1 Department of Pediatric Oncology, Dana-Farber Cancer Institute, Boston, MA 02115, USA
2 Department of Neurobiology, Harvard Medical School, Boston, MA 02115, USA
*Author for correspondence (e-mail: Rosalind_segal{at}dfci.harvard.edu)
Accepted 31 January 2002
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SUMMARY |
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Key words: Sonic hedgehog, Proteoglycans, Ext, Cerebellum, Granule cells, Mouse
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INTRODUCTION |
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How modulators and co-acting factors influence SHH responses is not yet known. We became interested in evaluating the effect of heparan sulfate proteoglycans (HSPGs) on SHH signaling when work in Drosophila suggested that long-range hedgehog (HH) signals depend upon the normal synthesis of HSPGs. Mutation of the HSPG synthetic enzyme Tout-velu (TTV) results in a phenotype similar to the HH mutation. The TTV phenotype has been ascribed to a disruption of the ability of HH to diffuse and establish a concentration gradient (Bellaiche et al., 1998; The et al., 1999
). Additionally, HH-HSPG interactions may modulate cellular responses to HH.
We asked whether HSPGs were important for vertebrate SHH signaling and if so, how. We evaluated the CNS expression of the Ttv orthologs, the exostosins (Exts) and found the highest level of Ext1 and Ext2 in the cerebellum. As cerebellar granule cells require SHH for proliferation (Dahmane and Ruiz-i-Altaba, 1999; Wallace, 1999
; Wechsler-Reya and Scott, 1999
), this seemed an ideal system for studying the interaction of SHH and HSPGs. We report here that SHH interacts with HSPGs through a highly conserved heparin-binding domain. This interaction is not required for binding to patched (PTCH) but is necessary for maximal proliferative response to SHH. The influence of HSPGs on SHH induced proliferation increases with age during the neonatal period and is temporally correlated with an increase in expression of Ext1 and Ext2, as well as increased binding of SHH to in situ HSPGs and a dramatic change in the SHH dose-response curve. This mature curve is bell-shaped, with peak proliferation elicited only by a sharply narrowed range of SHH concentrations. Together these data provide a molecular basis for SHH-heparin/HSPG interactions and identify HSPGs as important modulators of SHH-induced proliferation.
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MATERIALS AND METHODS |
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Transient transfection
Plasmids containing sequences for SHH:AP, AlaSHH:AP, GlnSHH:AP, Arg+SHH:AP and AP alone were transiently transfected into COS 7 cells by the Lipofectamine method (Gibco BRL, Rockville, MD). Plasmid DNA (4-8 µg) was mixed with 60 µl of lipofectamine per 100 mm tissue culture dish. Transfection proceeded for 9 hours. Transfected cells were maintained in serum free DMEM/F12 without supplements or antibiotics. Culture supernatants were collected every 24 hours and assayed for alkaline phosphatase activity. Protein was analyzed by western blotting with an antibody to alkaline phosphatase (Biomeda, Foster, CA).
Column chromatography
Wild-type or mutant SHH transfection supernatants (1 ml) were applied to a 3 ml heparin-agarose column (Sigma, St Louis, MO) in equilibration buffer (20 mM Tris pH 7.4, 150 mM NaCl and 0.1% Triton X-100). The column was washed with two volumes of equilibration buffer. Then, 20 ml of a salt gradient from 0 to 2 M NaCl in 20 mM Tris pH 7.4 and 0.1% Triton X-100 was applied. Fractions (0.5 ml) were obtained from the time of protein application until the end of the gradient. Elution of wild-type and mutant SHH was detected by alkaline phosphatase activity of each fraction. The peak of elution was determined by curve fitting the gradient profile to y=mx + b and deriving a value for y (molarity of NaCl) at the peak (x=fraction number) of elution.
Alkaline phosphatase assay
Determination of alkaline phosphatase activity was accomplished by incubation with 2 M diethanolamine (Sigma), 0.5 mM MgCl2, 0.5 mg/ml bovine serum albumin (BSA) and 12 mM p-nitrophenylphosphate (Sigma 104® phosphatase substrate). Reactions proceeded at 37°C for 20 minutes and the reaction product was quantitated by measuring sample absorbance at 405 nm.
Section binding assay
In situ HSPG binding was evaluated by methods based on those of Friedl (Friedl et al., 1997). Briefly, brains from postnatal day 3 and 6 BALB/c mice were removed and fixed in 4% paraformaldehyde for 24 hours and cryoprotected in 30% sucrose. Sections were treated, or not, with a combination of 1 mU/ml of heparinase I (Sigma) and 1 mU/ml heparinase III, overnight at 4°C (Sigma). Autofluorescence was diminished by treatment with 0.05% sodium borohydride for 10 minutes at room temperature, followed by treatment with 0.1 M glycine at 4°C overnight. Non-specific ligand binding was blocked with 1% BSA in phosphate-buffered saline (PBS) for 1 hour at room temperature. Equimolar amounts of SHH:AP or AlaSHH:AP were added for 1 hour at room temperature. Sections were washed with PBS containing 0.5 M NaCl to dissociate any low affinity interaction between ligands and HSPGs. Washed sections were incubated with rabbit anti-human alkaline phosphatase for 1 hour at room temperature (Biomeda). Ligand-antibody complexes were visualized with a Cy3-conjugated goat anti-rabbit IgG, for 1 hour at room temperature (Jackson Immunoresearch). High magnification views of binding were examined by DeltaVision® restoration fluorescence microscopy (Applied Precision, Issaquah, WA), viewed with a 60x objective. z-series comprising 20 0.2 µm serial optical sections were acquired and deconvolved with softWoRx imaging software (Applied Precision). Final images are single optical sections rendered in softWoRx volume viewer.
In situ hybridization
In situ hybridization was performed as described (Klein et al., 2001). Briefly, brains from postnatal day 8 BALB/c mice were removed and fixed in 4% paraformaldehyde for 24 hours and cryoprotected in 30% sucrose. Sagittal sections (15 µm) were obtained and treated with 20 µg/ml proteinase K for 10 minutes at room temperature. Sections were fixed in paraformaldehyde and washed in PBS. Hybridization was performed with digoxigenin (DIG)-labeled sense and antisense RNA probes for 20 hours at 65°C 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). Sections were washed with 0.2x SSC, 0.1% Tween 20 at 65°C and treated with 20% sheep serum to block non-specific binding. Hybridized DIG-labeled probes were visualized with an antibody to DIG according to manufacturers instructions (Boehringer Mannheim, Mannheim, Germany).
Probe preparation
Plasmid containing full-length mouse Ext1 was from Dominique Stickens and Glenn Evans. Plasmid containing rat Ext 2 probe was from IMAGE consortium (IMAGE clone ID: UI-R-EO-dd-h-11-0-UI.s1). Sequence and orientation of each probe was confirmed by direct sequencing. Sense and antisense digoxigenin-labeled riboprobes were synthesized using DIG/Genius labeling kit according to the manufacturers instructions (Boehringer Mannheim).
Northern blot analysis
RNA was obtained from pooled cerebella using Trizol according to manufacturers instructions (Gibco BRL). Total RNA (25 µg) was electrophoresed on a 1.2% agarose formaldehyde gel and transferred to GeneScreen membrane (NEN Life Science Products, Boston, MA). Prehybridization and hybridization was performed as previously described (Klein et al., 2001). 32P-labeled full-length Ext1 and 389 bp Ext2 antisense probes were generated by random primed DNA synthesis (Promega Life Sciences, Madison, WI).
Primary culture
Primary cultures of neonatal mouse cerebellum were established as previously described (Klein et al., 2001). Briefly, cerebella were dissected and meninges were removed. After incubation with 0.1% trypsin (Sigma) in HBSS with 125 units/ml DNase (Sigma), 0.5 mM EDTA for 20 minutes at 37°C, cells were pelleted in a clinical centrifuge. Cell pellets were washed three times with HBSS. The final cell suspension was passed through a 100 µM nylon mesh cell strainer (Falcon, Franklin Lakes, MI). Cells were diluted to 2x106 cells/ml in DMEM/F12 (Gibco BRL) supplemented with N2 (Gibco BRL), 20 mM KCl, 36 mM glucose and penicillin/streptomycin, SHH, SHH:AP or AlaSHH:AP as indicated, and plated at 2x105 cells/well onto a 96-well tissue culture dish coated with 15 µg/ml poly-ornithine (Sigma). Control cultures were treated with an equivalent volume of media conditioned by non-transfected COS cells. For heparinase treatment, a mixture of heparinase I 1 mU/ml (Sigma) and heparinase III 1 mU/ml (Sigma) was added at 24 hours post-plating.
Proliferation assay
At 36-40 hours post-plating cultures were treated with 5 µCi/well of [3H]thymidine (New England Nuclear, Boston, MA). After 4 hours at 37°C, Triton X-100 was added to a final concentration of 1% and cells were lysed for 10 minutes at room temperature. Ice-cold trichloroacetic acid was added to a final concentration of 10% and DNA was precipitated for 1 hour on ice. Precipitated DNA was collected by vacuum filtration through phosphocellulose membranes (Pierce, Rockford, IL). Filters were washed with ice-cold 10% TCA, dried with 100% ethanol and then solubilized in Scintisafe (New England Nuclear) and counted. Each experiment was performed in quadruplicate. Representative experiments are presented as mean DPM±s.e.m. Statistical significance was determined by two-tailed Students t-test.
Binding assay
Primary cerebellar cultures prepared as above were washed with PBS. Measurements of specific binding were conducted on unfixed cultures or cultures that had been fixed in 4% paraformaldehyde for 10 minutes at room temperature. In all cases, non-specific binding was reduced by treatment of cultures with 1% BSA in PBS for 1 hour on ice.
Total specific binding
Cultures treated or not with heparinase were incubated with 1 nM SHH:AP for 2 hours on ice in the absence or presence of 100 nM unconjugated SHH. Cultures were washed three times with buffer containing 20 mM Tris pH 7.4 and 0.75 M NaCl. Bound ligand was then measured by assessing alkaline phosphatase activity as described above. Binding experiments were carried out in quadruplicate and data are presented as mean±s.e.m.
Competition binding
Fixed cultures were incubated with increasing concentration of SHH:AP or AlaSHH:AP (0.7-35 nM) in the absence or presence of 100 nM unconjugated SHH. Cultures were washed, and cell-associated alkaline phosphatase activity was determined as above. Specific binding was derived by subtracting the cell-associated alkaline phosphatase activity measured in the presence of excess unconjugated SHH from the cell-associated alkaline phosphatase activity measured in the absence of unconjugated SHH. Each determination was done in triplicate and data are presented as the mean cell-associated AP activity±s.e.m.
Scatchard analysis
SHH:AP (1 nM) was added and incubated on ice for 2 hours in absence or presence of 5-500 nM unconjugated SHH. Concentration of bound SHH was calculated as the product of (cell-associated alkaline phosphatase activity/total applied alkaline phosphatase activity) and (concentration of total SHH). Free SHH was calculated as the difference between total applied SHH and bound SHH. Each determination was made in triplicate and data are presented as mean bound/free±s.e.m. versus the mean bound (nM). Values for Kd and Bmax were derived from a linear curve fit to the steepest region of the relationship.
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RESULTS |
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At higher magnification, SHH:AP binding localizes in a lattice-like pattern around granule cell bodies in the EGL (Fig. 5C). The pattern suggests that the bulk of the SHH:AP-HSPG interactions occur at the surface of granule cells and/or in the extracellular matrix. The ability of SHH:AP to bind to these sites is reduced by mutation of the Cardin-Weintraub sequence.
Comparison between the binding of SHH:AP to sections of P3 and P6 mouse cerebellum demonstrates that the ability of HSPGs in the granule cell layers to bind to SHH is developmentally regulated. The increased binding of SHH:AP to P6 relative to P3 sections parallels the increases in Ext1 and Ext2 expression and is temporally correlated with increased granule cell proliferation in vivo (Mares et al., 1970).
Loss of SHH-HSPG interactions decreases SHH-induced proliferation
We next asked whether interaction of SHH with HSPGs is important for biological effects of SHH. Granule cells proliferate postnatally (Altman, 1972a; Altman, 1972b
) and SHH is a potent mitogen for this proliferation (Dahmane and Ruiz-i-Altaba, 1999
; Wallace, 1999
; Wechsler-Reya and Scott, 1999
). We evaluated whether mutation of the Cardin-Weintraub sequence (AlaSHH:AP) affects the ability of SHH to promote proliferation of cerebellar cells. Granule cell cultures derived from P6 mice, the stage of maximal granule cell proliferation, exhibited a bell-shaped proliferation dose-response curve in response to SHH:AP (Fig. 6A, white squares) or SHH (Fig. 6B, white squares). Peak proliferative responses were observed for 1.5 µg/ml (35 nM) conjugate protein or 0.28 µg/ml (14 nM), SHH. When we compared the effects of equimolar amounts of SHH:AP and AlaSHH:AP, we found that loss of SHH-HSPG interactions was associated with a reduction in the proliferative responses (Fig. 6A, black diamonds compared to white squares). The peak of proliferation in response to AlaSHH:AP was decreased to 60% of the response to wild-type SHH:AP (n=8, P<0.02), but occurred at the same dose.
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The dependence of granule cell proliferation on SHH-HSPG interactions is developmentally regulated
Given the age-dependent changes in cerebellar Ext expression and SHH:AP-HSPG interactions, we asked whether granule cell proliferation also displays age-dependent changes in the requirement for SHH-HSPG interactions. When we examined primary cultures from P3 mice (when Ext expression and SHH:AP-HSPG binding are low), we observed a markedly different dose response to SHH when compared with that observed for cultures from P6 mice (when Ext expression and SHH-HSPG binding are high). Whereas P6 cultures exhibited a bell-shaped SHH dose-response curve, cultures derived from P3 mice displayed increasing proliferation in response to increasing doses of SHH:AP (Fig. 6C, white squares) or SHH (Fig. 6D, white squares). The magnitude of the peak response was less at P3 when compared with P6 but the range of effective SHH concentrations was broader.
In addition to age-dependent changes in the SHH dose-response curve, there was also an age-dependent change in the impact of SHH-HSPG interactions on these responses. In cultures derived from P3 mice, when Ext expression is low, mutation of the Cardin-Weintraub sequence had no effect on proliferative responses (Fig. 6C, compare black diamonds with white squares). Similarly, in P3 cultures, degradation of heparan sulfates with heparinase I and III had no effect on the dose response to SHH (Fig. 6D, compare black diamonds with white squares). Taken together, these data suggest that minimal SHH-HSPG interactions are taking place at P3. Of note, the height of peak proliferation in P3 cultures (twofold over control) was similar in magnitude to the peak proliferation observed in P6 cultures treated with AlaSHH:AP or SHH in the presence of heparinase. Thus, the magnitude of the peak of proliferation in response to SHH in the absence of interactions with HSPGs was the same regardless of age. We conclude that age-dependent changes in Ext expression are temporally correlated with: (1) increased synthesis of proteoglycan binding sites for SHH; (2) changes in the proliferation dose response of cerebellar granule cells to SHH; and (3) increasing influence of SHH-HSPG interactions on proliferation.
Mutation of the Cardin-Weintraub sequence does not alter binding to patched
HSPGs frequently act as co-receptors that facilitate high affinity binding of ligands to their specific receptors (Bernfield et al., 1999). As mutation of the SHH Cardin-Weintraub sequence abrogated SHH-HSPG interactions and reduced the proliferative response, we asked whether or not AlaSHH:AP retained its high-affinity binding to PTCH. Several observations suggested that disruption of SHH-HSPG interactions did not lead to decreased proliferative responses as a result of a loss in high-affinity binding. A decrease in receptor binding would have shifted the dose-response curve in P6 cultures to the right rather than diminish the peak. Additionally cultures derived from P3 mice were not affected by treatments that abrogated SHH interactions with HSPGs. This argues against an absolute requirement for SHH-HSPG interactions for receptor binding and activation.
We first characterized the high-affinity binding of SHH to primary cultures of cerebellar cells. Scatchard analysis of unconjugated SHH binding revealed a Kd of binding of 23 nM (Fig. 7A), and indicated that there were 50x103 high-affinity SHH-binding sites per granule cell. Previously measured values for the Kd of SHH binding have ranged between 0.46 and 7 nM in transfected cells expressing high levels of PTCH (Fuse et al., 1999
; Marigo et al., 1996
; Pathi et al., 2001
; Stone et al., 1996
) and are comparable with the values measured here in primary cultures. In addition the Scatchard analysis suggests that proliferative granule cells possess a relatively high number of receptors.
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To assess further whether HSPGs function as co-receptors for SHH, we examined the effect of heparinase on SHH binding. Disruption of the interaction of SHH with HSPGs by heparinase treatment did not diminish specific SHH:AP binding (Fig. 7C). Taken together, these data indicate that HSPGs function as modulators of SHH-induced proliferation but do not function as facilitators of SHH-PTCH binding.
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DISCUSSION |
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The Cardin-Weintraub sequence consists of a cluster of basic amino acids that allow for protein interaction with sulfates contained within the glycosaminoglycan side chains of proteoglycans. The SHH Cardin-Weintraub sequence occurs in the N terminus (amino acids 31-38 of murine SHH) of the biologically active fragment of SHH, a region of the molecule that is lacking in significant tertiary structure (Pepinsky et al., 2000). Freedom of movement in this domain could be important to its function (Cardin and Weintraub, 1989
). Mutation of the two conserved basic amino acid positions (33 and 37) within the Cardin-Weintraub sequence to either alanine (AlaSHH:AP) or glutamine (GlnSHH:AP) results in loss of high-affinity interaction with heparin. The finding that two out of five basic amino acids in a Cardin-Weintraub sequence are predominantly responsible for high-affinity interaction with heparin is similar to what has been found for the chemokine SDF-1
(Sadir et al., 2001
). Addition of a basic amino acid adjacent to the Cardin-Weintraub sequence (Arg+SHH:AP) had no effect on heparin binding. Together, these data suggest that interaction between SHH and heparin is mediated by the Cardin-Weintraub sequence, requires interactions other than hydrogen bonding and is not a simple function of total positive charge.
The Cardin-Weintraub sequence mediates interaction of SHH with endogenous HSPGs in the cerebellum. When we tested the ability of endogenous HSPGs in P6 cerebellar tissue to bind wild-type SHH:AP, binding was greatly diminished by treatment of tissue sections with heparinase, identifying the relevant proteoglycan as an HSPG. Binding was similarly diminished by mutation of the Cardin-Weintraub sequence (Fig. 5). These data identify the Cardin-Weintraub sequence as an essential domain for the binding of SHH to endogenous HSPGs as well as to heparin.
The identity of the relevant HSPG remains unclear. There are two families of pure heparan sulfate proteoglycans that can be distinguished by their core proteins. The syndecans possess membrane spanning core proteins, while the glypicans are characterized by core proteins that are GPI linked to the cell surface. There is some speculation that glypicans are the HSPGs that are most likely to bind hedgehog proteins (De Cat and David, 2001). However, so far, the proteoglycans that interact with hedgehog proteins have not been identified. Data presented here suggest that SHH-HSPG interactions are determinants of more than just appropriate SHH localization. Thus, more than one type of HSPG may interact specifically with SHH and perform multiple functions.
Interaction between SHH and HSPGs is critical for developmental regulation of proliferation
We have found that there is an age-dependent change in the effect of HSPGs on cerebellar granule cell proliferation in response to SHH. Primary cultures from P3 mice display a sigmoidal dose response curve to SHH, that is not affected by mutation of the Cardin-Weintraub sequence, nor by treatment of cultures with heparinase or sodium perchlorate. At this stage, expression of Ext1 and Ext2 are low, and SHH:AP binds at low levels to HSPGs in cerebellar slices. These correlations suggest that HSPGs that participate in SHH responses may not be synthesized during the early neonatal period.
By contrast, proliferation in cultures derived from P6 mice was modulated by SHH-HSPG interactions. Primary cultures derived from P6 mice display a bell-shaped dose-response curve to SHH. Mutation of the Cardin-Weintraub sequence, or treatment of cultures with heparinase or sodium perchlorate, reduces the peak proliferative response to SHH. This developmentally regulated dependence on HSPG interactions is accompanied by increased expression of Ext1 and Ext2, and the synthesis of HSPGs capable of binding to SHH (Fig. 5). Thus, at P6, the developmental stage when granule cell proliferation is maximal, HSPGs contribute to SHH-induced proliferation. Furthermore, SHH binds at highest levels to HSPGs in the EGL, the location of proliferating granule cell precursors. Thus, the regulated synthesis of HSPGs may allow optimal proliferation to occur at both the right time and place.
SHH is one of many growth factors that interact with low-affinity HSPG binding sites as well as with high-affinity primary receptors (Bernfield et al., 1999). Proteoglycans can modulate growth factor signaling by several possible mechanisms. They can increase the likelihood of ligand high-affinity receptor binding by limiting ligand diffusion to the two-dimensional space of the membrane surface rather than the three-dimensional extracellular space (Schlessinger et al., 1995
). They can promote the formation of ligand dimers (Moy et al., 1997
) and thereby enhance receptor activation. They can regulate internalization (Tyagi et al., 2001
) and modulate intracellular signaling (Delehedde et al., 2000
). Finally, they can possess independent signaling functions that are initiated by ligand interactions (Kinnunen et al., 1998
).
HSPGs do not appear to modulate SHH responses by altering binding of SHH to PTCH. The peak proliferative response in P6 cultures occurred at the same SHH concentration regardless of the presence or absence of intact SHH-HSPG interactions. This indicates that HSPGs do not alter the affinity of receptor binding, in which case treatment with heparinase or mutant SHH would have produced a shift in the dose-response curve to the right. Consistent with this, when directly tested, wild-type and mutant SHH bound with equal affinity to receptor sites on the cell surface. However, the present studies do suggest that SHH biological activity is not a simple function of receptor binding. The coordinated interaction between SHH and HSPGs allows for modulation of receptor signaling in a developmentally regulated fashion. The identification of the molecular basis for the interaction between SHH and HSPGs will facilitate the elucidation of the mechanism by which HSPGs modulate SHH responses.
Two previous reports have evaluated the receptor binding and biological activity of SHH mutants that delete the N-terminal half of what is identified here as a consensus sequence for heparin binding (Fuse et al., 1999; Katsuura et al., 1999
). Consistent with data presented here, both Katsuura et al. (
25-35) and Fuse et al. (
25-34) observed that this sequence was not essential for high-affinity binding to PTCH. Although the heparin binding of
25-35 was not evaluated, Fuse found that the
25-34 mutant was capable of binding heparin. However, no measurements of the affinity of this interaction were presented. It is therefore not possible to determine whether the deletion mutant had lost the higher affinity (0.75 M NaCl) heparin binding and retained only the lower affinity (0.5 M NaCl) binding in a manner similar to AlaSHH:AP and GlnSHH:AP mutants presented here. Alternatively, it is possible that the high affinity interaction between SHH and heparin is predominantly dependent on Lys37, which is preserved in the deletion mutant but not the AlaSHH:AP mutant described here, and not Arg33, which is altered in both mutants.
A comparison of the biological responses to the mutants is particularly revealing. Katsuura et al. (Katsuura et al., 1999), who evaluated induction of alkaline phosphatase activity, found that
25-35 SHH lost all biological activity. By contrast, Fuse et al. (Fuse et al., 1999
), who used a neural plate HNF3ß induction assay, found that
25-34 retained its biological activity. We observed that loss of SHH-HSPG interactions had no effect on SHH-induced proliferation at P3, but resulted in a dramatic decrement in the proliferative potency of SHH at P6. Thus, the modulation of SHH biological responses by HSPGs appears to be strongly context dependent.
The dose-response to SHH induced proliferation is developmentally regulated
Developmental regulation of SHH-induced proliferation is evident in the modulatory actions of HSPGs and also in changes in the shape of the dose-response curve. In cultures from P3 mice, proliferative responses of granule cells to SHH are characterized by a sigmoidal relationship between dose and proliferation. By contrast, cultures from P6 mice display a bell-shaped dose-response curve. While HSPGs modulate the magnitude of the peak response at P6, they do not appear to be responsible for the change in the shape of the dose-response curve. The morphogenetic effects of hedgehog proteins often display bell-shaped responses and depend upon the establishment of a SHH concentration gradient. Within these gradients, individual cell types are induced at limited locations, where the correct dose of SHH occurs (Ingham and McMahon, 2001).
Similarly, the bell-shaped proliferative response could constitute a mechanism for promoting granule cell proliferation exclusively in the EGL. SHH concentrations within the EGL may fall within the narrow proliferative range while those of the molecular layers and internal granule cell layers may be either too high or too low to induce proliferation. Thus the emergence of the bell shaped curve and the anatomic localization of modulatory factors such as HSPGs, SDF (Klein et al., 2001), laminin (Pons et al., 2001
) and Notch2 (Solecki et al., 2001
) may all work together to promote granule cell proliferation in the correct place and time.
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ACKNOWLEDGMENTS |
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