1 Center for Immunology and Inflammatory Diseases, Division Rheumatology, Allergy and Immunology, Massachusetts General Hospital, Harvard Medical School, Charlestown, MA 02129, USA
2 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston, MA 02115, USA
3 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
4 Department of Pathology, New England Regional Primate Research Center, Harvard Medical School, Southboro, MA 01772, USA
* These two authors contributed equally
Author for correspondence (e-mail: rklein{at}partners.org)
Accepted March 1, 2001
![]() |
SUMMARY |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Key words: Chemokine, SDF-1, Sonic hedgehog, Granule cell, Cerebellum, Mouse
![]() |
INTRODUCTION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Factors that localize granule cell precursors to the proliferative environment of the EGL would be expected to enhance their proliferation. The chemokine, stromal-cell-derived factor (SDF)-1 has been identified as one such factor, as targeted deletion of the gene encoding SDF-1
or its receptor, CXCR4, resulted in the premature migration of granule cell precursors out of the EGL (Ma et al., 1998; Zou et al., 1998). SDF-1
has been shown to be expressed in the pia mater during embryonic development and its receptor CXCR4 is expressed in the EGL during the same period (McGrath et al., 1999). Together these data suggest that SDF-1
and CXCR4 play a role in retaining granule cells in the EGL.
Chemokines are a superfamily of over 40 structurally homologous chemotactic cytokines that are involved in the trafficking of leukocytes to areas of inflammation (Luster, 1998). Chemokines induce cell migration and activation by binding to a subfamily of seven transmembrane-spanning receptors shown to be coupled to pertussis toxin sensitive Gi proteins (Premack and Schall, 1996). Most chemokine receptors bind more than one chemokine ligand and many chemokines bind more than one chemokine receptor, however, SDF-1
binds only CXCR4 and is its only ligand (Rossi and Zlotnik, 2000). CXCR4 is expressed on all leukocytes and more recently has been shown to be present on subpopulations of cortical neurons (Lavi et al., 1997; Westmoreland et al., 1998; Zhang et al., 1998; Banisadr et al., 2000). While the role of CXCR4 and SDF-1
in leukocyte trafficking and activation has been extensively studied, very little is known about their function in neuronal physiology. We and others have shown that human fetal cortical neurons express functional CXCR4 in vitro (Sanders et al., 1998; Klein et al., 1999). SDF-1
activates neuronal CXCR4 and induces increases in intracellular calcium. As in leukocytes, neuronal responses to SDF-1
are pertussis toxin sensitive, indicating coupling of neuronal CXCR4 to G
i (Klein et al., 1999). The expression of CXCR4 and the ability of SDF-1
to induce calcium transients has been observed in rat cerebellar granule cells (Limatola et al., 2000). SDF-1
has also been shown to induce chemotaxis of rat neuronal progenitor cells and to affect apoptotic responses to various stimuli in cultured rat neurons (Meucci et al., 1998; Kaul and Lipton, 1999; Zheng et al., 1999; Lazarini et al., 2000).
SDF-1 has multiple effects on hematopoietic cells, including localization, augmentation of proliferation and promotion of survival (Nagasawa et al., 1994; Nagasawa et al., 1996; Bleul et al., 1998; Bradstock et al., 2000; Lataillade et al., 2000; Nanki and Lipsky, 2000). We hypothesized that SDF-1
could play similar roles in both granule cell proliferation and in the maintenance of the EGL. We demonstrate here that SDF-1
is a chemoattractant for cerebellar granule precursor cells and acts synergistically with SHH to promote their proliferation. The interaction between SDF-1
and SHH occurs via a pertussis toxin-sensitive pathway, suggesting it occurs through G
i. CXCR4 expression in the neonatal cerebellum peaks during the first postnatal week, coinciding with peak SDF-1
-induced chemotactic and proliferative responses. These studies support a model of cerebellar development in which SDF-1
is essential for the maintenance of the EGL and the establishment of granule cell number.
![]() |
MATERIALS AND METHODS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Northern blot analysis
Whole litters of mouse pups were sacrificed at postnatal days 3, 5, 7, 9 and 12 and cerebella were rapidly removed and snap-frozen on dry ice. RNA was obtained from pooled cerebella using lysis in guanidium hydrochloride followed by pelleting through a CsCl2 gradient as previously described (Khan et al., 2000). 20 µg total RNA was electrophoresed on a 1.2% agarose formaldehyde gel and then capillary transferred to a GeneScreen membrane (NEN Life Science Products, Boston, MA). Prehybridization and probe preparations were caried out according to previously published procedures (Khan et al., 2000). A 584 bp coding fragment of the murine CXCR4 cDNA cloned into the BamHI site of T7T3 vector (Incyte Genomics, Inc., St. Louis, MO) was used for probe synthesis after confirmatory sequence was obtained. A 180 bp coding fragment of murine SDF-1 gene was obtained by PCR of total murine thymic RNA and cloned into the BamHI site of Bluescript (ks) (Promega Life Sciences, Madison, WI). 32P-labeled CXCR4 and SDF-1
probes were generated by random primed DNA synthesis of BamHI fragments according to the manufacturers instructions (Boehringer Mannheim, Mannheim, Germany). Glyceraldehylde-3-phosphate dehydrogenase (GAPDH) (kindly provided by M. Pyrstowsky) was used as a control for RNA loading. Signal was quantified using a phosphoimager (Molecular Imager System, Bio-Rad, Hercules, CA) and values for each sample were normalized based on the GAPDH signal.
In situ hybridization and immunohistochemical analyses
Tissue preparation
Brains from postnatal days 4 and 8 BALB/c mice were removed, postfixed in 4% paraformaldehyde for 24 hours and cryoprotected in 30% sucrose. Serial sagittal sections (15 µm) of cerebella were obtained using a Reichart Jung Cryostat.
Probe preparation
The orientation of CXCR4 and SDF-1 probes was determined by direct sequencing. Sense and antisense digoxigenin-labeled riboprobes were synthesized according to the manufacturers instructions (Boehringer Mannheim, Mannheim, Germany).
In situ hybridization
Formadehyde fixed tissue sections were digested with 20 µg/ml proteinase K for 10 minutes at room temperature. Sections were refixed in 4% paraformaldehyde and washed in PBS. In situ hybridization was conducted for 20 hours at 65°C using DIG-labeled cRNA probes in hybridization buffer (50% formamide, 5x SSC, 100 µg/ml yeast tRNA, 100 µg/ml heparin, 1x Denhardts, 0.1% Tween 20, 0.1% CHAPS, 5 mM EDTA). The sections were washed with 0.2x SSC, 0.1% Tween 20 at 65°C and then treated with blocking reagent (20% sheep serum in buffer) and then anti-DIG antibody followed by antibody detection according to the manufacturers protocol (Boehringer Mannheim, Mannheim, Germany).
Immunohistochemistry
Tissue sections were permeablized with 0.1% Triton X-100 (Sigma) and nonspecific antibody binding was blocked with 5% normal goat serum (Biofluids, Rockville, MD) for 1 hour at room temperature. Polyclonal antibodies specific for CXCR4 and SDF-1 were applied at 1 µg/ml in PBS containing 5% normal goat serum and 0.1% Triton X-100 overnight at 4°C. Sections were washed free of unbound antibody by repeated immersion in PBS containing 5% normal goat serum and 0.1% Triton X-100. Primary antibodies were detected with secondary goat anti-rabbit antibody conjugated to Cy3. Nuclei were counterstained with DAPI (Sigma) for 1 minute at room temperature. Sections were then washed with 20 mM Tris, and mounted using Immunomount (Shandon, Pittsburgh, PA).
Immunocytochemistry and confocal microscopy
Granule cells were prepared and seeded onto coverslips as described above and cultured for 1-2 days. Cells were fixed in 4% paraformaldehyde for 1 hour, washed with sterile PBS and then immunostained. Cells were treated for 30 minutes with a blocking buffer consisting of 10% normal goat serum and 5% mouse serum in sterile PBS. Cultures were assessed for purity using a monoclonal antibody to calbindin (a marker for Purkinje cells). Cultures were found to contain 3-4% Purkinje cells (
96% granule cells). Double and single-label immunofluorescence was performed using polyclonal antibodies to CXCR4 (1 µg/ml; kindly provided by Jose Gonzalo, Millenium Pharmaceuticals Inc., Cambridge, MA; Gonzalo et al., 2000) and monoclonal antibody to calbindin (1:200; Sigma) plus topro-3 (Molecular Probes, Eugene, OR) staining to identify cell nuclei. Primary antibodies were detected using secondary anti-rabbit or mouse antibodies conjugated with Texas Red (1:200; Sigma) or FITC (1:200; Sigma), respectively. Control coverslips were treated with blocking buffer (containing non-immune serum) and then secondary antibodies alone. Confocal microscopy was performed using a Leica TCS SP laser scanning microscope, fitted with a 100x Leica objective (PL APO, 1.4 NA), and using the Leica image software. Images were collected at 512x512 pixel resolution. The stained cells were optically sectioned in the z-axis and the images in the different channels (photo-multiplier tubes) were collected simultaneously. The step size in the z-axis was varied from 0.2 to 0.5 µm to obtain 30-50 slides per imaged field. The images were transferred to a Macintosh G3 computer and NIH Image v1.61 software was used to render the images.
Chemotaxis assay
The in vitro migration of granule cells in response to human SDF-1 (Peprotech Inc., Rocky Hill, NJ) was assessed using poly-D-lysine (PDL) or laminin (Sigma) (20 µg/ml) coated polyvinylcarbonate-free membranes (Neuroprobe Inc., Gaithersburg, MD) with 8-12 µm pore size in a modified Boyden chambers as previously described (Garcia-Zepeda et al., 1996). Most experiments were conducted using granule cells prepared from the cerebella of P7-10 BALB/c mouse pups or P3-4 and P7-8 rat pups. Briefly, 2-6x106 cells/ml in 50 µl serum-free DMEM were added to upper chambers. To observe chemotaxis, SDF-1
was added to lower chambers in doses from 0 ng/ml to 10,000 ng/ml in serum-free DMEM. To observe chemokinesis, SDF-1
was also added to upper chambers. In some experiments, cells were pretreated with 200 ng/ml pertussis toxin for 1 hour (Sigma) or preincubated with anti-CXCR4 polyclonal antibodies. Chemotaxis to the chemokine MIP-1
(Peprotech Inc.) was also assessed as a negative control. 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. Experiments were performed in triplicate and data are expressed as numbers of cells per high-power field (cells/HPF) ± s.e.m. Data were analyzed for statistical significance between groups using Students t-test.
Proliferation assay
Primary cultures of mouse cerebellum were established as previously described (Segal et al., 1995). Briefly, cerebella of P6 BALB/c mice were removed and placed in HBSS with 6 mg/ml glucose and 15 mM Hepes pH 7.4. Meninges were removed under 10x magnification and tissue was digested with 1 mg/ml trypsin with 125 U/ml DNase (Sigma), 0.15 mM EDTA and 15 mM Hepes pH 7.4 for 20 minutes at 37°C. Digestion was stopped by the addition of an equal volume of 20% fetal calf serum in DMEM. Tissue was pelletted in a clinical centrifuge by spinning for 4 minutes at 740 g. Cells were washed twice by resuspending in HBSS and spinning as above. The final cell suspension was passed through a 100 µm cell strainer. Cells were counted and diluted to a final concentration of 2x106 cells/ml with DMEM/F12 supplemented with N2 growth medium, 6 mg/ml glucose and 20 mM KCl. Cells were plated in the presence of all additives such as SHH (0.14 µg/ml or 7 nM; Curis Inc., Cambridge, MA), human SDF-1 (1000 ng/ml; Peprotech Inc.), pertussis toxin (25 ng/ml; Sigma), or forskolin (10 µM; Sigma) onto 96-well culture plates (Falcon) for approximately 20 hours before the addition of 5 µCi/well of [3H]thymidine (1 mCi/ml, NEN Inc., Boston, MA). After 4 hours of labeling, cells were lysed with 1% Triton X-100 for 10 minutes at room temperature. Subsequently, trichloroacetic acid (TCA) was added to 10% and incorporated thymidine was precipitated for 1 hour on ice. Precipitates were collected by vacuum filtration and filters were washed extensively with ice-cold TCA and dried with 100% ethanol. Dried filters were solubilized in Scint-Safe liquid scintillant (NEN Inc., Boston, MA) and incorporated counts were determined with a standard liquid scintillation counter. Experiments were performed in triplicate and data are expressed as fold-induction over control ± s.e.m. Data were analyzed for statistical significance between groups using Students t-test.
Calcium flux analysis
Peripheral blood mononuclear cells
Human peripheral blood mononuclear cells (PBMCs) were obtained from whole peripheral blood of healthy donors by passing blood through a Ficoll-Hypaque (Sigma) density gradient as previously described (Garcia-Zepeda et al., 1996). PBMCs were washed in HBSS and loaded with 5 µM fura-2 AM (Molecular Probes, Eugene, OR) for 60 minutes in a dark chamber at 37°C at 5x106 cells/ml in RPMI/1% heat-inactivated FCS. Loaded cells were washed twice and resuspended in calcium flux buffer as previously described (Garcia-Zepeda et al., 1996). Two milliliters of cells were placed in a continuously stirring cuvette at 37°C in a dual-wavelength excitation source fluorimeter (Photon Technology Inc, South Brunswick, NJ). Changes of cytosolic free calcium were determined after addition of SDF-1 (Peprotech Inc.) by monitoring the excitation fluorescence intensity emitted at 510 nm in response to sequential excitation at 340 nm and 380 nm. The data are presented as the relative ratio of fluorescence at 340 and 380 nm.
Granule cells
Granule cells were prepared as described above, and seeded onto 110 µm thick coverslips and cultured as above. Cells were washed with PBS and loaded with 5 µM fura-2 for one hour in a dark chamber at 37°C. Cells were then washed with PBS and kept at 37°C under serum-free medium until analyzed for calcium flux responses (up to 1 hour). For calcium flux analysis, coverslips were placed under buffer at 37°C and examined under an inverted microscope connected to a spectrofluorimeter. Groups of 10-20 neurons were analyzed for their response to stimulation with 100 ng/ml SDF-1 after predepolarization with 20 mM KCl or 100 µM glutamate. Additional experiments were performed after pretreatment of neurons with 200 ng/ml pertussis toxin or 10 µg/ml polyclonal antibodies to CXCR4. Calcium flux tracings were analyzed for the maximum increase in intracellular calcium according to the formula [Ca]i=224 nM [(R-Rmin/(Rmax-R)], assuming a Kd of 224 nM, as previously described (Rothenberg et al., 1996), where Rmax is the amount of calcium increase after treatment with the nonspecific calcium ionophore ionomycin (5 µg/ml) and Rmin is the level of calcium following calcium chelation with Tris-EDTA. Calcium concentrations are expressed as the mean level ± s.e.m.
![]() |
RESULTS |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
|
|
SDF-1 induces chemotaxis of granule cells
SDF-1-induced chemotaxis has been reported for various leukocyte populations and for rodent neural cell types. Given the high level of expression of SDF-1
observed in the pia mater overlying the CXCR4-positive EGL, we tested the ability of SDF-1
to induce chemotaxis in purified populations of granule cells in vitro. Utilizing a modified Boyden chamber microchemotaxis assay, we detected specific migration of granule cells toward SDF-1
. Dose response studies showed a biphasic curve that is characteristic for chemoattractant-induced migration observed in leukocytes with a peak at 1000 ng/ml of the chemoattractant (Fig. 3A). Granule cells did not demonstrate a significant chemotactic response in control experiments using the chemokine MIP-1
, whose receptors, CCR1 and CCR5, are not present on granule cells (Fig. 3A). The chemotactic response to SDF-1
was abolished by pretreatment of cells with pertussis toxin or with a polyclonal antibody to CXCR4 (Fig. 3B). In addition, chemokinesis controls, in which chemokine was added to both the upper and lower wells of the chemotaxis chamber were negative, demonstrating that SDF-1
induces directed cell migration rather than random movement (Fig. 3B). The effect of pertussis toxin on SDF-1
-induced chemotaxis of granule cells is consistent with coupling of neuronal CXCR4 to G
i, as it is in leukocytes.
|
SDF-1 and SHH are synergistic for granule cell proliferation
CXCR4 mRNA expression and the SDF-1-induced chemotactic response of granule cells are greatest in tissue and cells examined during the first postnatal week, the time of maximal granule cell proliferation (Miale and Sidman, 1961; Lodin and Srager, 1970). We questioned whether SDF-1
might also play a direct role in the establishment of granule cell number during cerebellar development. Several granule cell mitogens have been identified including EGF, FGF and SHH (Gao et al., 1991; Tao et al., 1996; Ye et al., 1996; Lin and Bulleit, 1997; Dahmane and Ruiz-i-Altaba, 1999; Traiffort et al., 1999; Wallace, 1999; Wechsler-Reya and Scott, 1999). SHH is the most potent of these (Dahmane and Ruiz-i-Altaba, 1999; Wechsler-Reya and Scott, 1999). SHH activity is known to be antagonized by protein kinase A, presumably in the setting of increased cAMP (Jiang and Struhl, 1995; Epstein et al., 1996; Hammerschmidt et al., 1996; Noveen et al., 1996; Hammerschmidt and McMahon, 1998). Based on our chemotaxis data indicating neuronal CXCR4 coupling to G
i, we reasoned that SDF-1
might decrease intracellular cAMP and enhance SHH-induced proliferation. Primary cultures of mouse cerebellum derived from P6 mice were treated for 24 hours with SHH at 0.14 µg/ml (7 nM) in the presence or absence of 1000 ng/ml SDF and or pertussis toxin or forskolin, and proliferation was measured by tritiated thymidine incorporation. Consistent with previous studies, nanomolar SHH produced a 2.5-fold increase in proliferation (Fig. 4). Proliferation was significantly enhanced (+50%) when primary cultures were coincubated with SDF-1
and SHH. SDF-1
enhancement of SHH-induced proliferation was blocked by treatment of cultures with pertussis toxin. Pertussis toxin alone had no effect on control or SHH-induced proliferation. In addition, as has been previously shown (Noveen et al., 1996), treatment with forskolin abolishes all SHH activity presumably by increasing cAMP activation of protein kinase A. SDF-1
had no proliferative activity on its own. These data demonstrate an interaction between SDF-1
and SHH that promotes granule cell proliferation in a pertussis toxin sensitive manner. The coincident expression of SDF-1
, CXCR4 and SHH during a period of maximal granule cell proliferation suggests that this is a relevant in vivo relationship.
|
|
![]() |
DISCUSSION |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Granule cell chemotaxis and maintenance of the EGL
A role for SDF-1 and its receptor CXCR4 in the formation and maintenance of the EGL was first suggested by targeted gene deletion of each of these genes. Normally, most granule cell precursor proliferation and migration occurs postnatally (Miale and Sidman, 1961; Lodin and Srager, 1970; Altman, 1972; Burgoyne and Cabray-Deakin, 1988; Hatten, 1993; Altman and Bayer, 1996; Doughty et al., 1998; Raetzman and Siegel, 1999). Deletion of SDF-1
or CXCR4 resulted in premature migration of granule cell precursors away from the proliferative environment of the EGL, with small numbers of granule cell precursors found ectopically, outside EGL, by E18 (Ma et al., 1998; Zou et al., 1998). The mechanisms responsible for these effects is not known. Based on SDF-1
s chemoattractant effects on leukocytes and some neural cell types (Aiuti et al., 1997; Tanabe et al., 1997; Lazarini et al., 2000), we hypothesized that chemoattraction of granule cells to a pial source of SDF-1
might serve to promote their localization to the EGL. This would be similar to the effect of SDF-1 on lymphocyte localization in secondary lymphoid organs. At these sites, subsets of B-lymphocytes that respond to SDF-1
localize to specific microenvironments where they proliferate and differentiate (Bleul et al., 1998). We report here that granule cells display dose-dependent chemotaxis to SDF-1
, which peaks at a concentration similar to the peak chemotactic dose observed for B-lymphocytes (Bleul et al., 1998). In addition, chemotactic responses were observed to be greater in cells obtained from animals at P3-4 compared with those from animals at P7-8, coinciding with the peak expression of CXCR4 as demonstrated by northern blot analysis. The localization of SDF-1
to the pia and CXCR4 to granule cells of the EGL suggests that, in vivo, SDF-1
attracts granule cells towards the pia mater. This supports the hypothesis that maintenance of granule cell residence in the EGL is a function of SDF-1
chemoattraction and provides a mechanism for the observation that granule cells migrate prematurely out of the EGL in animals lacking SDF-1
or CXCR4 (Ma et al., 1998; Zou et al., 1998).
Granule cell migration from the EGL peaks between P8 and P12 in vivo. This period coincides with the observed decline in the expression of CXCR4 and is consistent with SDF-1 playing an inhibitory role in granule cell emigration from the EGL. Other factors essential for granule cell migration, such as BDNF, display an opposite pattern of expression, increasing during the period of peak granule cell migration out of the EGL (Maisonpierre et al., 1990). Thus, the age-dependent decrease in expression of CXCR4 and increasing promigratory effects of BDNF may both contribute to the movement of granule cells out of the EGL.
SDF-1 enhances SHH-Induced granule cell proliferation
Previous experiments to explore possible interactions between SHH and the other known mitogens for granule cells have failed to demonstrate any additive or synergistic effects (Wechsler-Reya and Scott, 1999). We report here that SDF-1 directly enhances the proliferative effects of SHH without possessing any mitogenic activity of its own. We demonstrate that SDF-1
increases the proliferative effects of nanomolar SHH by approximately 50%; increasing a 2.5-fold mitogenic effect to a nearly 4-fold effect. Importantly this concentration of SHH may better approximate physiologic concentrations. Recent studies have shown that SDF-1
, acting together with other cytokines can induce the proliferation of CD4+ T lymphocytes and pluripotent hematopoietic stem cells (Lataillade et al., 2000; Nanki and Lipsky, 2000). Thus proliferative responses to SDF-1
of both immune and neural cell types can require receptor cross talk.
SDF-1 is likely to have a second, less direct, effect on granule cell proliferation through its maintenance of granule cell residence in the specialized proliferative environment of the EGL. Increased granule cell proliferation secondary to prolonged position within the EGL has recently been described in BDNF null mice (P. B. Borghesani and R. A. S., unpublished observations). Thus SDF-1
is likely to enhance granule cell proliferation both directly, by augmenting SHH effects and indirectly, by localizing granule cells to the EGL during the first postnatal week. The loss of direct and indirect pial-derived SDF-1
effects on granule cell proliferation could explain the decreased granule cell numbers in the cerebella of animals in whom SDF-1
or CXCR4 had been deleted (Ma et al., 1998; Zou et al., 1998), as well as the observation that ablation of the pia in neonatal animals results in thinning of the cerebellar cortex (Pehlemann et al., 1985). The decline in CXCR4 expression observed during the second postnatal week could contribute to the observed decrease in granule cell proliferation during the same time period (Altman and Bayer, 1996).
SDF-1 signaling in cerebellar granule cells
In some regards, SDF-1 signaling in cerebellar granule cells appears to resemble SDF-1
signaling in leukocytes. Calcium flux and chemotaxis are mediated through CXCR4 activation and can be blocked by pertussis toxin. However, granule cell SDF-1
responses differ from leukocyte responses in their requirement for membrane depolarization. No calcium flux in response to SDF-1
is observed in granule cells without their prior treatment with KCl. The KCl effect is likely to reflect a need for membrane depolarization as glutamate can substitute for KCl. Similar observations have been made for cortical neuron responses to SDF-1
(Klein et al., 1999). Notably, granule cell chemotaxis and proliferation assays are both performed with high potassium conditions (20 mM: see Materials and Methods). Activity dependent signaling during CNS development has been linked to trophic interactions between neurons and their targets as well as to regulation of apoptosis (Ghosh and Greenberg, 1995; Katz and Shatz, 1996). In the developing cerebellum, correctly located granule cells may be depolarized by glutamate derived from neighboring granule cells. This would allow for selective expansion of those granule cells that had achieved the correct position and relationships within the EGL through SDF-1
enhancement of SHH-induced proliferation.
Interaction between SDF-1 and SHH
It is well known that increased cAMP or PKA activity can antagonize SHH signaling (Hynes et al., 1995; Concordet et al., 1996; Epstein et al., 1996; Ungar and Moon, 1996), but no extracellular factors have yet been identified that modulate SHH effects through the regulation of cAMP or PKA. In the absence of SHH, its receptor, patched (PTC), inhibits signaling by a seven-transmembrane domain protein, smoothened (SMO; Stone et al., 1996; Chen and Struhl, 1998). Binding of SHH to PTC releases this inhibition. All SHH effects, including proliferation, appear to be transduced through SMO (Stone et al., 1996; van den Heuval and Ingham, 1996; Chen and Struhl, 1998). Whether SMO interacts with a G-protein remains unclear. Two reports have linked SMO signaling to the pertussis toxin sensitive G-protein, Gi (Hammerschmidt and McMahon, 1998; Decamp et al., 2000), while others have suggested that cAMP and PKA are active in a parallel pathway that can modulate SHH responses (Li et al., 1995; Jiang and Struhl, 1995; Goodrich and Scott, 1998). Similar to others, we have observed that forskolin can inhibit SHH-induced proliferation (Wechsler-Reya and Scott, 1999; Kenney and Rowitch, 2000). We did not, however, find that SHH-induced proliferation was sensitive to treatment with pertussis toxin. This is consistent with PKA acting in parallel to the SHH signaling pathway rather than SHH responses relying on G
i activation. However, SDF-1
enhancement of SHH-induced proliferation was sensitive to pertussis toxin and therefore constitutes a specific pathway that may modulate SHH signaling through the control of cAMP (Fig. 6).
|
![]() |
Conclusions |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
![]() |
ACKNOWLEDGMENTS |
---|
![]() |
REFERENCES |
---|
![]() ![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
Aiuti, A., Webb, I. J., Bleul, C., Springer, T. and Gutierrez-Ramos, J. C. (1997). The chemokine SDF-1 is a chemoattractant for human CD34+ hematopoietic progenitor cells and provides a new mechanism to explain the mobilization of CD34+ progenitors to peripheral blood. J. Exp. Med. 185, 111-120.
Altman, J. (1972). Postnatal development of the cerebellar cortex in the rat. J. Comp. Neurol. 145, 353-398.[Medline]
Altman, J. and Bayer, S. A. (1996). Development of the Cerebellar System in Relation to its Evolution, Structure and Function. New York: CRC Press.
Bajetto, A., Bonavia, R., Barbero, S., Picciolli, P., Costa, A., Florio, T. and Schettini, G. (1999). Glial and neuronal cells express functional chemokine receptor CXCR4 and its natural ligand, stromal cell-derived factor 1. J. Neurochem. 73, 2348-2357.[Medline]
Banisadr, G., Dicou, E., Berbar, T., Rostene, W., Lombet, A. and Haour, F. (2000). Characterization and visualization of [125I] stromal cell-derived factor-1alpha binding to CXCR4 receptors in rat brain and human neuroblastoma cells J. Neuroimmunol. 110, 151-160.[Medline]
Bleul, C. C., Schultze, J. L. and Springer, T. A. (1998). B lymphocyte chemotaxis regulated in association with microanatomic localization, differentiation state, and B cell receptor engagement. J. Exp. Med. 187, 753-762.
Bradstock, K. F., Makrynikola, V., Bianchi, A., Shen, W., Hewson, J. and Gottlieb, D. J. (2000). Effects of the chemokine stromal cell-derived factor-1 on the migration and localization of precursor-B acute lymphoblastic leukemia cells within bone marrow stromal layers. Leukemia 14, 882-888.[Medline]
Bruno, V., Copani, A., Besong, G., Scoto, G. and Nicoletti, F. (2000). Neuroprotective activity of chemokines against N-methyl-D-aspartate or beta-amyloid-induced toxicity in culture. Eur. J. Pharmacol. 399, 117-121.[Medline]
Burgoyne, R. and Cabray-Deakin, M. A. (1988). The cellular neurobiology of neuronal develoment: the cerebellar granule cell. Brain Res. Rev. 13, 77-101.
Chen, Y. and Struhl, G. (1998). In vivo evidence that Patched and Smoothened constitute distinct binding and transducing components of a Hedgehog receptor complex. Development 125, 4943-4948.
Concordet, J. P., Lewis, K. E., Moore, J. W., Goodrich, L. V., Johnson, R. L., Scott, M. P. and Ingham, P. W. (1996). Spatial regulation of a zebrafish patched homologue reflects the roles of sonic hedgehog and protein kinase A in neural tube and somite patterning. Development 122, 2835-2846.
Dahmane, N. and Ruiz-i-Altaba, A. (1999). Sonic hedgehog regulates the growth and patterning of the cerebellum. Development 126, 3089-3100.
DeCamp, D. L., Thompson, T. M., de Sauvage, F. J. and Lerner, M. R. (2000). Smoothened activates Galphai-mediated signaling in frog melanophores. J. Biol. Chem. 275, 26322-26327.
Doughty, M. L., Lohof, A., Campana, A., Delhaye-Bouchaud, N. and Mariani, J. (1998). Neurotrophin-3 promotes cerebellar granule cell exit from the EGL. Eur. J. Neurosci. 10, 3007-3011.[Medline]
Dutt, P., Wang, J. F. and Groopman, J. E. (1998). Stromal cell-derived factor-1 alpha and stem cell factor/kit ligand share signaling pathways in hemopoietic progenitors: a potential mechanism for cooperative induction of chemotaxis. J. Immunol. 161, 3652-3658.
Epstein, D. J., Marti, E., Scott, M. P. and McMahon, A. P. (1996). Antagonizing cAMP-dependent protein kinase A in the dorsal CNS activates a conserved Sonic hedgehog signaling pathway. Development 122, 2885-2894.
Gao, W. O., Heintz, N. and Hatten, M. E. (1991). Cerebellar granule cell neurogenesis is regulated by cell-cell interactions in vitro. Neuron 6, 705-715.[Medline]
Garcia-Zepeda, E. A., Combadiere, C., Rothenberg, M. E., Sarafi, M. N., Lavigne, F., Hamid, Q., Murphy, P. M. and Luster, A. D. (1996). Human monocyte chemoattractant protein (MCP)-4 is a novel CC chemokine with activities on monocytes, eosinophils, and basophils induced in allergic and nonallergic inflammation that signals through the CC chemokine receptors (CCR)-2 and -3. J. Immunol. 157, 5613-5626.[Abstract]
Garcia-Zepeda, E. A., Rothenberg, M. E., Ownbey, R. T., Celestin, J., Leder, P. and Luster, A. D. (1996). Human eotaxin is a specific chemoattractant for eosinophil cells and provides a new mechanism to explain tissue eosinophilia. Nat. Med. 2, 449-456.[Medline]
Ghosh, A. and Greenberg, M. E. (1995). Calcium signaling in neurons: molecular mechanisms and cellular consequences. Science 268, 239-247.[Medline]
Gonzalo, J. A., Lloyd, C. M., Peled, A., Delaney, T., Coyle, A. J. and Gutierrez-Ramos, J. C (2000). Critical involvement of the chemotactic axis CXCR4/stromal cell-derived factor-1 alpha in the inflammatory component of allergic airway disease. J. Immunol. 165, 499-508.
Goodrich, L. V. and Scott, M. P. (1998). Hedgehog and patched in neural development and disease. Neuron 21, 1243-1257.[Medline]
Hammerschmidt, M., Bitgood, M. J. and McMahon, A. P. (1996). Protein kinase A is a common negative regulator of Hedgehog signaling in the vertebrate embryo. Genes Dev. 10, 647-658.
Hammerschmidt, M. and McMahon, A. P. (1998). The effect of pertussis toxin on zebrafish development: a possible role for inhibitory G-proteins in hedgehog signaling. Dev. Biol. 194, 166-171.[Medline]
Hatten, M. (1993). The role of migration in central nervous system neuronal development. Curr. Opin. Neurobiol. 3, 38-44.[Medline]
Hynes, M., Porter, J. A., Chiang, C., Chang, D., Tessier-Lavigne, M., Beachy, P. A. and Rosenthal, A. (1995). Induction of midbrain dopaminergic neurons by Sonic hedgehog. Neuron 15, 35-44.[Medline]
Jiang, J. and Struhl, G. (1995). Protein kinase A and hedgehog signaling in Drosophila limb development. Cell 80, 563-572.[Medline]
Katz, L. C. and Shatz, C. J. (1996). Synaptic activity and the construction of cortical circuits. Science 274, 1133-1138.
Kaul, M. and Lipton, S. A. (1999). Chemokines and activated macrophages in HIV gp120-induced neuronal apoptosis. Proc Natl Acad Sci U S A 96, 8212-6821.
Kenney, A. M. and Rowitch, D. H. (2000). Sonic hedgehog promotes G(1) cyclin expression and sustained cell cycle progression in mammalian neuronal precursors. Mol. Cell Biol. 20, 9055-9067.
Khan, I. A., MacLean, J. A., Lee, F. S., Casciotti, L., DeHaan, E., Schwartzman, J. D. and Luster, A. D. (2000). IP-10 is critical for effector T cell trafficking and host survival in Toxoplasma gondii infection. Immunity 12, 483-494.[Medline]
Klein, R., Williams, K. C., Alvarez-Hernandez, X., Westmoreland, S., Force, T., Lackner, A. A. and Luster, A. D. (1999). Chemokine receptor expression and signaling in macaque and human fetal neurons and astrocytes: implications for the neuropathogenesis of AIDS. J. Immunol. 163, 1636-1646.
Lataillade, J. J., Clay, D., Dupuy, C., Rigal, S., Jasmin, C., Bourin, P. and Le Bousse-Kerdiles, M. C. (2000). Chemokine SDF-1 enhances circulating CD34(+) cell proliferation in synergy with cytokines: possible role in progenitor survival. Blood 95, 756-68.
Lavi, E., Strizki, J.M., Ulrich, A.M., Zhang, W., Fu, L., Wang, Q., Oconnor, M., Hoxie, J.A. and Gonzalez-Scarano, F. (1997). CXCR-4(fusin), a cor-receptor for the type 1 human immunodeficiency virus (HIV-1), is expressed in hte human brain in a variety of cell types, including microglia and neurons. Am. J. Pathol. 51, 1035.
Lazarini, F., Casanova, P., Tham, T. N., De Clercq, E., Arenzana-Seisdedos, F., Baleux, F. and Dubois-Dalcq, M. (2000). Differential signalling of the chemokine receptor CXCR4 by stromal cell-derived factor 1 and the HIV glycoprotein in rat neurons and astrocytes. Eur. J. Neurosci. 12, 117-125.[Medline]
Li, W., Ohlmeyer, J.T., Lane, M.E. and Kalderon, D (1995) Function of protein kinase A in hedgehog signal transduction and Drosophila imaginal disc development. Cell 80, 553-562.[Medline]
Limatola, C., Giovannelli, A., Maggi, L., Ragozzino, D., Castellani, L., Ciotti, M. T., Vacca, F., Mercanti, D., Santoni, A. and Eusebi, F. (2000). SDF-1alpha-mediated modulation of synaptic transmission in rat cerebellum. Eur. J. Neurosci. 12, 2497-2504.[Medline]
Lin, X. and Bulleit, R. F. (1997). Insulin-like growth factor I (IGF-I) is a critical trophic factor for developing cerebellar granule cells. Brain Res. Dev. Brain Res. 99, 234-242.[Medline]
Lodin, V. and Srager, J (1970). The cellular kinetics of the developing mouse cerebellum. I. The generation cycle, growth fraction and rate of proliferation of the external granular layer. Brain Res. 123, 323-342.
Luster, A. (1998). Chemokineschemotactic cytokines that mediate inflammation. New Eng. J. Med. 338, 436-445.
Ma, Q., Jones, D., Borghesani, P. R., Segal, R. A., Nagasawa, T., Kishimoto, T., Bronson, R. T. and Springer, T. A. (1998). Impaired B-lymphopoiesis, myelopoiesis, and derailed cerebellar neuron migration in CXCR4- and SDF-1-deficient mice. Proc. Natl Acad. Sci. USA 95, 9448-9453.
Maisonpierre, P. C., L. Belluscio, Friedman, B., Alderson, R. F., Wiegand, S. J., Furth, M. E., Lindsay, R. M. and Yancopoulos, G. D. (1990). NT-3, BDNF, and NGF in the developing rat nervous system: parallel as well as reciprocal patterns of expression. Neuron 5, 501-509.[Medline]
McGrath, K., Koniski, A. D., Maltby, K. M., McGann, J. K. and Palis, J. (1999). Embryonic expression and function of the chemokine SDF-1 and its receptor, CXCR4. Dev. Biol. 213, 442-456.[Medline]
Meucci, O., Fatatis, A., Simen, A. A., Bushell, T. J., Gray, P. W. and Miller, R. J. (1998). Chemokines regulate hippocampal neuronal signaling and gp120 neurotoxicity. Proc. Natl. Acad. Sci. USA 95, 14500-14505.
Meucci, O., Fatatis, A., Simen, A. A. and Miller, R. J. (2000). Expression of CX3CR1 chemokine receptors on neurons and their role in neuronal survival. Proc. Natl. Acad. Sci. USA 97, 8075-8080.
Miale, I. and Sidman, R. L. (1961). An autoradiographic analysis of histogenesis in the mouse cerebellum. Exp Neurol 4, 277-296.
Nagasawa, T., Kikutani, H. and Kishimoto, T. (1994). Molecular cloning and structure of a pre-B-cell growth-stimulating factor. Proc. Natl. Acad. Sci. USA 91, 2305-9230.[Abstract]
Nagasawa, T., Hirota, S., Tachibana, K., Takakura, N., Nishikawa, S., Kitamura, Y., Yoshida, N., Kikutani, H. and Kishimoto, T. (1996). Defects of B-cell lymphopoiesis and bone-marrow myelopoiesis in mice lacking the CXC chemokine PBSF/SDF-1. Nature 382: 635-638.[Medline]
Nanki, T. and Lipsky, P. E. (2000). Cutting edge: stromal cell-derived factor-1 is a costimulator for CD4+ T cell activation. J. Immunol. 164, 5010-5014.
Noveen, A., Jiang, T. X. and Chuong, C. M. (1996). cAMP, an activator of protein kinase A, suppresses the expression of sonic hedgehog. Biochem. Biophys. Res. Commun. 219, 180-185.[Medline]
Pehlemann, F. W., Sievers, J. and Berry, M. (1985). Meningeal cells are involved in foliation, lamination, and neurogenesis of the cerebellum: evidence from 6-hydroxydopamine-induced destruction of meningeal cells. Dev. Biol. 110, 136-146.[Medline]
Premack, B. and Schall, TJ (1996). Chemokine receptors: gateways to inflammation and infection. Nature Medicine 2, 1174-1178.[Medline]
Raetzman, L. T. and Siegel, R. E. (1999). Immature granule neurons from cerebella of different ages exhibit distinct developmental potentials. J. Neurobiol. 38, 559-570.[Medline]
Rossi, D. and Zlotnik, A. (2000). The biology of chemokines and their receptors. Annu. Rev. Immunol. 18, 217-242.[Medline]
Rothenberg, M. E., Ownbey, R., Mehlhop, P. D., Loiselle, P. M., van de Rijn, M., Bonventre, J. V., Oettgen, H. C., Leder, P. and Luster, A. D. (1996). Eotaxin triggers eosinophil-selective chemotaxis and calcium flux via a distinct receptor and induces pulmonary eosinophilia in the presence of interleukin 5 in mice. Mol Med 2, 334-348.[Medline]
Sanders, V., Pittman, C. A., White, G. W., Wiley, C. A. and Achim, C. L. (1998). Chemokines and receptors in HIV encephalitis. AIDS 12, 1021.[Medline]
Segal, R. A., Pomeroy, S.L. and Stiles, C.D. (1995). Axonal growth and fasciculation linked to differential expression of BDNF and NT3 receptors in developing cerebellar granule cells. J. Neurosci. 15, 4970-4981.[Abstract]
Stone, D. M., Hynes, M., Armanini, M., Swanson, T. A., Gu, Q., Johnson, R. L., Scott, M. P., Pennica, D., Goddard, A., Phillips, H., Noll, M., Hooper, J. E., de Sauvage, F. and Rosenthal, A. (1996). The tumour-suppressor gene patched encodes a candidate receptor for Sonic hedgehog [see comments]. Nature 384, 129-134.[Medline]
Tanabe, S., Heesen, M., Yoshizawa, I., Berman, M. A., Luo, Y., Bleul, C. C., Springer, T. A.Okuda, K., Gerard, N. and Dorf, M. E. (1997). Functional expression of the CXC-chemokine receptor-4/fusin on mouse microglial cells and astrocytes. J. Immunol. 159, 905-911.[Abstract]
Tao, Y., Black, I. B. and DiCicco-Bloom, E. (1996). Neurogenesis in neonatal rat brain is regulated by peripheral injection of basic fibroblast growth factor (bFGF). J. Comp. Neurol. 376, 653-663.[Medline]
Traiffort, E., Charytoniuk, D., Watroba, L., Faure, H., Sales, N. and Ruat, M. (1999). Discrete localizations of hedgehog signalling components in the developing and adult rat nervous system. Eur. J. Neurosci. 11, 3199-3214.[Medline]
Ungar, A. R. and Moon, R. T. (1996). Inhibition of protein kinase A phenocopies ectopic expression of hedgehog in the CNS of wild-type and cyclops mutant embryos. Dev. Biol. 178, 186-191.[Medline]
van den Heuvel, M. and Ingham, P. W. (1996). Smoothened encodes a receptor-like serpentine protein required for hedgehog signalling. Nature 382, 547-551.[Medline]
Wallace, V. A. (1999). Purkinje-cell-derived Sonic hedgehog regulates granule neuron precursor cell proliferation in the developing mouse cerebellum. Curr. Biol. 9, 445-448.[Medline]
Wechsler-Reya, R. J. and Scott, M. P. (1999). Control of neuronal precursor proliferation in the cerebellum by Sonic Hedgehog [see comments]. Neuron 22, 103-114.[Medline]
Westmoreland, S., Rottman, J.B., Williams, K.C., Lackner, A.A. and Sasseville, V.G. (1998). Chemokine receptor expression on resident and inflammatory cells in the brains of macaques with ismian immunodeficiency virus encephalitis. Am. J. Pathol. 152, 659.[Abstract]
Ye, P., Xing, Y., Dai, Z. and DErcole, A. J. (1996). In vivo actions of insulin-like growth factor-I (IGF-I) on cerebellum development in transgenic mice: evidence that IGF-I increases proliferation of granule cell progenitors. Brain. Res. Dev. Brain. Res. 95, 44-54.[Medline]
Zhang, L., He, T., Talal, A., Wang, G., Frankel, S.S. and Ho, D.D. (1998). In vivo distribution of human immunodeficiency/simian immunodeficiency virus coreceptors CXCR4, CCR3 and CCR5. J. Virol. 72, 5035.
Zheng, J., Thylin, M. R., Ghorpade, A., Xiong, H., Persidsky, Y., Cotter, R., Niemann, D., Che, M., Zeng, Y. C., Gelbard, H. A., Shepard, R. B., Swartz, J. M. and Gendelman, H. E. (1999). Intracellular CXCR4 signaling, neuronal apoptosis and neuropathogenic mechanisms of HIV-1-associated dementia. J. Neuroimmunol. 98, 185-200.[Medline]
Zou, Y. R., Kottmann, A. H., Kuroda, M., Taniuchi, I. and Littman, D. R. (1998). Function of the chemokine receptor CXCR4 in haematopoiesis and in cerebellar development [see comments]. Nature 393, 595-599.[Medline]
Zujovic, V., Benavides, J., Vige, X., Carter, C. and Taupin, V. (2000). Fractalkine modulates TNF-alpha secretion and neurotoxicity induced by microglial activation. Glia 29, 305-315.[Medline]