1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, 44 Binney Street, Boston, MA 02115, USA
2 Division of Neuroscience, The Childrens Hospital, Boston, MA 02115, USA
3 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
4 Center for Immunology and Inflammatory Diseases, Massachusetts General Hospital, Charlestown, MA 02129, USA
* The first three authors contributed equally
The last two authors contributed equally
Author for correspondence (e-mail: Rosalind_segal{at}dfci.harvard.edu)
Accepted 17 December 2001
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SUMMARY |
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Movies available on-line
Key words: BDNF, Granule cells, Cell migration, CNS, Mouse
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INTRODUCTION |
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The processes of granule cell differentiation and migration depend on many extracellular factors (Hatten, 1999). Among these are the neurotrophins. Brain-derived neurotrophic factor (BDNF) is expressed in cerebellar granule cells (Rocamora et al., 1993
; Wetmore et al., 1990
) and the level of BDNF expression increases during development. Granule cells also express the high affinity BDNF receptor, TrkB (NTRK2; Klein et al., 1990
; Segal et al., 1995
). Activated Trk receptors can be detected in the EGL of wild-type mice, and receptor activation is reduced in Bdnf/ mice, indicating that TrkB receptors function during early granule cell development (Schwartz et al., 1997
). Thus BDNF is one of the factors that could regulate early granule cell development.
It is well established that BDNF promotes granule cell survival (Lindholm et al., 1993; Schwartz et al., 1997
; Segal et al., 1992
) and can stimulate axonal outgrowth by these cells (Gao et al., 1995
; Segal et al., 1995
). In accordance with these well established functions of BDNF, analysis of Bdnf/ mice revealed impaired survival of granule cells (Schwartz et al., 1997
). However, analysis of the mutant mice also resulted in a surprising finding although there is increased granule cell apoptosis in the absence of BDNF, the EGL is actually thicker in mutant than in wild-type littermates (Jones et al., 1994
; Schwartz et al., 1997
). There are several potential explanations for this finding. The layering defect could result from delayed differentiation of granule cells. This explanation accords well with the known ability of neurotrophins to promote neuronal differentiation (Henderson, 1996
). Alternatively, this defect in layer formation could reflect a novel role for BDNF in migration of granule cells.
Here we show that BDNF stimulates migration of granule cells. In the absence of BDNF, migration of granule cells is impaired both in vivo and in vitro. The impaired migration of granule cells can be rescued by exogenous BDNF, indicating that it is a direct result of the lack of BDNF. Acute addition of BDNF immediately stimulates granule cells to begin migration, indicating that BDNF is a motogenic factor. Furthermore, BDNF produces a directional cue that prompts the radial migration of granule cells. The accumulating evidence that BDNF exerts motogenic and chemotactic effects and thereby regulates migration adds to a growing and diverse list of neurotrophin functions.
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MATERIALS AND METHODS |
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In vivo BrdU labeling and immunohistochemistry
Mice were injected subcutaneously with 50 mg/kg of 5-bromo-2'-deoxyuridine (Sigma) and the animals were sacrificed at the indicated times. The pups were perfused with 4% paraformaldehyde and 14 µm sagittal cryostat sections were prepared. These were permeabilized in 0.4% Triton X-100, treated for 1 hour at 37°C with 2 M HCl followed by two 10-minute washes in 0.1 M sodium borate and stained with anti-BrdU antibody (Boehringer Mannheim) at 1:50, and visualized with Cy3- or HRP-linked secondary antibodies (Jackson Laboratories). In migration experiments the number of labeled cells/mm in each cortical layer was counted in five non-adjacent mid-sagittal sections on folia 6 within the primary fissure and on folia 9 in the secondary fissure. Four sets of Bdnf littermates (counted at 42 and 96 hours post-labeling) were used.
In vitro migration assays
Radial migration assays were carried out using primary glial and neuronal cells purified from P5-P7 wild-type and Bdnf/ cerebella as described by Hatten (Hatten, 1985) with a few modifications (Rio et al., 1997
). Cerebella were collected and the meninges were removed. Tissue blocks were incubated in 1% trypsin (Sigma) with 0.1% DNaseI (DNase, Worthington) in PBS for 10 minutes at 37°C and triturated in Ca2+/Mg2+-free HBSS (Gibco BRL) with 0.1% DNase using fire-polished Pasteur glass pipettes. The cell suspension was layered on top of a two step Percoll gradient (35/60%, Pharmacia Biotech) in PBS. After a 3,000 rpm centrifugation at 4°C for 10 minutes, the glia rise to the top of the gradient, and the neurons are found in the 35-60% interface. Cell fractions were washed once in PBS and twice in basal medium Eagle (BME) before use. Previous studies have demonstrated that the granule cells prepared and plated in this way are derived from the EGL, and predominantly express the early transcription factor ATOH1 (MATH1) (Hatten, 1985
; Kenney and Rowitch, 2000
).
For glial cultures, cells were resuspended in BME with 10% fetal bovine serum (FBS), 0.1% glucose, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin. Cells were preplated for 25 minutes on an uncoated tissue culture flask to remove contaminating fibroblasts, and transferred to a tissue culture flask coated with 25 µg/ml of poly-D-lysine (Collaborative) for 1 hour to allow glial attachment; the flask was rinsed and left in the same medium. Glial cells were used for no more than two passages.
For granule cell cultures the cells were resuspended in the same media as above. Glia were removed by plating the cells on tissue culture dishes coated with 25 µg/ml poly-D-lysine 3 times for approximately 1 hour each. After each preplating period, the non-adherent cells were gently removed. The purified neurons were plated on top of the glial cells at a ratio of 5-10 neurons per glial cell and cultures were maintained for 20-24 hours prior to time-lapse analysis.
Time-lapse video microscopy and quantification of migration
Cultures, supplemented with 10 mM Hepes and covered with a layer of mineral oil to prevent evaporation, were placed in a Leiden microincubator (Medical Systems Corp.) and kept at 35°C (Narishige MS-C temperature controller). Granule cells, identified by their characteristic cell body and nucleus size and shape, were chosen for imaging if they were attached to a glial cell with a radial process greater than 50 µm. In each experiment, 20 different fields containing at least one neuron attached to a radial glial fiber were recorded. The following variables were measured: (1) the total number of neurons; (2) the percentage of neurons that attached to radial glia; (3) the migratory index, the percentage of neurons attached to radial glia that migrated at more than 10 µm/hour; (4) the average speed of migration of the migrating neurons. Speed of migration was calculated by measuring the position of individual granule cells along glial fibers every 4 minutes over 120-180 minutes using Openlab 2.06 imaging software. Data shown represent the average speed of migrating cells over the course of 120-180 minutes of observation.
Chemotaxis assay
Purified populations of granule cells were obtained from post-natal day 8 (P8) Bdnf+/+ and Bdnf/ littermates. Purified cells were resuspended at 2-5x106 cells/ml in serum-free DMEM (supplemented with N2 growth medium (Gibco, Grand Island, NY) and 20 mM KCl), for use in chemotaxis assays. The in vitro migration of granule cells was assessed using laminin (Sigma; 20 µg/ml) coated polyvinylcarbonate-free membranes (Neuroprobe Inc., Gaithersburg, MD) with 12 µm pore size in modified Boyden chambers as previously described (Garcia-Zepeda et al., 1996; Klein et al., 2001
). Briefly, 50 µl of a solution containing 7x106 cells/ml in serum-free DMEM was placed in the upper chamber. BDNF (30 ng/ml) in serum-free DMEM was added to lower chambers alone, or to both chambers. After overnight incubation at 37°C in 8% CO2, the upper surface of membranes were scraped free of cells and debris, membranes were air-dried, then fixed and stained using Dif-quik cell fixation and staining kit (Dade Behring Inc., Newark, DE). Cells that had migrated through pores and adhered to the membrane were analyzed under high-power light microscopy and counted in five adjacent high-power fields (area of HPF=0.78 mm2; area of each filter=7.07 mm2). Experiments were performed in duplicate or triplicate and data are expressed as numbers of cells per high-power field (cells/HPF)±s.e.m. Using cells from wild-type animals in control medium 535 cells per HPF migrated into the lower chamber, corresponding to 1.4% of plated cells. In these short term assays BDNF does not affect proliferation or cell number (P. R. B. and R. A. S., unpublished observations). Data were analyzed for statistical significance between groups using Students t-test.
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RESULTS |
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To confirm that the difference in migration is due to a lack of BDNF, we tested the ability of exogenous BDNF to rescue this defect. BDNF (5 ng/ml) was added to the granule cell/glial cell co-cultures at the time of plating and migration was recorded 24 hours later. Exogenous BDNF increased the migratory index in both wild-type and mutant cultures, resulting in the complete elimination of any difference in migration between the two genotypes (Fig. 4). BDNF did not affect the ability of granule cells of either genotype to attach to glial fibers, nor did it affect the average speed of migration. Furthermore, in this short time period, BDNF did not alter the ability of granule cells to survive. Thus BDNF specifically rescued the defect in initiation of migration that was seen in mutant cells.
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In all the experiments thus far, granule cells were stimulated by exogenous BDNF applied uniformly. To determine whether a gradient of BDNF, as occurs in vivo, stimulates directed movement of granule cells, we analyzed the chemotactic effects of BDNF on wild-type granule cells using the Boyden chamber assay. Addition of BDNF to the lower compartment only, establishing a BDNF concentration gradient, increased migration approximately two-fold when compared to medium alone (Fig. 6B). Furthermore, larger numbers of granule cells migrated in response to a gradient of BDNF than in response to a uniform distribution of BDNF, consistent with prior studies (Lu et al., 2001). This indicates that BDNF can function as a chemotactic factor for wild-type granule cells that are competent to migrate.
We conclude that BDNF has a dual role in stimulating granule cell migration. Endogenous BDNF, acting in a paracrine or autocrine fashion, promotes the initiation of migration. Purified granule cells from animals that lack BDNF exhibit defective migration, and this defect can be rescued by acute application of exogenous factor. In addition, a gradient of BDNF stimulates directed movement of granule cells. Thus BDNF is both a motogenic and a chemotactic factor for granule cells.
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DISCUSSION |
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During prenatal development, granule cell precursors migrate from the rhombic lip to the EGL. Then, in a second wave of migration, granule cells attach to radial glia and migrate inwards from the EGL to the IGL. During this radial migration, the granule cells traverse three distinct cerebellar laminae. First they begin to migrate and leave the EGL, second they cross the molecular layer and finally they arrive in the IGL. Using BrdU labeling to visualize a cohort of granule cells, we find that there is a significant delay in the migration of granule cells out of the EGL in Bdnf/ mice (Fig. 1). This in vivo defect in the initiation of radial migration can be recapitulated in vitro, both in a radial migration assay and in a Boyden chamber migration assay. Exogenous BDNF rescues the migration defect of mutant cells. Granule cells of the mutant mice have been subject to chronic BDNF deprivation, and so could exhibit an aberrant response to BDNF. Therefore, an important feature of the data (Fig. 4) is that exogenous BDNF also stimulates radial migration of wild-type cells, by increasing the migratory index beyond the basal level. This gain of function experiment complements the results obtained with the mutant cells, and indicates a specific role for BDNF in promoting the initiation of migration. The rapidity with which BDNF stimulates migration provides further evidence that this is a direct effect. This motogenic or chemokinetic effect was observed in cells exposed to a uniform distribution of BDNF. Taken together, these data indicate a specific role for BDNF in inducing granule cells to initiate radial migration.
In vivo, BDNF mRNA and protein is expressed at higher levels in the IGL than in the EGL (Fig. 7) (Rocamora et al., 1993). Thus the developing granule cells migrate up a BDNF concentration gradient. This gradient may provide one of the directional cues responsible for the inward migration of granule cells along radial glia. Using a Boyden chamber assay, we have shown that a gradient of BDNF can directly induce granule cell chemotaxis. In agreement with our results, others have also found that BDNF acts as a chemoattractant for purified wild-type granule cells (Lu et al., 2001
). Thus, BDNF provides granule cells with both an impetus to initiate migration and a directional cue.
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Together the data presented here indicate that BDNF both stimulates migration of granule cells, and helps guide the migrating granule cells from the EGL to the IGL. These direct effects of BDNF on granule cell migration explain the layering defect of the BDNF mutant animals, in that the granule cells are impaired in their ability to initiate movement from the EGL to the IGL. Factors, such as Stromal cell derived factor, SDF (Ma et al., 1998; Klein et al., 2001
), Ephrins (Lu et al., 2001
) or MIA (Mason et al., 2001
), are likely to work in concert with BDNF to regulate the radial migration of granule cells from the EGL to the IGL. We hypothesize that these other factors provide some redundancy, enabling the granule cells in Bdnf/ mice to migrate eventually.
Since radial migration is a widespread phenomenon in the developing CNS, BDNF may have a general function in stimulating migration of early neuronal cells. In the cortex, BDNF, acting via the TrkB receptor, has been implicated as a chemotactic factor for early neurons (Behar et al., 1997), perhaps by regulating expression of reelin (Ringstedt et al., 1998
). The related neurotrophin, NGF, can induce expression of cdk5 (Harada et al., 2001
), a molecule critical for neuronal migration (Harada et al., 2001
). However, these changes in gene expression are likely to be slow responses to neurotrophins. A novel finding here is the acute nature of the response to BDNF, suggesting that BDNF stimulates the migratory machinery, and does not merely regulate expression of proteins involved in migration. Future studies will be needed to determine if BDNF is required for radial migration of cortical neurons, in a manner analogous to the results shown here for cerebellar granule cells.
Neurotrophins activate two types of receptors, the Trk family of receptor tyrosine kinases and the p75 NTR (Kaplan and Miller, 2000). Stimulation of migration by BDNF apparently relies on activation of the TrkB receptors rather than the p75 NTR. The EGL is enlarged in animals with mutations of Trkb (Ntrk2) and Trkc (Ntrk3) (Minichello and Klein, 1996
), but not in p75NTR mutant animals (A. R. C. and R. A. S., unpublished observations). Consistent with this interpretation, K252a, a pharmacological inhibitor of kinases including the Trks, hinders migration in Boyden chambers and in organotypic cultures (data not shown). In vivo, a gradient of BDNF may lead to the preferential activation of TrkB receptors located at the leading edge of the migrating granule cells, with lower levels of activated receptors at other subcellular locations.
The signaling pathways activated by Trk and responsible for neurotrophin-induced migration of granule cells have not yet been defined. However, studies examining the signaling pathways that enable NGF to function as a chemotactic agent for mast cells (Sawada et al., 2000), and numerous studies on cell migration in invertebrates suggest that activation of phosphatidylinositol 3 kinase or of small GTPases (Montell, 1999
) are likely candidates. Differential localization of activated receptors and of critical signaling intermediates (Haugh et al., 2000
) within granule cells could provide a mechanism enabling BDNF both to stimulate migration and to provide a directional cue for movement.
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ACKNOWLEDGMENTS |
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