in the corpus callosum by lysolecithin injection and showed that intranasal heparin-binding epidermal growth factor (HB-EGF) administration induces a significant increase in SVZ cell proliferation together with a stronger SVZ cell mobilization toward the lesions. Besides, HB-EGF causes a shift of SVZ-derived progenitor cell differentiation toward the astrocytic lineage. However, due to the threefold increase in cell recruitment by EGF treatment, the absolute number of SVZ-derived oligodendrocytes in the lesion of treated mice is higher than in controls. These results suggest that enhancing SVZ cell proliferation could be part of future strategies to promote SVZ progenitor cell mobilization toward brain lesions.     

Postnatal deletion of Numb/Numblike reveals repair and remodeling capacity in the subventricular neurogenic niche

Neural stem cells are retained in the postnatal subventricular zone (SVZ), a specialized neurogenic niche with unique cytoarchitecture and cell-cell contacts. Although the SVZ stem cells continuously regenerate, how they and the niche respond to local changes is unclear. Here we generated nestin-creER(tm) transgenic mice with inducible Cre recombinase in the SVZ and removed Numb/Numblike, key regulators of embryonic neurogenesis from postnatal SVZ progenitors and ependymal cells. This resulted in severe damage to brain lateral ventricle integrity and identified roles for Numb/Numblike in regulating ependymal wall integrity and SVZ neuroblast survival. Surprisingly, the ventricular damage was eventually repaired: SVZ reconstitution and ventricular wall remodeling were mediated by progenitors that escaped Numb deletion. Our results show a self-repair mechanism in the mammalian brain and may have implications for both niche plasticity in other areas of stem cell biology and the therapeutic use of neural stem cells in neurodegenerative diseases.     

In vivo fate analysis reveals the multipotent and self-renewal features of embryonic AspM expressing cells

Radial Glia (RG) cells constitute the major population of neural progenitors of the mouse developing brain. These cells are located in the ventricular zone (VZ) of the cerebral cortex and during neurogenesis they support the generation of cortical neurons. Later on, during brain maturation, RG cells give raise to glial cells and supply the adult mouse brain of Neural Stem Cells (NSC). Here we used a novel transgenic mouse line expressing the CreER(T2) under the control of AspM promoter to monitor the progeny of an early cohort of RG cells during neurogenesis and in the post natal brain. Long term fate mapping experiments demonstrated that AspM-expressing RG cells are multi-potent, as they can generate neurons, astrocytes and oligodendrocytes of the adult mouse brain. Furthermore, AspM descendants give also rise to proliferating progenitors in germinal niches of both developing and post natal brains. In the latter--i.e. the Sub Ventricular Zone--AspM descendants acquired several feature of neural stem cells, including the capability to generate neurospheres in vitro. We also performed the selective killing of these early progenitors by using a Nestin-GFP(flox)-TK allele. The forebrain specific loss of early AspM expressing cells caused the elimination of most of the proliferating cells of brain, a severe derangement of the ventricular zone architecture, and the impairment of the cortical lamination. We further demonstrated that AspM is expressed by proliferating cells of the adult mouse SVZ that can generate neuroblasts fated to become olfactory bulb neurons.     

Retinoic acid regulates postnatal neurogenesis in the murine subventricular zone-olfactory bulb pathway

Neurogenesis persists throughout life in the rodent subventricular zone (SVZ)-olfactory bulb pathway. The molecular regulation of this neurogenic circuit is poorly understood. Because the components for retinoid signaling are present in this pathway, we examined the influence of retinoic acid (RA) on postnatal SVZ-olfactory bulb neurogenesis. Using both SVZ neurosphere stem cell and parasagittal brain slice cultures derived from postnatal mouse, we found that RA exposure increased neurogenesis by enhancing the proliferation and neuronal differentiation of forebrain SVZ neuroblasts. The RA precursor retinol had a similar effect, which was reversed by treating cultures with the RA synthesis inhibitor disulfiram. Electroporation of dominant-negative retinoid receptors into the SVZ of slice cultures also blocked neuroblast migration to the olfactory bulb and altered the morphology of the progenitors. Moreover, the administration of disulfiram to neonatal mice decreased in vivo cell proliferation in the striatal SVZ. These results indicate that RA is a potent mitogen for SVZ neuroblasts and is required for their migration to the olfactory bulb. The regulation of multiple steps in the SVZ-olfactory bulb neurogenic pathway by RA suggests that manipulation of retinoid signaling is a potential therapeutic strategy to augment neurogenesis after brain injury.     

Myosin IIA Dependent Retrograde Flow Drives 3D Cell Migration

Epithelial cell migration is an essential part of embryogenesis and tissue regeneration, yet their migration is least understood. Using our three-dimensional (3D) motility analysis, migrating epithelial cells formed an atypical polarized cell shape with the nucleus leading the cell front and a contractile cell rear. Migrating epithelial cells exerted traction forces to deform both the anterior and posterior extracellular matrix toward the cell body. The cell leading edge exhibited a myosin II-dependent retrograde flow with the magnitude and direction consistent with surrounding network deformation. Interestingly, on a two-dimensional substrate, myosin IIA-deficient cells migrated faster than wild-type cells, but in a 3D gel, these myosin IIA-deficient cells were unpolarized and immobile. In contrast, the migration rates of myosin IIB-deficient cells were similar to wild-type cells. Therefore, myosin IIA, not myosin IIB, is required for 3D epithelial cell migration.     

Four distinct phases of basket/stellate cell migration after entering their final destination (the molecular layer) in the developing cerebellum

In the adult cerebellum, basket/stellate cells are scattered throughout the ML, but little is known about the process underlying the cell dispersion. To determine the allocation of stellate/basket cells within the ML, we examined their migration in the early postnatal mouse cerebellum. We found that after entering the ML, basket/stellate cells sequentially exhibit four distinct phases of migration. First, the cells migrated radially from the bottom to the top while exhibiting saltatory movement with a single leading process (Phase I). Second, the cells turned at the top and migrated tangentially in a rostro-caudal direction, with an occasional reversal of the direction of migration (Phase II). Third, the cells turned and migrated radially within the ML at a significantly reduced speed while repeatedly extending and withdrawing the leading processes (Phase III). Fourth, the cells turned at the middle and migrated tangentially at their slowest speed, while extending several dendrite-like processes after having completely withdrawn the leading process (Phase IV). Finally, the cells stopped and completed their migration. These results suggest that the dispersion of basket/stellate cells in the ML is controlled by the orchestrated activity of external guidance cues, cell-cell contact and intrinsic programs in a position- and time-dependent manner.     

Dynamics of cell movement during the wound repair of human surface respiratory epithelium.

Epithelial wound repair represents an important process by which the epithelial barrier integrity recovers after wounding. To evaluate and quantify the dynamics of surface airway cell movement during the wound repair process, we developed an in vitro wounding model of human respiratory cells in culture and we analyzed the wound repair by using videomicroscopic and image analysis techniques. We observed that wound closure occurred within 6 hours, due to the spreading and migration of the cells surrounding the wounded surface. The migration rate of the cells at the leading edge of the wound surface increased progressively up to 26 microns/h during the repair process which was characterized by a uniform centripetal direction of cell movement. The distance travelled by these cells was 2.5 fold longer than the distance travelled by ciliated cells which were located far from the wound area. These results suggest that cell migration after wounding is an important process by which the respiratory epithelial barrier integrity is maintained.     

EGF-Induced Expansion of Migratory Cells in the Rostral Migratory Stream

The presence of neural stem cells in the adult brain is currently widely accepted and efforts are made to harness the regenerative potential of these cells. The dentate gyrus of the hippocampal formation, and the subventricular zone (SVZ) of the anterior lateral ventricles, are considered the main loci of adult neurogenesis. The rostral migratory stream (RMS) is the structure funneling SVZ progenitor cells through the forebrain to their final destination in the olfactory bulb. Moreover, extensive proliferation occurs in the RMS. Some evidence suggest the presence of stem cells in the RMS, but these cells are few and possibly of limited differentiation potential. We have recently demonstrated the specific expression of the cytoskeleton linker protein radixin in neuroblasts in the RMS and in oligodendrocyte progenitors throughout the brain. These cell populations are greatly altered after intracerebroventricular infusion of epidermal growth factor (EGF). In the current study we investigate the effect of EGF infusion on the rat RMS. We describe a specific increase of radixin⁺/Olig2⁺ cells in the RMS. Negative for NG2 and CNPase, these radixin⁺/Olig2⁺ cells are distinct from typical oligodendrocyte progenitors. The expanded Olig2⁺ population responds rapidly to EGF and proliferates after only 24 hours along the entire RMS, suggesting local activation by EGF throughout the RMS rather than migration from the SVZ. In addition, the radixin⁺/Olig2⁺ progenitors assemble in chains in vivo and migrate in chains in explant cultures, suggesting that they possess migratory properties within the RMS. In summary, these results provide insight into the adaptive capacity of the RMS and point to an additional stem cell source for future brain repair strategies.     

Regulation of interkinetic nuclear migration by cell cycle-coupled active and passive mechanisms in the developing brain

A hallmark of neurogenesis in the vertebrate brain is the apical-basal nuclear oscillation in polarized neural progenitor cells. Known as interkinetic nuclear migration (INM), these movements are synchronized with the cell cycle such that nuclei move basally during G1-phase and apically during G2-phase. However, it is unknown how the direction of movement and the cell cycle are tightly coupled. Here, we show that INM proceeds through the cell cycle-dependent linkage of cell-autonomous and non-autonomous mechanisms. During S to G2 progression, the microtubule-associated protein Tpx2 redistributes from the nucleus to the apical process, and promotes nuclear migration during G2-phase by altering microtubule organization. Thus, Tpx2 links cell-cycle progression and autonomous apical nuclear migration. In contrast, in vivo observations of implanted microbeads, acute S-phase arrest of surrounding cells and computational modelling suggest that the basal migration of G1-phase nuclei depends on a displacement effect by G2-phase nuclei migrating apically. Our model for INM explains how the dynamics of neural progenitors harmonize their extensive proliferation with the epithelial architecture in the developing brain.