α6β1 integrin directs migration of neuronal precursors in adult mouse forebrain

Available online at www.sciencedirect.com R

Experimental Neurology 183 (2003) 273–285

www.elsevier.com/locate/yexnr

␣6␤1 integrin directs migration of neuronal precursors in adult mouse forebrain J.G. Emsleya,1 and T. Hagga,b,* b

a Department of Anatomy and Neurobiology, Dalhousie University, Halifax, Nova Scotia, Canada Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, Louisville, KY 40292, USA

Received 7 June 2002; revised 18 November 2002; accepted 31 March 2003

Abstract 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 ␣6␤1 integrin, and its ligand, laminin, in controlling guidance of the migrating neuroblasts in adult mice. Immunostaining for the ␣6␤1 integrin was present in neuroblasts and their processes in the anterior/rostral SVZ and the RMS. Inhibition of the endogenous ␣6 or ␤1 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 ␣ chains that bind ␣6␤1 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. © 2003 Elsevier Science (USA). All rights reserved. Keywords: Chemoattractive; Extracellular matrix; Guidance molecules; Laminin; Neostriatum; Neuroblast; Rostral migratory stream; Subventricular zone

Introduction The adult SVZ contains neural precursors whose progeny predominantly become neuroblasts, which migrate within the RMS towards the olfactory bulb, where they become neurons (Reynolds and Weiss, 1992; Richards et al., 1992; Luskin, 1993; Lois and Alvarez-Buylla, 1993, 1994; Morshead et al., 1994; Doetsch and Alvarez-Buylla, 1996; Lois et al., 1996; Wichterle et al., 1997; Doetsch et al., 1997; Luskin, 1998). This migration is highly cohesive

* Corresponding author. Kentucky Spinal Cord Injury Research Center, Department of Neurological Surgery, University of Louisville, 511 S. Floyd Street, MDR Rm. 616, Louisville, Kentucky 40292. Fax: ⫹1-502852-5148. E-mail address: [email protected] (T. Hagg). 1 Current address: MGH-HMS Center for Nervous System Repair, Harvard Medical School, Massachusetts General Hospital, Edwards 4, (EDR 410), 50 Blossom Street, Boston, MA 02114.

and persistent, e.g., neuroblasts continue to migrate after removal of the olfactory bulb (Kirschenbaum et al., 1999). The neuroblasts express the polysialylated form of neural cell adhesion molecule (PSA-NCAM), which appears to be important for the mobility of these cells (Kiss, 1998; Chazal et al., 2000). The lipid phosphatase PTEN inhibits migration (Li et al. 2002). A potential inducer of migration is Migration-Inducing Activity (Mason et al., 2001). Ephrins (Conover et al., 2000) and Slit (Hu, 1999; Wu et al., 1999) are repellants, perhaps involved in containing the migratory stream. Little is known about guidance molecules with chemoattractant properties. Such molecules might be useful therapeutically to redirect neuroblasts to areas where new cells are needed. Integrins are receptors for extracellular matrix molecules and are important for guidance of migration of a variety of cells (Hynes, 1992; Clarke and Brugge, 1995), including neural precursors during development (Galileo et al., 1992;

0014-4886/03/$ – see front matter © 2003 Elsevier Science (USA). All rights reserved. doi:10.1016/S0014-4886(03)00209-7

274

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Bronner-Fraser, 1993). Integrins are heterodimers of one alpha and one beta subunit and are distributed throughout the CNS (Pinkstaff et al., 1998, 1999). Integrins link the extracellular matrix to cytoskeletal proteins to orient cells, as well as activate intracellular signaling pathways (Hynes, 1992; Kanner et al., 1993; Schwartz and Denninghoff, 1994; Clarke and Brugge, 1995; Wei et al., 1998; Giancotti and Ruoslahti, 1999; Pinkstaff et al., 1999). Integrin ␣6␤1 is present in neonatal rat SVZ neural precursors in vitro (Jacques et al., 1998) and is the only integrin enriched in embryonic, hematopoietic and neural stem cells (Ramalho-Santos et al., 2002). The ␤1 integrin is critical for neuroblast migration to the chick optic tectum (Galileo et al., 1992; Zhang and Galileo, 1998) and ␣6␤1, but not other integrins, plays a role in neuroblast migration in vitro (Jacques et al., 1998). The ␣6␤1 integrin is also involved in migration of Schwann cells (Dubovy et al., 2001) and lymphocytes (Gimond et al., 1998). In contrast to most other integrins, which bind multiple ligands, ␣6␤1 only seems to bind laminins (Aumailley et al., 1990; Kortesmaa et al., 2000; Talts et al., 2000). Laminins are well-known for promoting neurite outgrowth, but they also regulate migration of many non-neuronal cell types (Calof and Lander, 1991; Wei et al., 1998; Dubovy et al., 2001; Fujiwara et al., 2001). Laminins are large heterotrimer proteins composed of an ␣, ␤, and ␥ chain. The globular domain at the end of the laminin ␣ chain binds to ␣1, 2, 3, 6 or 7 integrin subunits, in combination with ␤1 or ␤4 integrins (Sonnenberg et al., 1990; Engvall and Wewer, 1996; Gimond et al., 1998; Fujiwara et al., 2001). A small laminin peptide representing the E8 laminin domain that binds ␣6␤1 integrin (Muruyama et al., 1996) enhances chain migration of SVZ derived neuronal precursors in vitro (Jacques et al., 1998). Laminin-1 is expressed during CNS development and migration, but this subsides over time (Liesi, 1985; Zhou, 1990), laminin ␣1 chain is present in the blood vessel basement membrane, as well as in reactive glia (Liesi et al., 1984), and laminin ␣2 is present in neurons. Several laminin isoforms have been described in the adult CNS but not in the SVZ or RMS (Hagg et al., 1989, 1997). Here, we characterized the endogenous guidance role of ␣6␤1 integrin in the migration of neuroblasts in the RMS of adult mice by inhibition with subunit specific antibodies and local administration of laminin peptides or intact laminin.

Materials and methods Animals and housing All procedures were performed on adult (2–3 month old) male C57BL/6 mice (Charles River, Quebec, Canada) and were conducted in accordance with Dalhousie University and Canadian Council on Animal Care guidelines. Efforts were undertaken to reduce the number of animals used in this study. Mice were housed in group cages (3–5 animals per cage), under a standard 12 hour light cycle, with food and water

available ad libitum. All surgical and euthanasia procedures were performed with an anesthetic mixture of ketamine (60 mg/kg) and xylazine (12 mg/kg) in 0.9% saline. Delivery of neutralizing antibodies against integrin subunits Biologically active neutralizing antibodies were used to block the activity of either the ␣6 or ␤1 integrin subunits (Sonnenberg et al., 1987, 1988; Gao et al., 1995; Wilkins et al., 1996). A 1 ␮L bolus of 1 ␮g rat anti-human ␣6, mouse anti-human ␤1, or purified control rabbit, rat or mouse IgG (all from Chemicon, Temecula, CA) was delivered into the cortex directly above the RMS at the following coordinates, in mm from Bregma: RC 1.7, ML, ⫺ 0.7, and DV⫺4.0 (Franklin and Paxinos, 1997). Antibodies were injected over a two-minute period via a 470 ␮m diameter needle of a 1 ␮L Hamilton syringe, which was left in the brain for an additional two minutes before retraction. Mice were left for three days (a sufficient amount of time during which the antibodies could reach the RMS and biological effects on the rapidly migrating neuroblasts could be expected), and these mice were then euthanized and analyzed. Mice with injection tracts penetrating the RMS were excluded. Production and administration of a laminin peptide A 15 amino acid polypeptide (designated P3, with amino acid sequence VSWFSRHRYSPFAVS), representing the E8 domain of the laminin ␣ chain and capable of binding ␣6␤1 integrin (Sonnenberg et al., 1990; Muruyama et al., 1996; Jacques et al., 1998), was commercially synthesized (Sigma Genosys, The Woodlands, TX). The peptide was dissolved in vehicle containing 0.02% acetic acid to ensure solubility (BDH, Toronto, Ontario), rat serum albumin to protect the peptide and serve as a control for non-specific protein actions (1 mg/mL; Sigma, Oakville, Ontario), and gentamicin antibiotic (25 ␮g/mL; Sigma). Peptide (2.5 ␮g/day) or control vehicle was loaded into Alzet 1002 mini-osmotic pumps (0.25 ␮L/hour; Alza, Palo Alto, CA) and was delivered for 14 days via a 150 ␮m diameter metal infusion cannula (Plastics One, Roanoke, Virginia), stereotactically placed into the neostriatum to a depth of 3.0 mm from the skull surface, 1.1 mm rostral and 1.3 mm lateral to Bregma (Franklin and Paxinos, 1997), away from the SVZ/RMS region. The cannula was affixed to the skull with cyanoacrylate glue (Loctite 454, Loctite Corp, Hartford, CT), and the pump was inserted subcutaneously between the scapulae. The pump and cannula were then rinsed with 0.1 ␮g/mL gentamicin in 0.9% saline. The infusion lasted for 14 days, to ensure sufficient diffusion of the peptide or control vehicle to the SVZ and RMS, and to observe potential biological effects. Laminin tract injection Using a modification of the method described in (Zhou and Azmitia, 1988), a narrow 2 mm long tract containing 1

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

␮L of purified mouse laminin (isolated from EngelbrethHolm-Swarm (EHS) mouse sarcoma; Chemicon; 0.025 ␮g or 0.25 ␮g in 0.05 M Tris HCl, pH 7.4 with 2 mM EDTA (Sigma)) or vehicle was deposited above the RMS in adult mice. The needle of a Hamilton syringe was inserted, in mm from Bregma, to the following coordinates: RC 1.7, ML ⫺0.7, and DV ⫺ 4.0 (Franklin and Paxinos, 1997), just touching the dorsal boundary of the RMS, and left for three minutes. The needle was then drawn dorsally, over a three minute period to DV ⫺ 2.0 mm, while dispensing the solution, thereby leaving a narrow, 2 mm long tract perpendicular to the axis of the RMS. After two more minutes, the needle was slowly retracted. Mice were left for seven days (deemed a sufficient amount of time for cells of the RMS to respond to the deposited laminin or vehicle), and were then euthanized and analyzed. Mice with an injection that penetrated the RMS were excluded. 5-bromo-2⬘-deoxyuridine (BrdU) administration To label proliferating cells within each of the experimental paradigms, mice received intraperitoneal (i.p.) injections of the nucleotide BrdU (200 mg/kg; Sigma) dissolved in 0.9% saline (Miller and Nowakowski, 1988). Animals receiving a single bolus of blocking antibodies received daily BrdU injections for the duration of the experiment (for three days after surgery). To label the majority of proliferating cells in animals that received laminin peptide or vehicle infusion via the osmotic pumps, mice received single daily BrdU injections on days three to five after surgery, two daily injections on days six through 13, and six hourly injections on the day prior to sacrifice. Injections started on day 3, to reduce labeling of inflammatory cells that would infiltrate around the intracerebral injection sites. Mice receiving an injected laminin tract were injected with BrdU once per day for five days, starting three days after surgery. Histology On the day following the last BrdU administration, animals were again anaesthetized and transcardially perfused with 20 mL cold phosphate-buffered saline and 20 mL cold 4% paraformaldehyde in 0.1 M phosphate buffer (pH 7.4). Brains were post-fixed for 24 h, cryoprotected in 30% sucrose in 0.1 M phosphate buffer, and coronal, horizontal, or sagittal sections of 30 ␮m were cut on a freezing microtome and collected in 0.12 M Millonig’s phosphate buffer containing 0.06% sodium azide (Millonig, 1961). Every sixth section of tissue was used for each immunocytochemical procedure. DNA denaturation for BrdU immunocytochemistry followed the procedure described in Kuhn et al. (1996). A monoclonal mouse anti-BrdU antibody was used to label proliferating cells (1:1,000; MAB 3424, Chemicon) followed by a secondary horse anti-mouse antibody (1:600; Vector). Labeling of ␣6␤1 (1:3,000, mouse monoclonal, MAB 1410, Chemicon), laminin A-B chain (1:100, mouse

275

monoclonal, MAB 1904, Chemicon), laminin ␣2 chain (1:100, mouse monoclonal, Alexis, San Diego, CA), PSANCAM (1:5; mouse monoclonal supernatant; Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA), a marker for neuroblasts in the adult CNS neurogenic regions (Dodd et al., 1988; Schubert et al., 2000), and Tuj1 (1:3,000; mouse monoclonal, Babco, Richmond, CA), directed against class III ␤-tubulin, a marker for immature neurons (Lee et al., 1990; Alexander et al., 1991; Menezes and Luskin, 1994; Gates et al., 1995), was followed with appropriate secondary antibodies (horse anti-mouse IgG or goat anti-rabbit IgG, 1:400 (Vector)), or goat anti-mouse IgM for PSA-NCAM; 1:400 (Cappel, West Chester, PA). Fluorescent double-labeling of cells was performed with sheep polyclonal anti-BrdU antibodies (1:1,000; Research Diagnostics; Flanders, NJ), antibodies against Tuj1 (1:1,000 mouse monoclonal or 1:1000 rabbit polyclonal, both from Babco) or ␣6␤1 (1:500; mouse monoclonal; MAB 1410, Chemicon). Appropriate fluorescent secondary antibodies were conjugated with Alexa 488 (1:500; donkey anti-sheep or goat anti-mouse) and Alexa 546 (1:800; goat anti-mouse or goat anti-rabbit; Molecular Probes; Eugene, OR). Analysis All tissue was blindly analyzed with the observer unaware of control or experimental groups. Cell profiles were counted at 400X total magnification (40X objective), and total neuroblast number was estimated as the number of profiles counted ⫻ [section thickness/(section thickness ⫹ average neuroblast diameter)] ⫻ section interval (Abercrombie, 1946). In the peptide infusion experiment, cells were considered to be en route to the infusion site if they were found anywhere between their normal location in the SVZ or RMS and the infusion site itself. The area of the infusion site was taken as a 100 ␮m radius around the tip of the infusion cannula. In the experiments where neutralizing antibodies were delivered, the degree of dispersion of migrating neuroblasts was assessed by counting the number of Tuj1 positive cells found along a 500 ␮m stretch of the RMS and up to 100 ␮m outside the rostral migratory stream in a total of six sections per animal. In the laminin treatment experiments, the number of Tuj1 cells in the RMS between the rostral pole of the SVZ and the (rostral) laminin tract (six sections per animal) was assessed, as was the thickness of the RMS. Confocal micrographs were produced with a Zeiss LSM 510 laser scanning confocal microscope. Double-labeling was verified by scanning through consecutive confocal image scans, as well as by rotating these projected image stacks in various planes. All other photomicrographs were produced with a Spot CCD camera attached to a Zeiss Axioplan 2 microscope. Data are presented as means ⫾ SEM, and differences between groups were assessed using the nonparametric Mann Whitney U-Test with a minimum significance level of P ⬍ 0.05.

276

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Fig. 1. ␣6␤1 integrin is present in neuroblasts of the adult mouse RMS. (A) Immunostaining for ␣6␤1 integrin is associated with many cells of the RMS, here visualized in sagittal sections. The expression of ␣6␤1 integrin appears less or in fewer cells than that of other markers for migrating immature neurons, such as Tuj1 (B). (C) In cross-sections through the RMS, many cells are double-labeled for ␣6␤1 integrin (green) and Tuj1 (red). Scale bar in A for A and B ⫽ 100 ␮m, in C ⫽ 10 ␮m.

Results The ␣6␤1 integrin is associated with neuroblasts of the rostral migratory stream Immunostaining for the ␣6␤1 integrin was detectable in cell bodies and their thin, faintly labeled processes to varying degrees along the RMS, from the anterior SVZ to the olfactory bulb (Fig. 1A). In some areas many cells were stained whereas in others only few had clearly detectable staining. Overall, clear immunostaining for ␣6␤1 appeared to be present in only a portion of the cells compared to markers for immature neuronal markers such as Tuj1 (Fig. 1B) or PSA-NCAM (not shown). Expression of the ␣6␤1 integrin was also present in glial cells within the striatum, particularly in cells most closely associated with the injury that was caused by injection cannulas. In addition, there were occasionally

␣6␤1 positive glial cells within the cerebral cortex, and this was also evident in tissue from untreated mice. Confocal imaging of cells double labeled for ␣6␤1 and Tuj1 in cross-section through RMS areas containing many ␣6␤1-positive cells clearly revealed an overlap between these markers, evidence that many of the neuroblasts express this integrin in vivo (Fig. 1C). The distribution of the integrin positive cells throughout different regions and in cross-sections of the RMS tube did not reveal any distinct pattern or structure. Blockade of endogenous integrins disrupts the rostral migratory stream We assessed the endogenous role of integrins in guiding neuroblast migration to the olfactory bulb by injecting biologically active neutralizing antibodies against the ␣6 or ␤1 integrin subunits above the RMS (Fig. 2A). Injection of rat,

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

277

Fig. 2. Blockade of endogenous integrin subunits disrupts the rostral migratory stream. (A) As shown in the schematic diagram (after Franklin and Paxinos, 1997), antibodies against specific integrin subunits were infused into the parenchyma above the RMS. Tissue was analyzed seven days after injections. Tuj1 labeling shows that a bolus injection of control IgG did not affect the integrity of the stream (B), whereas neutralizing antibodies against the ␣6 integrin subunit altered distribution of neuroblasts within the stream (C). Antibodies against the ␤1 integrin subunit produced marked disruption of the stream, with chains of neuroblasts losing their cohesion, and cells drifting off from their normal migration route (D). Scale bar in B for B–D ⫽ 50 ␮m.

mouse or rabbit IgG controls had no detectable effect on the integrity and direction of the stream, as shown by Tuj1 immunoreactivity three days later (Fig. 2B; n ⫽ 4, 4 and 8, respectively). In sharp contrast, antibodies against the ␣6 (Fig. 2C; n ⫽ 8) and ␤1 integrin (Fig. 2D; n ⫽ 8) subunit led to a disruption of the stream in each case. Injection of ␣6 antibodies appeared to cause a disappearance of most of the Tuj1-stained neuroblasts below the injection site (which was above the RMS). Blocking the ␤1 integrin subunit also led to dispersal of the neuroblasts as well as the loss of cohesive patterns of chains within the leading portion of the stream (Fig. 2D). This distribution pattern appeared different from that seen in mice injected with ␣6 antibody. Analysis of adjacent sections did not reveal clear clusters of cells outside the RMS, suggesting that the cells had dispersed themselves widely. With the blocking antibodies, there was a similar dispersed distribution pattern for BrdU-labeled or PSA-NCAM stained neuroblasts (not shown). To quantify the degree to which cells were dispersed by the neutralizing antibodies, the number of Tuj1 positive cells outside the

RMS was calculated as a percentage of the normal number of Tuj1 positive cells in the RMS proper. This percentage was greater in animals receiving the ␣6 antibody versus rat IgG injected controls (95 ⫾ 24% versus 17 ⫾ 6%, SEM, P ⬍ 0.01). Similarly, the dispersion of neuroblasts was significantly greater in ␤1 antibody injected animals (50 ⫾ 17%) versus mouse IgG injected controls (17 ⫾ 6%, P ⬍ 0.05). There were no significant differences in dispersion of neuroblasts between the control groups. The number of Tuj1 positive cells within the analyzed 500 ␮m stretch of the RMS plus the surrounding regions (dispersed cells) was not significantly different among any of the control (mouse IgG, 107 ⫾ 15; rat IgG, 157 ⫾ 15) and experimental groups (anti ␣6, 105 ⫾ 19; anti ␤1, 135 ⫾ 26). The morphology of the Tuj1- or PSA-NCAM-positive cell bodies in the remainder of the RMS appeared normal. The morphology of the BrdU-positive nuclei was not noticeably different between the antibody treated and control groups. Only an occasional condensed or fragmented nucleus, potentially indicative of cell death, was observed in and around the RMS, with no

278

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Fig. 3. Cell proliferation following laminin peptide or control infusion. (A) A peptide corresponding to the ␣6␤1 integrin binding domain of laminin was infused into the neostriatum for two weeks, as shown in the schematic diagram (after Franklin and Paxinos, 1997). (B) A photomicrograph of a BrdU-labeled horizontal section, with rostral at the top and caudal at the bottom. Infusion of control vehicle or peptide led to increased BrdU labeling throughout the neostriatum on the infusion side. Scale bar for B ⫽ 500 ␮m.

differences between the animal groups. This is consistent with the findings by others that very few TUNEL-positive cells exist in the normal RMS (Brunjes and Armstrong, 1996; Linnarsson et al., 2000). An ␣6␤1 integrin binding laminin peptide redirects neuroblasts into the neostriatum Laminin is the natural ligand for the ␣6␤1 integrin and we infused a 15 amino acid peptide corresponding to the ␣6␤1 integrin binding domain (designated P3; Muruyama et al., 1996; Jacques et al., 1998), to test its effect on the migrating neuroblasts (schematically illustrated in Fig. 3A). We examined BrdU-positive cells in the SVZ and within the neostriatum in both P3 (n ⫽ 6; Fig. 3B) and vehicle-infused (n ⫽ 7) mice. BrdU labeled cells were present in the neostriatum when vehicle or P3 was infused, and it is likely that many of these cells

were infiltrating leukocytes. The thickness of the SVZ (in both BrdU and Tuj1 stained sections) was not significantly different between any of the control and experimental groups. In addition, there was no significant difference in the thickness of the SVZ when the infused (P3 or vehicle) sides were compared with their contralateral, non-infused sides. Numerous PSA-NCAM positive cells were present between the SVZ and the infusion site in P3-infused animals (Fig. 4B and C) but not in vehicle-infused animals (Fig. 4A). In some cases chains of cells were evident, apparently deriving either from the SVZ (closest to the ventricle) or actually connecting portions of the RMS (located more lateral in coronal sections) with the infusion site. In addition, many PSA-NCAM positive cells were clustered around the infusion site (lateral to the RMS), and were only found on the medial, and not the lateral, side of the infusion site. The number of PSANCAM positive cells between the SVZ and infusion site was ⬃15 times greater after P3 infusion (n ⫽ 6, P ⬍ 0.001) than with vehicle (n ⫽ 7, Fig. 5A). There were ⬃25 times more PSA-NCAM positive cells in the region of the infusion site in P3-infused animals compared to vehicle-infused animals (P ⬍ 0.001). The overall number of PSA-NCAM positive cells drawn away from the SVZ or RMS also was much greater (P ⬍ 0.001). Labeling for the immature neuronal marker Tuj1 revealed a pattern of staining very similar to that seen with antibodies against PSA-NCAM. Briefly, chains of Tuj1 positive cells were evident between the SVZ/RMS and the infusion site in P3 (Fig. 4E) but not vehicle-infused animals (Fig. 4D). As with PSA-NCAM labeling, Tuj1 positive cells were only seen at the medial side of the P3 infusion site. Tuj1 immunostaining revealed (more clearly than with PSA-NCAM staining) that many of the neuroblast located between the RMS and infusion site had very long processes often oriented and extending towards the infusion site (Fig. 4F). Approximately 16 times more Tuj1 positive cells were present between the SVZ and infusion site in P3-infused (P ⬍ 0.001) versus vehicle-infused controls (Fig. 5B), and ⬃21 times more at the infusion site (P ⬍ 0.001). The overall number of redirected Tuj1 positive cells was ⬃18 times greater (P ⬍ 0.001). There were no significant differences between the number of Tuj1 and PSA- NCAM positive neuroblasts. To evaluate whether the Tuj1-positive cells that were located around the P3 infusion site were neuroblasts, we analyzed confocal images of BrdU-Tuj1 double-labeled sections (Fig. 6A). Many proliferated (BrdU-positive) cells per section were also positive for Tuj1, suggesting that they were newly formed neurons or neuronal precursors. As seen in the normal RMS (Fig. 6B), some of the cells had Tuj1 but no BrdU, suggesting that some of these cells, at least within this BrdU injection paradigm, had not recently proliferated, and may have reached these locations in the days when no BrdU was given (days 1 and 2). Also, as in the normal RMS, some cells were positive for BrdU but not Tuj1. Such cells

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

279

Fig. 4. Laminin peptide agonist of ␣6␤1 integrin draws neuroblasts into the neostriatum. Migrating neuroblasts were immunostained with antibodies to either PSA-NCAM (A–C) or Tuj1 (D–F). Compared to vehicle infused controls (A and D), infusion of a 15 amino acid peptide into the neostriatum led two weeks later to the presence of PSA-NCAM (B) or Tuj1 (E) positive neuroblasts between the SVZ/RMS and infusion site (asterisks). Neuroblasts en route from the RMS towards the infusion site extended their processes over considerable distances as seen at higher magnification in C and F. Scale bar in A for A, B, D and E ⫽ 50 ␮m; in C, for C and F ⫽ 20 ␮m.

could include leukocytes that had proliferated and infiltrated the brain during the time of infusion. A tract of intact laminin diverts neuroblasts from the rostral migratory stream We assessed whether a narrow 2 mm long tract of intact laminin, starting close to and perpendicular to the axis of the

RMS, could guide migrating neuroblasts away from the RMS over a 7 day period (Fig. 7A). We expected that the intact laminin would diffuse less through the brain tissue than the peptide and thereby would create a more restricted distribution of ligand. BrdU incorporation revealed that in vehicle (n ⫽ 6) as well as in laminin-injected (n ⫽ 7, 0.025 ␮g; n ⫽ 8, 0.25 ␮g) animals, there was cellular proliferation

280

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Fig. 5. Laminin peptide agonist of ␣6␤1 integrin draws a significant number of neuroblasts towards and to the infusion site. (A) After two weeks, significantly more PSA-NCAM positive neuroblasts were found between and at the SVZ/RMS and the infusion site in mice infused with P3 peptide compared to vehicleinfused controls (P ⬍ 0.001). (B) The number of Tuj1 positive cells either between or at the infusion site was similarly greater than in controls (P ⬍ 0.001).

around the injection site and along the length of the cannula tract (Fig. 7B). BrdU-positive cells also were seen dorsal to the tract. The number of Tuj1 positive cells within the RMS between the rostral SVZ and the tract was significantly greater in animals receiving a high concentration of laminin (76 ⫾ 6) versus controls (49 ⫾ 5, P ⬍ 0.01). The RMS in that region was thicker (55 ⫾ 5 ␮m; n ⫽ 8; P ⬍ 0.025) with the higher dose of laminin than in controls (32 ⫾ 6 ␮m; n ⫽ 6), but not with a low dose of laminin (44 ⫾ 3 ␮m; n ⫽ 6). The ability of laminin to guide neuroblasts into the tract was assessed with Tuj1 immunoreactivity. In all but one of the six mice injected with vehicle no neuroblasts were located within the tract (identified by the disturbance of the brain tissue) (Fig. 7C). In one of the six vehicle-injected controls, there were 180 Tuj1 positive neuroblasts in the tract up to a 200 ␮m distance. In six out of eight high dose (0.25 ␮g) and five out of seven low dose (0.025 ␮g) laminin-injected mice, neuroblasts were seen in the injection tract (Fig. 7D). There were anywhere from 10 to 494 (average of 169) Tuj1-positive cells seen within the

high-dose laminin tract of each animal, and 15 to 303 (average of 119) positive cells in the low-dose laminin group. The greatest distance migrated by neuroblasts within the laminin tract ranged from 100 to 950 ␮m (540 ⫾ 146 ␮m, SEM, n ⫽ 5 sagittally-sectioned specimens). Finally, as with Tuj1 positive cells diverted to the P3 peptide infusion site, many Tuj1 positive cells in the laminin tract were also positive for BrdU (Fig. 6C).

Discussion These results provide evidence for a critical role of ␣6␤1 integrin and its only known natural ligand, laminin, in controlling the direction of migrating neuroblasts in the adult CNS. They show that ␣6␤1 integrin is expressed by migrating neuroblasts of the RMS; that neutralizing antibodies against the ␣6 or ␤1 integrin subunits can disrupt neuroblast migration, suggesting an endogenous role for ␣6␤1 integrin in guiding migration; and that laminin is a chemoattractant for neuroblasts of

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

281

the SVZ/RMS, drawing neuroblasts away from their normal course of migration in a restricted fashion when injected as a tract of intact laminin, and in a dispersed fashion when provided as a more soluble peptide. The ␣6␤1 integrin is present in rostral migratory stream neuroblasts Double-immunostaining demonstrated that ␣6␤1 integrin is present in many, but not all, Tuj1-positive neuroblasts of the adult mouse RMS. It is possible that the responsiveness to guidance cues of a subset of neuroblasts would affect the direction of all of the neuroblasts as they use one another as a substrate (Lois et al., 1996; Wichterle et al., 1997). The density of positive cells was variable over the length and width of the RMS with no clear pattern that would suggest association of integrin-positive neuroblasts with certain structures. We attempted to determine whether laminins are present in the adult mouse RMS and did not detect immunostaining for laminin ␣1 or ␣2 chain, other than in basement membranes of blood vessels. However, these antibodies might not be suitable for use in mice, as we have clearly detected laminin ␣2 chain in glial cells and their fine processes of the RMS of adult rabbits immunostained in a previous study (not shown; Hagg et al., 1997). Thus, the presence of laminin isoforms in and along the mouse RMS remains to be determined and, until such a time, the idea that laminin is the endogenous ligand for neuroblast ␣6␤1 integrin will remain speculative. Other integrin subunit combinations are present in neuroblasts from neonatal rat forebrain in vitro (Jacques et al., 1998), including ␣5␤1, ␣v␤1, ␣v␤5 and ␣v␤8, where they regulate proliferation but not migration. It remains to be determined whether these integrins are also present in the adult RMS. These integrins do not interact with laminins, but are receptors for other extracellular matrix molecules such as fibronectin. In fact, the RMS travels within a glial tube, which is highly enriched with other extracellular matrix molecules, including tenascin-C and chondroitin sulfate proteoglycan (Thomas et al., 1996; Peretto et al., 1997). An endogenous role for ␣6␤1 integrin in guiding migration Three days after injection of neutralizing antibodies, inhibition of the ␣6 integrin subunit caused a disappearance from the RMS of the neuroblasts closest to the injection site.

Fig. 6. Proliferation and Tuj1 immunostaining of normal and diverted neuroblasts. (A) Some migrating neuroblasts (arrow; Tuj1, red) en route to the P3 peptide infusion site also are positive for the proliferation maker BrdU (green). The arrowhead shows a Tuj1 positive cell not positive for BrdU. (B) en face coronal view of the normal RMS, showing recently proliferated migrating neuroblasts (arrow; Tuj1, red; BrdU, green). (C) A confocal image of a laminin tract containing cells labeled for Tuj1 (red) and BrdU (green). Scale bar in A for A and B, 20 ␮m and for C, 10 ␮m.

282

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Fig. 7. A tract of intact laminin draws neuroblasts away from the rostral migratory stream. A) A thin tract of laminin was injected above the RMS, as shown schematically (after Franklin and Paxinos, 1997). B) Immunostaining for BrdU illustrates the position of the tract relative to the RMS, and shows proliferated cells (likely of various types) within the laminin tract (arrows). Tuj1 staining reveals that, compared to vehicle injected controls (C), laminin can divert neuroblasts from their normal migration pathway (D). Scale bar in B ⫽ 100 ␮m, in C for C and D ⫽ 50 ␮m.

Inhibition of the ␤1 integrin subunit also caused a disruption of the cohesive nature of the RMS. The distribution pattern of BrdU, Tuj1 and PSA-NCAM in and around the RMS of these mice was very similar, suggesting that the neuroblasts had not just lost their markers. The neuroblasts had not stopped migrating, as they would have remained in the antibody-treated regions. The combined number of Tuj1 positive neuroblasts within plus dispersed from the RMS was not different from the number in the normal RMS. The morphology of the remaining cells in the injected RMS region was normal and there was little evidence of nuclear condensation or fragmentation (in BrdU stained sections) characteristic of cells undergoing cell death (see also Brunjes and Armstrong, 1996; Linnarsson et al., 2000). This suggests that the neuroblasts had not died in response to the antibodies, consistent with the finding that they are not lethal in vitro (Jacques et al., 1998). It remains to be determined whether dispersed neuroblasts do eventually die. Other types of cells die after losing contact with the extracellular matrix, a process termed anoikis (Frisch and Screaton, 2001). Thus, the neuroblasts had most likely mi-

grated away from the RMS because they had lost directional guidance by ␣6␤1 integrin activation. This is consistent with the observation that ␣6␤1 activation induces cohesive chain migration of neuroblasts in vitro (Jacques et al., 1998). The wide dispersion of the neuroblasts through the surrounding brain regions would be analogous to findings that endogenous and transplanted neuroblasts can migrate over great distances within the adult CNS (Lois and Alvarez-Buylla, 1994; Fricker et al., 1999; Shin et al., 2000). The subtle differences in the effects of antibodies against the individual subunits could be explained by the fact that ␤1 associates with other ␣ subunits to form receptors for other extracellular matrix molecules, whereas ␣6 only dimerizes with ␤1 or ␤4 to form receptors for laminins (Hynes, 1992). This raises the possibility that other extracellular matrix molecules, such as fibronectin (e.g., binding to ␣5␤1 and ␣v␤1), are involved in directing migration of the neuroblasts. In addition, the ␤ subunits associate more with the cytoskeleton (Chan et al., 1992), which could also contribute to the differential effects of inhibiting the individual subunits.

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Laminin and laminin peptide redirect neuroblast migration from the adult mouse RMS Administration of laminin or the laminin peptide redirected numerous cells to locations where they normally are not seen. Laminin is the only known ligand for ␣6␤1 integrin, and the peptide represents the non-RGD binding domain of laminin that binds ␣6␤1 integrin. This suggests that laminin-␣6␤1 binding is involved in directional guidance of neuroblasts in the adult CNS. It remains to be determined whether and which proportion of the re-directed neuroblasts express this integrin. Such chemoattractant properties of the laminin-integrin interaction most likely involve modification of the cytoskeleton, including alignment of actin filaments, a mechanism that directs other migrating cells (Hynes, 1992). With the higher dose of injected intact laminin, more neuroblasts were detected in the RMS, raising the possibility that laminin affected their proliferation. In contrast, the peptide did not affect the number of neuroblasts, suggesting that a different domain of laminin would affect proliferation. Various domains of intact laminin have different biological activities, including an effect on proliferation (Drago et al., 1991; Paulsson, 1992; Frade et al., 1996). On the other hand, the intact laminin may have retained neuroblasts around the area of the tract due to its chemoattractive properties. That would be consistent with the findings that laminin does not cause proliferation of SVZ neuroblasts in vitro (Jacques et al., 1998). In one animal with a control vehicle injection above the RMS, many neuroblasts had migrated over a short distance along the injury tract. Laminins can be upregulated in reactive astrocytes in response to CNS injury (Liesi et al., 1984) and such laminin may have attracted the neuroblasts in that animal. This mechanism may also underlie the attraction of neuroblasts to injured regions of the CNS (Magavi et al., 2000). On the other hand, this one case raises the possibility that other mechanisms contribute to the effects of the laminin tract injection. Because none of the controls in the P3 peptide infusion experiment showed signs of aberrant migration, such mechanisms could include ones related to the injection tract or location. The number of neuroblasts attracted by laminin or laminin peptide was in the range of 100, which is at least an order of magnitude lower than the number of neuroblasts that continued to travel to the olfactory bulb. It is possible that the relatively low doses of exogenous laminin had to compete with putatively higher concentrations of endogenous laminin in the RMS. We cannot exclude the possibility that many cells that had been drawn away from the RMS had died before the analysis. The smaller number of aberrantly migrating neuroblasts may also only represent the less abundant ␣6␤1 positive cells. The diverted neuroblasts were found up to approximately 1 mm into the laminin tract. A normal RMS neuroblast can travel up to 720 ␮m per day (Lois and Alvarez-Buylla,

283

1994). Therefore, during the 7-day experiment neuroblasts could have reached up to five times farther than they appeared to have done in the laminin tract. It is possible that the start of the diverted migration was delayed or that the cells migrate slowly outside their normal environment. The latter would be indicative of a difference between endogenous and transplanted neural precursors, which have a great capacity for migration (Aboody et al., 2000; Fricker-Gates et al., 2000). Alternatively, migrating neuroblasts may have returned to the RMS, as they normally are capable of reversing their direction of movement en route to the olfactory bulb (Kakita and Goldman, 1999). The pattern of migration seen with the soluble laminin peptide was suggestive of migration to a point source from restricted regions of the RMS and dorsal SVZ. This is different from the altered migration seen with intrastriatal infusion of TGF␣ (Fallon et al., 2000), where cells migrated as a wave towards the infusion site. This difference may be related to the fact that the EGF receptor for TGF␣ would activate ERK, whereas integrins activate ERK (Barberis et al., 2000; Wary et al., 1998) and modify the cytoskeleton. The pattern of diverted migration was more global and dispersed with the infusion of the laminin peptide and more restricted to the tract with the injection of intact laminin. This is probably caused by the lesser solubility of the 800 kDa intact laminin and therefore lesser diffusion away from the injection site than with the small laminin peptide, which would have diffused widely to establish a concentration gradient. This difference may in future be useful in repair strategies, e.g., to contain endogenous or transplanted neural precursors in certain regions with intact laminin, or to attract neural precursors into larger areas, using laminin peptides. Conclusions These results provide evidence that laminin and integrins are critical for directional guidance of neuroblast migration in the adult CNS in vivo. Specifically, the ␣6␤1 integrin appears to maintain the direction and cohesiveness of neuroblasts in the RMS as they travel from the SVZa to the olfactory bulb. Moreover, exogenous laminin or ␣6␤1 integrin binding laminin peptides can be used to draw endogenous neuroblasts to CNS regions where they normally are not found. Such approaches may prove effective within therapeutic neural repair strategies.

Acknowledgments We are grateful for the excellent technical assistance of Jessica Pastorius, Teena Chase, Xin Lu, and Min Huang. We are very thankful for the gift of some of the antibodies by Carol Birmingham from Chemicon International Inc. The monoclonal antibody 5A5 anti-PSA-NCAM was raised from a cell line originally developed by Dr. Thomas Jessell, and provided via the Developmental Studies Hybridoma

284

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285

Bank maintained by the University of Iowa. We thank Drs. Wolfram Tetzlaff (UBC) and Eileen Denovan-Wright (Dalhousie) for very helpful discussions. This work was supported by an operating grant (RO14547) from the Canadian Institutes for Health Research in partnership with Nova Neuron Inc., by a Fellowship from the Nova Scotia Health Research Foundation (J.G.E.) and by a CIHR Investigator Award (T.H.).

References Abercrombie, M., 1946. Estimation of nuclear population from microtome sections. Anat. Rec. 94, 239 –247. Aboody, K.S., Brown, A., Rainov, N.G., Bower, K.A., Liu, S., Yang, W., Small, J.E., Herrlinger, U., Ourednik, V., Breakefield, X., Snyder, E.Y., 2000. Neural stem cells display extensive tropism for pathology in adult brain: evidence from intracranial gliomas. Proc. Natl. Acad. Sci. U.S.A. 97, 12846 –12851. Alexander, J.E., Hunt, D.F., Lee, M.K., Shabanowitz, J., Michel, H., Berlin, S.C., MacDonald, T.L., Sundberg, R.J., Rebhun, L.J., Frankfurter, J.E., 1991. Characterization of posttranslational modifications in neuron-specific class III Beta-tubulin by mass spectrometry. Proc. Natl. Acad. Sci. U.S.A. 88, 4685– 4689. Aumailley, M., Timpl, R., Sonnenberg, A., 1990. Antibody to integrin ␣6 subunit specifically inhibits cell-binding to laminin fragment 8. Exp. Cell. Res. 188, 55– 60. Barberis, L., Wary, K.K., Fiucci, G., Liu, F., Hirsch, E., Brancaccio, M., Altruda, F., Tarone, G., Giancotti, F.G., 2000. Distinct roles of the adaptor protein Shc and focal adhesion kinase in integrin signaling to ERK. J. Biol. Chem. 275, 36532–36540. Bronner-Fraser, M., 1993. Mechanisms of neural crest cell migration. Bioessays 15, 221–230. Brunjes, P.C., Armstrong, A.M., 1996. Apoptosis in the rostral migratory stream of the developing rat. Brain Res. Dev. Brain Res. 92, 219 –222. Calof, A.L., Lander, A.D., 1991. Relationship between neuronal migration and cell-substratum adhesion: laminin and merosin promote olfactory neuronal migration but are anti-adhesive. J. Cell. Biol. 115, 779 –794. Chan, B.M., Kassner, P.D., Schiro, J.A., Byers, H.R., Kupper, T.S., Hemler, M.E., 1992. Distinct cellular functions mediated by different VLA integrin alpha subunit cytoplasmic domains. Cell 68, 1051–1060. Chazal, G., Durbec, P., Jankovski, A., Rougon, G., Cremer, H., 2000. Consequences of neural cell adhesion molecule deficiency on cell migration in the rostral migratory stream of the mouse. J. Neurosci. 20, 1446 –1457. Clarke, E.A., Brugge, J.S., 1995. Integrins and signal transduction pathways: the road taken. Science 268, 233–239. Conover, J.C., Doetsch, F., Garcia-Verdugo, J.-M., Gale, N.W., Yancopoulos, G.D., Alvarez-Buylla, A., 2000. Disruption of Eph/ephrin signaling affects migration and proliferation in the adult subventricular zone. Nat. Neurosci. 3, 1091–1097. Dodd, J., Morton, S.B., Karagogeos, D., Yamamoto, M., Jessell, T.M., 1988. Spatial regulation of axonal glycoprotein expression on subsets of embryonic spinal neurons. Neuron 1, 105–116. Doetsch, F., Garcı´a-Verdugo, J.M., Alvarez-Buylla, A., 1997. Cellular composition and three-dimensional organization of the subventricular germinal zone in the adult mammalian brain. J. Neurosci. 17, 5046 – 5061. Doetsch, F., Alvarez-Buylla, A., 1996. Network of tangential pathways for neuronal migration in adult mammalian brain. Proc. Natl. Acad. Sci. U.S.A. 93, 14895–14900. Drago, J., Nurcopmbe, V., Bartlett, P.F., 1991. Laminin through its long arm E8 fragment promotes the proliferation and differentiation of murine epithelial cells in vitro. Exp. Cell Res. 192, 256 –265.

Dubovy, P., Svizenska, I., Klusakova, I., Zitkova, A., Houst’Ava, L., Haninec, P., 2001. Laminin molecules in freeze-treated nerve segments are associated with migrating Schwann cells that display the corresponding alpha6beta1 integrin receptor. Glia 33, 36 – 44. Engvall, E., Wewer, U.M., 1996. Domains of laminin. J. Cell. Biochem. 61, 493–501. Fallon, J., Reid, S., Kinyamu, R., Opole, I., Opole, R., Baratta, J., Korc, M., Endo, T.L., Duong, A., Nguyen, G., Karkehabadhi, M., Twardzik, D., Loughlin, S., 2000. In vivo induction of massive proliferation, directed migration, and differentiation of neural cells in the adult mammalian brain. Proc. Natl. Acad. Sci. U.S.A. 97, 14686 –14691. Frade, J.M., Martinez-Morales, J.R., Rodriguez-Tebar, A., 1996. Laminin-1 selectively stimulates neuron generation from cultured retinal neuroepithelial cells. Exp. Cell Res. 222, 140 –149. Franklin, K.B.J., Paxinos, G., 1997. The mouse brain in stereotaxic coordinates. Academic Press, San Diego. Fricker-Gates, R.A., Winkler, C., Kirik, D., Rosenblad, C., Carpenter, M.K., Bjo¨ rklund, A., 2000. EGF infusion stimulates the proliferation and migration of embryonic progenitor cells transplanted in the adult rat striatum. Exp. Neurol. 165, 237–247. Fricker, R.A., Carpenter, M.K., Winkler, C., Greco, C., Gates, M.A., Bjo¨ rklund, A., 1999. Site-specific migration and neuronal differentiation of human neural progenitor cells after transplantation in the adult rat brain. J. Neurosci. 19, 5990 – 6005. Frisch, S.M., Screaton, R.A., 2001. Anoikis mechanisms. Curr. Opin. Cell Biol. 13, 555–562. Fujiwara, H., Kikkawa, Y., Sanzen, N., Sekiguchi, K., 2001. Purification and characterization of human laminin-8. Laminin-8 stimulates cell adhesion and migration through alpha3beta1 and alpha6beta1 integrins. J. Biol. Chem. 276, 17550 –17558. Galileo, D.S., Majors, J.S., Horwitz, A.F., Sanes, J.R., 1992. Retrovirally introduced antisense integrin mRNA inhibits neuroblast migration in vivo. Neuron 9, 1117–1131. Gao, J.X., Wilkins, J., Issekutz, A.C., 1995. Migration of human polymorphonuclear leukocytes through a synovial fibroblast barrier is mediated by both beta 2 (CD11/CD18) integrins and the beta 1 (CD29) integrins VLA-5 and VLA-6. Cell. Immunol. 163, 178 –186. Gates, M.A., Thomas, L.B., Howard, E.M., Laywell, E.D., Sajin, B., Faissner, A., Gotz, B., Silver, J., Steindler, D.A., 1995. Cell and molecular analysis of the developing and adult mouse subventricular zone of the cerebral hemispheres. J. Comp. Neurol. 361, 249 –266. Giancotti, F.G., Ruoslahti, E., 1999. Integrin signaling. Science 285, 1028 – 1032. Gimond, C., Baudoin, C., van der Neut, R., Kramer, D., Calafat, J., Sonnenberg, A., 1998. Cre-loxP-mediated inactivation of the alpha6A integrin splice variant in vivo: evidence for a specific functional role of alpha6A in lymphocyte migration but not in heart development. J. Cell. Biol. 143, 253–266. Hagg, T., Portera-Cailliau, C., Jucker, M., Engvall, E., 1997. Laminins of the adult mammalian CNS; laminin-␣2 (merosin M-) chain immunoreactivity is associated with neuronal processes. Brain Res. 764, 17–27. Hagg, T., Muir, D., Engvall, E., Varon, S., Manthorpe, M., 1989. Lamininlike antigen in rat CNS neurons: distribution and changes upon brain injury and nerve growth factor treatment. Neuron 3, 721–732. Hu, H., 1999. Chemorepulsion of neuronal migration by Slit2 in the developing mammalian forebrain. Neuron 23, 703–711. Hynes, R.O., 1992. Integrins: versatility, modulation, and signaling in cell adhesion. Cell 69, 11–25. Jacques, T.S., Relvas, J.B., Nishimura, S., Pytela, R., Edwards, G.M., Streuli, C.H., ffrench-Constant, C., 1998. Neural precursor cell chain migration and division are regulated through different ␤1 integrins. Development 125, 3167–3177. Kakita, A., Goldman, J.E., 1999. Patterns and dynamics of SVZ cell migration in the postnatal forebrain: monitoring living progenitors in slice preparations. Neuron 23, 461– 472.

J.G. Emsley, T. Hagg / Experimental Neurology 183 (2003) 273–285 Kanner, S., Grosmaire, L., Ledbetter, J., Damle, N., 1993. Beta 2-integrin LFA-1 signaling through phospholipase C-gamma 1 activation. Proc. Natl. Acad. Sci. U.S.A. 90, 7099 –7103. Kiss, J.A., 1998. A role of adhesion molecules in neuroglial plasticity. Mol. Cell. Endocrinol. 140, 89 –94. Kortesmaa, J., Yurchenco, P., Tryggvason, K., 2000. Recombinant laminin-8 (alpha(4) beta(1) gamma(1)). Production, purification, and interactions with integrins. J. Biol. Chem. 275, 14853–14859. Kirschenbaum, B., Doetsch, F., Lois, C., Alvarez-Buylla, A., 1999. Adult subventricular zone neuronal precursors continue to proliferate and migrate in the absence of the olfactory bulb. J. Neurosci. 19, 2171– 2180. Kuhn, H.G., Dickinson-Anson, H., Gage, F.H., 1996. Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation. J. Neurosci. 16, 2027–2033. Lee, M.K., Tuttle, J.B., Rebhun, L.I., Cleveland, D.W., Frankfurter, A., 1990. The expression and posttranslational modification of a neuronspecific beta-tubulin isotype during chick embryogenesis. Cell. Motil. Cytoskeleton. 17, 118 –132. Li, L., Liu, F., Salmonsen, R.A., Turner, T.K., Litofsky, N.S., Di Cristofano, A., Pandolfi, P.P., Jones, S.N., Recht, L.D., Ross, A.H., 2002. PTEN in neural precursor cells: regulation of migration, apoptosis, and proliferation. Mol. Cell Neurosci. 20, 21–29. Liesi, P., 1985. Do neurons in the CNS migrate in laminin? EMBO J. 4, 1163–1170. Liesi, P., Kaakola, S., Dahl, D., Vaheri, A., 1984. Laminin is induced in astrocytes of adult brain by injury. EMBO J. 3, 683– 686. Linnarsson, S., Willson, C.A., Ernfors, P., 2000. Cell death in regenerating populations of neurons in BDNF mutant mice. Brain Res. Mol. Brain Res. 75, 61– 69. Lois, C., Garcı´a-Verdugo, J.M., Alvarez-Buylla, A., 1996. Chain migration of neuronal precursors. Science 271, 978 –981. Lois, C., Alvarez-Buylla, A., 1994. Long-distance neuronal migration in the adult mammalian brain. Science 264, 1145–1148. Lois, C., Alvarez-Buylla, A., 1993. Proliferating subventricular zone cells in the adult mammalian forebrain can differentiate into neurons and glia. Proc. Natl. Acad. Sci. U.S.A. 90, 2074 –2077. Luskin, M.B., 1998. Neuroblasts of the postnatal mammalian forebrain: their phenotype and fate. J. Neurobiol. 36, 221–233. Luskin, M.B., 1993. Restricted proliferation and migration of postnatally generated neurons derived from the forebrain subventricular zone. Neuron 11, 173–189. Magavi, S.S., Leavitt, B.R., Macklis, J.D., 2000. Induction of neurogenesis in the neocortex of adult mice. Nature 405, 951–955. Mason, H.A., Ito, S., Corfas, G., 2001. Extracellular signals that regulate the tangential migration of olfactory bulb neuronal precursors: inducers, inhibitors, and repellents. J. Neurosci. 21, 7654 –7663. Menezes, J.R., Luskin, M.B., 1994. Expression of neuron-specific tubulin defines a novel population in the proliferative layers of the developing telencephalon. J. Neurosci. 14, 5399 –5416. Miller, M.W., Nowakowski, R.S., 1988. Use of bromodeoxyuridine-immunohistochemistry to examine the proliferation, migration and time of origin of cells in the central nervous system. Brain Res. 457, 44 –52. Millonig, G., 1961. Advantages of phosphate buffer for OsO4 solutions in fixation. J. Appl. Physics 32, 1637. Morshead, C.M., Reynolds, B.A., Craig, C.G., McBurney, M.W., Staines, W.A., Morasutti, D., Weiss, S., van der Kooy, D., 1994. Neural stem cells in the adult mammalian forebrain: a relatively quiescent subpopulation of subependymal cells. Neuron 13, 1071–1082. Muruyama, O., Nishida, H., Sekiguchi, K., 1996. Novel peptide ligands for integrin ␣6␤1 selected from a phage display library. J. Biochem. 120, 445– 451. Paulsson, M., 1992. The role of laminin in attachment, growth, and differentiation of cultured cells: a brief review. Cytotechnology 9, 99 – 106. Peretto, P., Merighi, A., Fasolo, A., Bonfanti, L., 1997. Glial tubes in the rostral migratory stream of the adult rat. Brain Res. 42, 9 –21.

285

Pinkstaff, J.K., Detterich, J., Lynch, G., Gall, C.M., 1999. Integrin subunit gene expression is regionally differentiated in adult brain. J. Neurosci. 19, 1541–1556. Pinkstaff, J.K., Lynch, G., Gall, C.M., 1998. Localization and seizureregulation of integrin ␤1 mRNA in adult rat brain. Mol. Brain Res. 55, 265–276. Ramalho-Santos, M., Yoon, S., Matsuzaki, Y., Mulligan, R.C., Melton, D.A., 2002. “Stemness:” transcriptional profiling of embryonic and adult stem cells. Science 298, 597– 600. Reynolds, B.A., Weiss, S., 1992. Generation of neurons and astrocytes from isolated cells of the adult mammalian central nervous system. Science 255, 1707–1710. Richards, L.J., Kilpatrick, T.J., Bartlett, P.F., 1992. De novo generation of neuronal cells from the adult mouse brain. Proc. Natl. Acad. Sci. U.S.A. 89, 8591– 8595. Schubert, W., Coskun, V., Tahmina, M., Rao, M.S., Luskin, M.B., Kaprielian, Z., 2000. Characterization and distribution of a new cell surface marker of neuronal precursors. Dev. Neurosci. 22, 154 –166. Schwartz, M., Denninghoff, J., 1994. Alpha v integrins mediate the rise in intracellular calcium in endothelial cells on fibronectin event though they play a minor role in adhesion. J. Biol. Chem. 269, 11133–11137. Shin, J.J., Fricker-Gates, R.A., Perez, F.A., Leavitt, B.R., Zurakowski, D., Macklis, J.D., 2000. Transplanted neuroblasts differentiate appropriately into projection neurons with correct neurotransmitter and receptor phenotype in neocortex undergoing targeted projection neuron degeneration. J. Neurosci. 20, 7404 –7416. Sonnenberg, A., Linders, C.J.T., Modderman, P.W., Damsky, C.H., Aumailley, M., Timpl, R., 1990. Integrin recognition of different cellbinding fragments of laminin (P1, E3, E8) and evidence that ␣6␤1 but not ␣6␤4 functions as a major receptor for fragment E8. J. Cell. Biol. 110, 2145–2155. Sonnenberg, A., Modderman, P.W., Hogervorst, F., 1988. Laminin receptor on platelets is the integrin VLA-6. Nature 336, 487– 489. Sonnenberg, A., Janssen, H., Hogervorst, F., Calafat, J., Hilgers, J., 1987. A complex of platelet glycoproteins Ic and IIa identified by a rat monoclonal antibody. J. Biol. Chem. 262, 10376 –10383. Talts, J.F., Sasaki, T., Miosge, N., Gohring, W., Mann, K., Mayne, R., Timpl, R., 2000. Structural and functional analysis of the recombinant G domain of the laminin alpha chain and its proteolytic processing in tissues. J. Biol. Chem. 275, 35192–35199. Thomas, L.B., Gates, M.A., Steindler, D.A., 1996. Young neurons from the adult subependymal zone proliferate and migrate along an astrocyte, extracellular matrix-rich pathway. Glia 17, 1–14. Wary, K.K., Mariotti, A., Zurzolo, C., Giancotti, F.G., 1998. A requirement for calveolin-1 and associated kinase Fyn in integrin signaling and anchorage-dependent cell growth. Cell 94, 625– 634. Wei, J., Shaw, L.M., Mercurio, A.M., 1998. Regulation of Mitogenactivated protein kinase activation by the cytoplasmic domain of the alpha6 integrin subunit. J. Biol. Chem. 273, 5903–5907. Wichterle, H., Garcı´a-Verdugo, J.M., Alvarez-Buylla, A., 1997. Direct evidence for homotypic, glia-independent neuronal migration. Neuron 18, 779 –791. Wilkins, J.A., Li, A., Ni, H., Stupack, D.G., Shen, C., 1996. Control of beta1 integrin function. Localization of stimulatory epitopes. J. Biol. Chem. 271, 3046 –3051. Wu, W., Wong, K., Chen, J.H., Jiang, Z.H., Dupuis, S., Wu, J., Rao, Y., 1999. Directional guidance of neuronal migration in the olfactory system by the protein Slit. Nature 400, 331–336. Zhang, Z., Galileo, D.S., 1998. Retroviral transfer of antisense integrin ␣6 or ␣8 sequences results in laminar redistribution or clonal cell death in developing brain. J. Neurosci. 18, 6928 – 6938. Zhou, F.C., 1990. Four patterns of laminin-immunoreactive structure in developing rat brain. Dev. Brain Res. 55, 191–201. Zhou, F.C., Azmitia, E.C., 1988. Laminin facilitates and guides fibre growth of transplanted neurons in adult brain. J. Chem. Neuroanat. 1, 133–146.