Amphiregulin-EGFR Signaling Mediates the Migration of Bone Marrow Mesenchymal Progenitors toward PTH-Stimulated Osteoblasts and Osteocytes

Intermittent administration of parathyroid hormone (PTH) dramatically increases bone mass and currently is one of the most effective treatments for osteoporosis. However, the detailed mechanisms are still largely unknown. Here we demonstrate that conditioned media from PTH-treated osteoblastic and osteocytic cells contain soluble chemotactic factors for bone marrow mesenchymal progenitors, which express a low amount of PTH receptor (PTH1R) and do not respond to PTH stimulation by increasing cAMP production or migrating toward PTH alone. Conditioned media from PTH-treated osteoblasts elevated phosphorylated Akt and p38MAPK amounts in mesenchymal progenitors and inhibition of these pathways blocked the migration of these progenitors toward conditioned media. Our previous and current studies revealed that PTH stimulates the expression of amphiregulin, an epidermal growth factor (EGF)-like ligand that signals through the EGF receptor (EGFR), in both osteoblasts and osteocytes. Interestingly, conditioned media from PTH-treated osteoblasts increased EGFR phosphorylation in mesenchymal progenitors. Using several different approaches, including inhibitor, neutralizing antibody, and siRNA, we demonstrate that PTH increases the release of amphiregulin from osteoblastic cells, which acts on the EGFRs expressed on mesenchymal progenitors to stimulate the Akt and p38MAPK pathways and subsequently promote their migration in vitro. Furthermore, inactivation of EGFR signaling specifically in osteoprogenitors/osteoblasts attenuated the anabolic actions of PTH on bone formation. Taken together, these results suggest a novel mechanism for the therapeutic effect of PTH on osteoporosis and an important role of EGFR signaling in mediating PTH''s anabolic actions on bone.     

Early specification of GAD67 subventricular derived olfactory interneurons

Olfactory bulb interneurons are continuously generated in the subventricular zone (SVZ) and migrate along the rostral migratory stream (RMS) into the olfactory bulb (OB) where the majority becomes local GABAergic interneurons. We previously showed that SVZ-derived progenitor cells expressed glutamic acid decarboxylase 65 kDa (GAD65) very early in the migratory pathway. However, only approximately half of OB GABAergic interneurons use GAD65, an equal number express the 67 kDa GAD enzyme. To investigate the differentiation of these GABAergic interneurons we examined their migration in a transgenic mouse expressing green fluorescent protein (GFP) under the control of the GAD67 promoter. In adult, GFP was expressed by a subpopulation of migratory cells in the SVZ and along the RMS. Using Doublecortin (DCX) as a marker of migrating neuroblasts and bromodeoxyuridine (BrdU) incorporation, we show that these GAD67-GFP neurons co-express DCX and incorporate BrdU indicating they are newly born migratory neuroblasts. This is similar to GAD65 transgene expression, and in contrast to dopaminergic interneuron transgene expression which occurs only after cells reach the olfactory bulb. Although the GAD65/67 transgenes are expressed early in migration, there is minimal protein production in the cells prior to reaching the OB. These results suggest that migrating SVZ-derived neuroblasts acquire GABAergic identity prior to reaching their final location in the olfactory bulb.     

Neurogenesis and neuronal migration in the neonatal rat forebrain anterior subventricular zone do not require GFAP-positive astrocytes

A prolific neuronal progenitor cell population in the anterior portion of the neonatal rat forebrain subventricular zone, the SVZa, is specialized for the production of olfactory bulb interneurons. At all ages, SVZa-derived cells traverse a tangential migratory pathway, the rostral migratory stream (RMS), while en route to the olfactory bulb. Unlike other neuronal progenitor cells of the forebrain, migrating progeny of SVZa progenitors express neuronal-specific proteins and continue to divide into adulthood. Recent studies indicate that in the adult, migrating SVZa-derived cells are ensheathed by astrocytes, although the function of these astrocytes has not been determined. To explore the possible role(s) of astrocytes in the rat SVZa and RMS, we examined the expression of astroglial-specific genes in the postnatal SVZa and RMS using RT-PCR, in situ hybridization, and immunohistochemistry during (Postnatal Days 1-10) and after the period of peak olfactory bulb interneuron generation. We also examined the expression of neuronal-specific genes throughout the rostral-caudal extent of the postnatal subventricular zone to determine if differential cell type-specific gene expression could distinguish the neurogenic SVZa as a region distinct from the remainder of the SVZ. We found little to no astrocyte-specific gene expression in the P0-P7 SVZa, although the neuron-specific isoforms of tubulin (T alpha 1 and beta-III tubulin) were expressed abundantly in the SVZa and RMS. In contrast, astrocyte-specific genes were strongly expressed in the SVZ posterior to the SVZa. GFAP expressions begins to appear in some restricted areas of the rostral migratory stream after the first postnatal week. These data suggest that astroglia are not involved in the generation or migration of most olfactory bulb interneurons. Moreover, the scarcity of glial markers in the neonatal SVZa indicates that the forebrain subventricular zone includes a distinct neurogenic anterior region containing predominantly committed neuronal progenitor cells.     

ADAM17 Is Critical for Multipolar Exit and Radial Migration of Neuronal Intermediate Progenitor Cells in Mice Cerebral Cortex

The radial migration of neuronal progenitor cells is critical for the development of cerebral cortex layers. They go through a critical step transforming from multipolar to bipolar before outward migration. A Disintegrin and Metalloprotease 17 (ADAM17) is a transmembrane protease which can process many substrates involved in cell-cell interaction, including Notch, ligands of EGFR, and some cell adhesion molecules. In this study, we used in utero electroporation to knock down or overexpress ADAM17 at embryonic day 14.5 (E14.5) in neuronal progenitor cells to examine the role of ADAM17 in cortical embryonic neurogenesis. Our results showed that the radial migration of ADAM17-knocked down cells were normal till E16.5 and reached the intermediate zone (IZ). Then most transfected cells stopped migration and stayed at the IZ to inner cortical plate (CP) layer at E18.5, and there was higher percentage of multipolar cells at IZ layer in the ADAM17-knocked down group compared to the cells in control group. Marker staining revealed that those ADAM17-knocked down cells differentiated normally from neural stem cells (NSCs) to neuronal intermediate progenitor cells (nIPCs) but did not differentiate into mature neurons. The migration and multipolar exit defects caused by ADAM17 knockdown could be partially rescued by over-expressing an shRNA resistant ADAM17, while overexpressing ADAM17 alone did not affect the radial migration. Taken together, our results showed for the first time that, ADAM17 is critical in regulating the multipolar-stage exit and radial migration of the nIPCs during telencephalon cortex development in mice.     

Continual expression of Rab5(Q79L) causes a ligand-independent EGFR internalization and diminishes EGFR activity

The amount of cell-surface Epidermal Growth Factor Receptor (EGFR) available to secreted ligand (EGF) dictates a cell's ability to mediate cell proliferation, differentiation or migration. Multiple factors regulate EGFR cell-surface expression including the rates of protein synthesis and protein degradation, and the endocytic trafficking of both stimulated and unstimulated EGFR. Rab5 is a 25 kDa protein that is localized to the plasma membrane and the early endosome. Its exact molecular function, however, remains controversial. We have used stable and transient expression systems in HeLa cells to examine the consequence of continual, overexpression of wild-type and activated mutants of rab5 on EGFR localization and signaling. Continual expression of constitutively activated mutants of rab5 causes a ligand-independent redistribution of EGFRs into intracellular vesicles that can not be blocked with an antagonistic antibody. The net result is a decrease in the level of cell-surface EGFRs available for ligand stimulation. Thus, rab5 activation regulates EGFR signaling by facilitating the internalization of the unliganded EGFR.     

Akt1 interacts with epidermal growth factor receptors and hedgehog signaling to increase stem/transit amplifying cells in the embryonic mouse cortex

A subset of precursors in the embryonic mouse cortex and in neurospheres expresses a higher level of the serine/threonine kinase Akt1 than neighboring precursors. We reported previously that the functional significance of high Akt1 expression was enhanced Akt1 activity, resulting in an increase in survival, proliferation, and self-renewal of multipotent stem/transit amplifying cells. Akt1 can interact with a number of signaling pathways, but the extrinsic factors that are required for specific effects of elevated Akt1 expression have not been identified. In this study we addressed the contributions of signaling via epidermal growth factor (EGF) and hedgehog (Hh) receptors. In EGF receptor-null precursors or following transient inhibition of EGF receptor tyrosine kinase activity, elevating Akt1 by retroviral transduction could still increase survival and proliferation but could not increase self-renewal. We also found that elevated Akt1 expression induced the expression of EGF receptors (EGFRs) in wild-type precursors. Several extrinsic factors, including Shh, can induce EGFR expression by cortical precursors, and we found that elevating Akt1 allowed them to respond to a subthreshold concentration of Shh to induce EGFRs. In precursors that lack the Hh receptor smoothened, however, elevating Akt1 did not increase EGFR expression or self-renewal, though it could still stimulate proliferation. These findings suggest that a subset of precursors in the embryonic cortex that express an elevated level of Akt1 can respond to lower concentrations of Shh than neighboring precursors, resulting in an increase in their expression of EGFRs. Signaling via EGFRs is required for their self-renewal.     

Populations of radial glial cells respond differently to reelin and neuregulin1 in a ferret model of cortical dysplasia

Radial glial cells play an essential role during corticogenesis through their function as neural precursors and guides of neuronal migration. Both reelin and neuregulin1 (NRG1) maintain the radial glial scaffold; they also induce expression of Brain Lipid Binding Protein (BLBP), a well known marker of radial glia. Although radial glia in normal ferrets express both vimentin and BLBP, this coexpression diverges at P3; vimentin is expressed in the radial glial processes, while BLBP appears in cells detached from the ventricular zone. Our lab developed a model of cortical dysplasia in the ferret, resulting in impaired migration of neurons into the cortical plate and disordered radial glia. This occurs after exposure to the antimitotic methylazoxymethanol (MAM) on the 24th day of development (E24). Ferrets treated with MAM on E24 result in an overall decrease of BLBP expression; radial glia that continue to express BLBP, however, show only mild disruption compared with the strongly disrupted vimentin expressing radial glia. When E24 MAM-treated organotypic slices are exposed to reelin or NRG1, the severely disrupted vimentin+ radial glial processes are repaired but the slightly disordered BLBP+ processes are not. The realignment of vimentin+ processes was linked with an increase of their BLBP expression. BLBP expressing radial glia are distinguished by being both less affected by MAM treatment and by attempts at repair. We further investigated the effects induced by reelin and found that signaling was mediated via VLDLR/Dab1/Pi3K activation while NRG1 signaling was mediated via erbB3/erbB4/Pi3K. We then tested whether radial glial repair correlated with improved neuronal migration. Repairing the radial glial scaffold is not sufficient to restore neuronal migration; although reelin improves migration of neurons toward the cortical plate signaling through ApoER2/Dab1/PI3K activation, NRG1 does not.     

Basolateral EGF Receptor Sorting Regulated by Functionally Distinct Mechanisms in Renal Epithelial Cells

Proliferation of epithelial tissues is controlled by polarized distribution of signaling receptors including the EGF receptor (EGFR). In kidney, EGFRs are segregated from soluble ligands present in apical fluid of nephrons by selective targeting to basolateral membranes. We have shown previously that the epithelial‐specific clathrin adaptor AP1B mediates basolateral EGFR sorting in established epithelia. Here we show that protein kinase C (PKC)‐dependent phosphorylation of Thr654 regulates EGFR polarity as epithelial cells form new cell–cell junctional complexes. The AP1B‐dependent pathway does not override a PKC‐resistant T654A mutation, and conversely AP1B‐defective EGFRs sort basolaterally by a PKC‐dependent mechanism, in polarizing cells. Surprisingly, EGFR mutations that interfere with these different sorting pathways also produce very distinct phenotypes in three‐dimensional organotypic cultures. Thus EGFRs execute different functions depending on the basolateral sorting route. Many renal disorders have defects in cell polarity and the notion that apically mislocalized EGFRs promote proliferation is still an attractive model to explain many aspects of polycystic kidney disease. Our data suggest EGFR also integrates various aspects of polarity by switching between different basolateral sorting programs in developing epithelial cells. Fundamental knowledge of basic mechanisms governing EGFR sorting therefore provides new insights into pathogenesis and advances drug discovery for these renal disorders.     

Arachidonic acid promotes migration and invasion through a PI3K/Akt-dependent pathway in MDA-MB-231 breast cancer cells

Arachidonic acid (AA) is a common dietary n−6 cis polyunsaturated fatty acid that under physiological conditions is present in an esterified form in cell membrane phospholipids, however it might be present in the extracellular microenvironment. AA and its metabolites mediate FAK activation, adhesion and migration in MDA-MB-231 breast cancer cells. However, it remains to be investigated whether AA promotes invasion and the signal transduction pathways involved in migration and invasion. Here, we demonstrate that AA induces Akt2 activation and invasion in MDA-MB-231 cells. Akt2 activation requires the activity of Src, EGFR, and PIK3, whereas migration and invasion require Akt, PI3K, EGFR and metalloproteinases activity. Moreover, AA also induces NFκB-DNA binding activity through a PI3K and Akt-dependent pathway. Our findings demonstrate, for the first time, that Akt/PI3K and EGFR pathways mediate migration and invasion induced by AA in MDA-MB-231 breast cancer cells.     

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.     

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.     

Rate and pattern of migration of lineally-related olfactory bulb interneurons generated postnatally in the subventricular zone of the rat

A spatially discrete region of the anterior part of the postnataltelescephalic subventricular zone, referred to as the SVZa generatesvast numbers of lineally-related neurons destined for the olfactorybulb (Luskin, 1993). The cells originating in the SVZa migrateto the olfactory bulb along a highly restricted pathway whichis in a direction orthogonal to the orientation of radial glialfibers. In this study we analysed the number, distribution,orientation and rate of migration of SVZa-derived cells as theyapproach the olfactory bulb. In order to track the SVZa-derivedcells, a retroviral lineage tracer, encoding the reporter geneE.coli ß-galactosidase (lacZ) was injected preciselyinto the rat SVZa at postnatal day 1 (Pl). The lacZ-positivecells were visualized 1, 2 and 3 days later by X-Gal histochemistryin cryostat sections. As the number of SVZa-derived cells inthe pathway increased with survival time, their distributionchanged systematically. The distribution pattern of lacZ-positivecells by 2 and 3 days postinjection suggested that some of theprogeny of infected progenitor cells were undergoing neurogenesisas they proceeded to the olfactory bulb; a large percentageof the lacZ-positive cells were substantially displaced fromthe SVZa injection site. To investigate whether lacZ-positivecells migrate in a directed fashion, their orientation preferencewas scored. For the majority of lacZ-positive cells (>94%),their leading process was directed toward the olfactory bulb,possibly reflecting a response to migratory cues present alongthe pathway. The estimated average rate of cell migration tothe olfactory bulb was 23 µm/h, which is approximatelytwice the speed of radially directed neuronal migration fromthe telencephalic ventricular zone to the cortical plate (O'Rourkeet al, 1992). Collectively, these results suggest that SVZa-derivedintemeurons en route to the olfactory bulb may employ a novelmode of tangential migration.     

Epidermal growth factor receptor-dependent and -independent cell-signaling pathways originating from the urokinase receptor

Urokinase-type plasminogen activator (uPA) and vitronectin activate cell-signaling pathways by binding to the uPA receptor (uPAR). Because uPAR is glycosylphosphatidylinositol-anchored, the signaling receptor is most likely a uPAR-containing multiprotein complex. This complex may be heterogeneous within a single cell and among different cell types. The goal of this study was to elucidate the role of the EGF receptor (EGFR) as a component of the uPAR-signaling machinery. uPA activated extracellular signal-regulated kinase (ERK) in COS-7 cells and in COS-7 cells that overexpress uPAR, and this response was blocked by the EGFR inhibitor, tyrphostin AG1478, implicating the EGFR in the pathway that links uPAR to ERK. By contrast, Rac1 activation, which occurred as a result of uPAR overexpression, was EGFR-independent. COS-7 cell migration was stimulated, in an additive manner, by uPAR-dependent pathways leading to ERK and Rac1. AG1478 inhibited only the ERK-dependent component of the response. CHO-K1 cells do not express EGFR; however, these cells demonstrated ERK activation in response to uPA, indicating the presence of an EGFR-independent alternative pathway. As anticipated, this response was insensitive to AG1478. When CHO-K1 cells were transfected to express EGFR or a kinase-inactive mutant of EGFR, ERK activation in response to uPA was unchanged; however, the EGFR-expressing cells acquired sensitivity to AG1478. We conclude that the EGFR may function as a transducer of the signal from uPAR to ERK, but not Rac1. In the absence of EGFR, an alternative pathway links uPAR to ERK; however, this pathway is apparently silenced by EGFR expression.     

MAPK-upstream protein kinase (MUK) regulates the radial migration of immature neurons in telencephalon of mouse embryo

The radial migration of differentiating neurons provides an essential step in the generation of laminated neocortex, although its molecular mechanism is not fully understood. We show that the protein levels of a JNK activator kinase, MUK/DLK/ZPK, and JNK activity increase potently and temporally in newly generated neurons in developing mouse telencephalon during radial migration. The ectopic expression of MUK/DLK/ZPK in neural precursor cells in utero impairs radial migration, whereas it allows these cells to leave the ventricular zone and differentiate into neural cells. The MUK/DLK/ZPK protein is associated with dotted structures that are frequently located along microtubules and with Golgi apparatus in cultured embryonic cortical cells. In COS-1 cells, MUK/DLK/ZPK overexpression impairs the radial organization of microtubules without massive depolymerization. These results suggest that MUK/DLK/ZPK and JNK regulate radial cell migration via microtubule-based events.     

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     

Activation of the Ras/Mitogen-Activated Protein Kinase Pathway by Kinase-Defective Epidermal Growth Factor Receptors Results in Cell Survival but Not Proliferation

Signalling by the epidermal growth factor (EGF) receptor (EGFR) has been studied intensively, but for most cell types the analysis is complicated by the fact that EGFR not only homodimerizes but can also form heterodimers with other EGFR family members. Heterodimerization is a particular problem in the study of EGFR mutants, where the true phenotype of the mutants is confounded by the contribution of the heterodimer partner to signal transduction. We have made use of the murine hemopoietic cell line BaF/3, which does not express EGFR family members, to express wild-type (WT) EGFR, three kinase-defective EGFR mutants (V741G, Y740F, and K721R), or a C-terminally truncated EGFR (CT957) and have measured their responses to EGF. We found that under the appropriate conditions EGF can stimulate cell proliferation of BaF/3 cells expressing WT or CT957 EGFRs but not that of cells expressing the kinase-defective mutants. However, EGF promotes the survival of BaF/3 cells expressing either of the kinase-defective receptors (V741G and Y740F), indicating that these receptors can still transmit a survival signal. Analysis of the early signalling events by the WT, V741G, and Y740F mutant EGF receptors indicated that EGF stimulates comparable levels of Shc phosphorylation, Shc–GRB-2 association, and activation of Ras, B-Raf, and Erk-1. Blocking the mitogen-activated protein kinase (MAPK) signalling pathway with the specific inhibitor PD98059 abrogates completely the EGF-dependent survival of cells expressing the kinase-defective EGFR mutants but has no effect on the EGF-dependent proliferation mediated by WT and CT957 EGFRs. Similarly, the Src family kinase inhibitor PP1 abrogates EGF-dependent survival without affecting proliferation. However blocking phosphatidylinositol-3-kinase or JAK-2 kinase with specific inhibitors does arrest growth factor-dependent cell proliferation. Thus, EGFR-mediated mitogenic signalling in BaF/3 cells requires an intact EGFR tyrosine kinase activity and appears to depend on the activation of both the JAK-2 and PI-3 kinase pathways. Activation of the Src family of kinases or of the Ras/MAPK pathway can, however, be initiated by a kinase-impaired EGFR and is linked to survival.     

Initial stages of radial glia astrocytic transformation in the early postnatal anterior subventricular zone

In the early postnatal subventricular zone (SVZ), two seemingly unrelated events occur simultaneously: a massive tangential migration of neuroblasts towards the olfactory bulb, known as the rostral migratory stream (RMS), and the outward movement of radial glia (RG) undergoing astrocytic transformation. Because of the orthogonal arrangement between these two sets of cells, little, if any, relevance has been ascribed for their possible interactions. By depositing DiI at the pial surface we have studied RG transformation within the SVZ/RMS, from birth up to the end of the first postnatal week. While still within the SVZ/RMS, RG morphology changed from simple bipolar to highly complex branched profiles, attaining their highest degree of complexity at the interface of the SVZ with the overlying white matter. At this interface cell bodies of radial glia accumulate and their processes run tangentially, surrounding the SVZ/RMS. Processes of RG surrounding the SVZ/RMS could also be observed by immunostaining for vimentin, GFAP, and nestin. In contrast, in the white matter all DiI-labeled RG presented a simple bipolar profile. These results indicate that the outward radial migration of the transforming RG does not occur uniformly. Instead, the different morphologies and cell densities that RG assume when they cross the SVZ/RMS and overlying white matter imply different migratory behaviors. Finally, our data suggest that RG provide a cellular scaffold to the early postnatal SVZ/RMS, much in the same way as astrocytes in the adult RMS.