1 Developmental Biology Unit, Institute of Child Health, University College London, London, UK
2 Neural Development Unit, Institute of Child Health, University College London, London, UK
* Present address: Department of Anatomy, National University of Singapore, Singapore
Author for correspondence (e-mail: a.copp{at}ich.ucl.ac.uk)
Accepted 12 February 2002
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SUMMARY |
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Key words: Chlorate, Glycosaminoglycans, Heparan sulphate, Neural tube defects, Patched, Proteoglycans, Sonic hedgehog, Extracellular matrix
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INTRODUCTION |
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Heparan sulphate is a glycosaminoglycan (GAG) of repeating disaccharide subunits, comprising glucosamine and glucuronic/iduronic acid, covalently linked to a core protein backbone (Bernfield et al., 1999). A family of HSPGs exists, some of which are localised to the cell surface through a transmembrane core protein (syndecans) or by a glycosylphosphatidylinositol linkage (glypicans), while others form part of the extracellular matrix (perlecan and agrin). HSPGs bind to and regulate the activity of many important signalling molecules. For example, analysis of the Drosophila tout-velu mutant and the Ext1-deficient mouse, both of which lack heparan sulphate co-polymerase, shows that HSPGs are essential for the function of several members of the hedgehog family of signalling proteins (Bellaiche et al., 1998
; Lin et al., 2000
; The et al., 1999
). Similarly, examination of the Drosophila sugarless and sulfateless mutants provides evidence for a role of HSPGs in fibroblast growth factor (FGF) and Wnt signalling (Häcker et al., 1997
; Lin et al., 1999
). Furthermore, tissue-specific developmental defects are seen after modification of the number and position of the sulphate groups on heparan sulphate. For example, mice homozygous for the Hs2st gene trap mutation are unable to add 2-O sulphate groups to heparan sulphate and exhibit renal, eye and skeletal defects (Bullock et al., 1998
), while targeted disruption of the Ndst1 gene, which encodes N-deacetylase N-sulphotransferase 1, leads to pulmonary hypoplasia, atelectasis and respiratory distress syndrome (Ringvall et al., 2000
).
HSPGs are found in the basement membrane of the neuroepithelium of the closing neural tube, as well as in the adjacent tissues of the posterior neuropore region (Copp and Bernfield, 1988; OShea, 1987
). Moreover, degradation of heparan sulphate by heparitinase disrupts cranial neurulation in cultured rat embryos, leading to the development of exencephaly (Tuckett and Morriss-Kay, 1989
). The purpose of the present study was to determine whether HSPGs, and specifically the sulphation pattern of heparan sulphate, could also regulate spinal neurulation. We cultured E8.5 mouse embryos in the presence of an inhibitor of sulphation, chlorate, to examine the importance of the sulphate group in HSPG function. The sulphate donor for GAG sulphation is 3'-phosphoadenosine 5'-phosphosulphate (PAPS), which is synthesised by PAPS synthetase. Chlorate acts as a sulphate analogue and competes with sulphate in PAPS synthesis, resulting in inhibition of GAG sulphation (Conrad, 1998
; Greve et al., 1988
). We find that chlorate acts to hasten the closure of the spinal neural tube, and we present evidence suggesting that this effect may be mediated, in part, via inhibition of Shh signalling.
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MATERIALS AND METHODS |
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After culture, embryos were selected for further analysis only if they exhibited vigorous blood flow in the yolk sac circulation, and if the heart beat was regular and above 100 per minute. They were dissected from the yolk sac and amnion, inspected for closure of the cranial and spinal neural tube, then scored using the Morphological Scoring System of Brown and Fabro (Brown and Fabro, 1981). Crown-rump length, head length and posterior neuropore length (the distance between the rostral end of the posterior neuropore and the tip of the tail bud) were measured using an eyepiece graticule attached to a Zeiss SV6 stereomicroscope. Some embryos were sonicated and used to determine total protein content using the BCA Protein Assay Kit (Pierce and Warriner), whereas others were fixed in 4% paraformaldehyde in phosphate-buffered saline (PBS) before embedding in paraffin wax and sectioning at 6 µm, transversely through the posterior neuropore region. Sections were stained with Haematoxylin and Eosin or used for immunolocalisation of chondroitin sulphate and heparan sulphate. Other embryos were processed for whole-mount mRNA in situ hybridisation for Shh and patched 1 (Ptch).
Immunohistochemistry
Mouse monoclonal antibodies 10E4 (Seikagaku) and CS-56 (Sigma) were used to detect heparan sulphate and chondroitin sulphate respectively (Avnur and Geiger, 1984; David et al., 1992
). A monoclonal mouse IgM antibody (DAKO) raised against Aspergillus niger glucose oxidase (an enzyme neither present nor inducible in mammalian tissues) was used as a negative control. Rehydrated tissue sections were incubated with 3% hydrogen peroxide and the blocking solutions from the HistoMouse-SP kit (Zymed) to reduce nonspecific background staining, then incubated with 10 µg/ml primary antibody for an hour at room temperature. The signal was amplified using a biotinylated anti-mouse secondary antibody and a streptavidin-horseradish peroxidase conjugate and detected using 3,3'-diaminobenzidine tetrahydrochloride.
Labelling and analysis of GAGs
GAGs were labelled using carrier-free [35S]sulphate and analysed by anion exchange chromatography (Solursh and Morriss, 1977; Copp and Bernfield, 1988
). Embryos were stabilised in culture for 3 hours, then carrier-free [35S]sulphate (Amersham Pharmacia) was added to the culture medium to a final concentration of 100 µCi/ml (final concentration of sulphate: 0.75 µM) and culture was continued for a further 5 hours. After removal of yolk sac and amnion, embryos were washed using ice-cold DMEM and PBS in order to remove unincorporated label. Embryos were stored in TE (50 mM Tris, pH 7.5; 2 mM EDTA) at 70°C.
Labelled GAGs were extracted by sonicating embryos in TE on ice. An aliquot was removed for scintillation counting and protein quantification, then 50 µg each of carrier hyaluronan and chondroitin-6-sulphate (Sigma) were added and GAGs were precipitated overnight at 20°C using three volumes of 1.3% potassium acetate in 95% ethanol. GAGs were pelleted by centrifugation at 14,000 g for 15 minutes, then re-suspended in de-ionised water. Pronase (Protease XIV, Sigma) was added (2 mg) and samples were incubated at 55°C overnight to degrade proteins. The enzyme was heat-inactivated, the GAGs were re-precipitated using potassium acetate and ethanol, and the pellet was dissolved in de-ionised water.
Labelled GAGs were separated by anion exchange chromatography on DEAE cellulose (DE52; Whatman) using a linearly increasing elution gradient of sodium chloride solution (up to 0.7 M), at a flow rate of 15 ml/hour. Total elution volume was 25 ml and fractions were collected at 2-minute intervals. Radioactivity in each fraction was measured using a Wallac 1410 liquid scintillation counter. Recovery from the anion exchange column was 99.3±0.4%. The efficiency of scintillation counting was 87.1%.
In situ hybridisation
Whole-mount in situ hybridisation using digoxigenin-labelled riboprobes was performed as described (Tautz and Pfeiffle, 1989), using plasmids for Shh (Echelard et al., 1993
) and Ptch (Goodrich et al., 1996
). Briefly, embryos fixed in 4% paraformaldehyde and stored in 100% methanol were rehydrated, incubated in 6% hydrogen peroxide for 1 hour, and then digested with Proteinase K (10 µg/ml) for 2 minutes. The embryos were re-fixed in 0.2% glutaraldehyde and 4% paraformaldehyde, and hybridised overnight at 70°C. After high stringency washes, the riboprobes were localised using a sheep anti-digoxigenin antibody and detected by incubation in nitroblue tetrazolium/5-bromo-4-chloro-3-indolyl phosphate. Transverse paraffin wax-embedded sections (10 µm) were prepared through the posterior neuropore region.
Statistical analysis
Embryonic parameters were compared between treatment groups by Students t-test or one-way analysis of variance, with subsequent pairwise comparisons against the PBS-treated control group by Dunnetts test. Statistical significance level was P<0.05.
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RESULTS |
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All embryos exposed to chlorate concentrations of 30 mM or less had a vigorous yolk sac circulation and regular heart beat. Moreover, comparison of crown-rump length, head length, Brown and Fabro morphological score and protein content showed no statistically significant differences between embryos treated with chlorate and those in the control group, cultured in the absence of chlorate (Fig. 1). All embryos had between 19 and 23 pairs of somites with no significant difference in somite number between treatment groups (Fig. 2A). No obvious difference in gross morphology was found between the two groups, apart from the posterior neuropore length, which differed significantly as described in the following sections.
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In contrast to chlorate, there was no significant effect of 1 mM ß-D-xyloside on posterior neuropore closure (not shown), whereas cranial neurulation was frequently disturbed, as described previously (Morriss-Kay and Crutch, 1982). Moreover, caudal somitogenesis was disturbed in ß-D-xyloside-treated embryos, but not in chlorate-treated embryos. Hence, chlorate and ß-D-xyloside have differing effects on neurulation in the spinal and cranial regions.
To confirm that the effect of chlorate is due to competitive inhibition of GAG sulphation, embryos were cultured in the presence of PBS, chlorate, or chlorate plus sulphate. Exogenous sulphate in the culture medium is predicted to compete out the effect of chlorate in inhibiting GAG sulphation. As in the dose-response experiment (Fig. 2B), we found that 30 mM chlorate leads to a reproducible shortening of the posterior neuropore (Fig. 3A). Moreover, exogenous sulphate (10 mM) was able to block the effect of chlorate in inducing premature neuropore closure (P<0.01). There was no difference in posterior neuropore length between embryos cultured in the presence of chlorate plus sulphate and those exposed to PBS (P>0.05). Hence, acceleration of posterior neuropore closure by chlorate is due to competitive inhibition of GAG sulphation.
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Effect of chlorate on GAG sulphation
We studied the effect of chlorate on GAG sulphation by culturing embryos in the presence of [35S]sulphate followed by extraction of 35S-labelled GAGs and analysis by anion exchange chromatography. Because the molar concentration of sulphate in these cultures was 1.3x104 times lower than in the blocking experiments (Fig. 3A), the addition of [35S]sulphate was not expected to block the effect of chlorate.
Embryos cultured in the absence of chlorate gave an elution profile (red line, Fig. 4A) in which heparan sulphate eluted at a sodium chloride concentration of 0.33±0.01 M (mean±s.e.m.) and was sensitive to heparitinase digestion (not shown), while chondroitin sulphate eluted at a sodium chloride concentration of 0.43±0.01 M and was sensitive to digestion by chondroitinase ABC. By contrast, in cultures of chlorate-treated embryos (green line, Fig. 4A), sulphation of chondroitin sulphate was completely abolished, while the degree of sulphation of heparan sulphate was reduced by 46.0%, giving rise to Peak HS'. This incomplete abolition of heparan sulphation probably results from chlorate (30 mM) inhibiting O-sulphation more effectively than N-sulphation (Safaiyan et al., 1999).
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We treated samples of material from Peak HS' with chondroitinase, heparitinase or PBS before analysis using anion exchange chromatography (Fig. 4B). Chondroitinase had no effect on Peak HS', whereas heparitinase reduced its height to give Peak HS'', thus confirming that Peak HS' consists of under-sulphated heparan sulphate.
Requirement for both N- and O-sulphate groups in heparan sulphate
Heparan sulphate contains both N- and O-linked sulphate groups. To determine which is needed for regulating spinal neurulation, embryos were cultured in the presence of chlorate plus either de-N- or de-O-sulphated heparan sulphate. Neither heparan sulphate species was able to prevent the neuropore closure effect of chlorate (Fig. 5), suggesting that both N- and O-sulphate groups are needed for heparan sulphate to regulate spinal neurulation.
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Formation of the chlorate-induced neuroepithelial morphology could be blocked by addition of exogenous sulphate to the culture medium (Fig. 6G), yielding a posterior neuropore morphology similar to that seen in embryos exposed only to PBS. This result correlates with the finding that exogenous sulphate blocks chlorate-induced premature posterior neuropore closure, and is consistent with competitive inhibition of GAG sulphation by chlorate.
Supplementation of the culture medium with exogenous heparan sulphate also prevents chlorate-induced changes in posterior neuropore morphology (Fig. 6C,H). This suggests that the exogenous heparan sulphate in the culture medium is an effective substitute for normally sulphated heparan sulphate synthesised endogenously by the embryo, and correlates with the finding that exogenous heparan sulphate blocks chlorate-induced premature posterior neuropore closure. By contrast, addition of exogenous chondroitin-6-sulphate to the culture medium does not prevent the chlorate-induced change in neuroepithelial morphology (Fig. 6I). In these embryos, the median hinge point is absent, the paired dorsolateral hinge points exhibit accentuated bending and neuroepithelial rigidity is lost in the region between the dorsolateral hinge points, as in embryos exposed to chlorate alone (Fig. 6F).
Chlorate diminishes immunostained GAGs in the mouse embryo
We assessed the presence of sulphated GAGs after chlorate treatment by immunostaining transverse sections of the posterior neuropore region with anti-heparan sulphate (10E4) and anti-chondroitin sulphate (CS-56) antibodies. Embryos cultured in the presence of PBS show strong staining for both heparan sulphate (Fig. 7A) and chondroitin sulphate (Fig. 7D) in the neuroepithelial basement membrane, extending from the median hinge point to the neural fold apices. Staining is also seen in the basement membrane of the surface ectoderm and gut endoderm, and in the extracellular matrix of the paraxial mesoderm. By contrast, staining for both sulphated GAGs is greatly reduced in chlorate-treated embryos. Staining for heparan sulphate is very weak throughout the section (Fig. 7B), whereas staining for chondroitin sulphate, although very much reduced in the neuroepithelium and neuroepithelial basement membrane, shows a less marked reduction than heparan sulphate in the underlying mesoderm (Fig. 7E). Importantly, chlorate-treated embryos exposed to exogenous heparan sulphate (Fig. 7C) exhibit an intensity of heparan sulphate staining that is intermediate between PBS-treated (Fig. 7A) and chlorate-treated embryos (Fig. 7B), with strongest staining in the surface ectodermal basement membrane and in the neural folds. This finding demonstrates that exogenous heparan sulphate, which is able to block the effect of chlorate on posterior neuropore closure, gains access to the cells of the posterior neuropore.
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Effect of chlorate on sonic hedgehog signalling
Shh has been suggested to participate in the regulation of spinal neurulation by contributing to the formation of the median hinge point, while inhibiting bending at paired dorsolateral hinge points (Ybot-Gonzalez et al., 2002). To determine whether absence of the median hinge point and the accentuated bending at dorsolateral hinge points in chlorate-treated embryos may be related to disruption of Shh signalling, we compared the expression patterns of Shh and Ptch by in situ hybridisation. Shh is a ligand of Ptch and Shh signalling up-regulates Ptch expression (Goodrich et al., 1996
; Marigo et al., 1996
). Hence, an abnormal pattern of Ptch expression may indicate disruption of Shh signalling.
The expression patterns of Shh and Ptch transcripts in embryos cultured in the presence of PBS are essentially as described (Echelard et al., 1993; Goodrich et al., 1996
). In the posterior neuropore region, Shh transcripts are detected in the notochord and in the ventral part of the hindgut (Fig. 8A), while Ptch transcripts are present in the neuroepithelium, particularly at the median hinge point, with staining intensity decreasing progressively towards the paired dorsolateral hinge points (Fig. 8C). The notochord is only weakly positive for Ptch, which is also seen in the hindgut (both dorsal and ventral parts) and weakly in mesoderm immediately lateral to gut and notochord.
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DISCUSSION |
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Heparan sulphate and the role of actin microfilaments in neurulation
Actin microfilament bundles are found in neuroepithelial cells (Baker and Schroeder, 1967; Karfunkel, 1974
; Morriss-Kay and Tuckett, 1985
; Morriss and New, 1979
; Nagele and Lee, 1980
; Sadler et al., 1982
; Ybot-Gonzalez and Copp, 1999
) and their contraction has been postulated to cause neuroepithelial cell wedging, leading to elevation and bending of the neural folds (Karfunkel, 1974
). Moreover, HSPGs are known to co-localise with, and bind to, actin and other components of the cytoskeleton in epithelial cells, thus participating in organisation of the cytoskeleton (Bernfield et al., 1999
; Carey et al., 1994
; Carey et al., 1996
; Fernandez-Borja et al., 1995
). Indeed, apical microfilament bundles in the cranial neuroepithelium were found to be poorly organised when cranial neural tube closure was inhibited by ß-D-xyloside treatment of cultured rat embryos, which blocks the attachment of GAGs to their proteoglycan core proteins (Morriss-Kay and Crutch, 1982
). The breadth of the apical region of neuroepithelial cells was widened in these embryos, suggesting that the microfilament bundles were not under tension. Moreover, exposure of cultured mouse embryos undergoing spinal neurulation to cytochalasin D also resulted in disassembly of the apical microfilaments in the neuroepithelium (Ybot-Gonzalez and Copp, 1999
). This led to loss of rigidity of the neural plate, resembling the morphology seen in chlorate-treated embryos. This suggests that microfilament assembly and contraction in the neuroepithelium require heparan sulphate, and that this heparan sulphate needs to be normally sulphated. Importantly, however, formation of the median and dorsolateral hinge points during mouse spinal neurulation was not inhibited by cytochalasin D (Ybot-Gonzalez and Copp, 1999
), indicating that additional factors must also act to prevent median hinge point formation in the chlorate-treated embryos.
Heparan sulphate and the cell cycle
Besides apical contraction, neuroepithelial cells may adopt a wedge shape by broadening of the cell base. This appears to be an important mechanism for hinge point formation during mouse spinal neurulation, in the absence of a requirement for actin microfilaments (Ybot-Gonzalez and Copp, 1999). Interkinetic nuclear migration results in the cell nucleus occupying a basal position during S- and early G2-phases of the cell cycle, and this is accompanied by cell wedging. Indeed, more cells are in G2-phase in the median hinge point than in non-bending regions of the neural plate of the chick embryo, and these cells have a prolonged cell cycle (Smith and Schoenwolf, 1987
; Smith and Schoenwolf, 1988
). Similarly, cells in the median hinge point have a higher S-phase labelling index but lower mitotic index than elsewhere in the spinal neuroepithelium of the mouse embryo (Gerrelli and Copp, 1997
).
Nuclear heparan sulphate is known to influence the cell cycle (Fedarko et al., 1989; Fedarko and Conrad, 1986
; Ishihara and Conrad, 1989
). Heparan sulphate has been localised in the nuclei of hepatoma cells in culture, and these heparan sulphate molecules contain highly sulphated residues (Fedarko and Conrad, 1986
). As the hepatoma cells become confluent and stop dividing, the amount of nuclear heparan sulphate increases up to threefold (Ishihara and Conrad, 1989
). Heparan sulphate, extracted from confluent cell cultures and added to growing cells, is taken up and transported to the cell nuclei (Fedarko et al., 1989
), leading to inhibition of cell division. By contrast, heparan sulphate obtained from cells in the logarithmic phase of growth is less effectively taken up and does not affect cell division. Thus, prolongation of the cell cycle in median hinge point cells during normal neurulation could require the presence of nuclear heparan sulphate. Accordingly, perturbation of sulphation of heparan sulphate by chlorate could cause cells in the median hinge point to progress through the cell cycle, resulting in loss of basal localisation of the cell nuclei and disruption of cell wedging.
Heparan sulphate on the cell surface and in the extracellular matrix is also able to regulate cell cycling through its interaction with growth factors (Conrad, 1998). Binding of FGF to high-affinity FGF receptors leads to dimerisation and mutual tyrosine phosphorylation of these receptors, resulting in biological effects such as cell proliferation (Schlessinger et al., 1995
). The growth factor-receptor interaction is facilitated by heparan sulphate. However, heparan sulphate that inhibits FGF-stimulated cell proliferation has also been described. For example, inhibitory heparan sulphate prevents the human breast cancer cell line MDA-MB-231 from responding to FGF2 (Delehedde et al., 1996
). MDA-MB-231 cells do not normally have a mitogenic response to FGF2, but blocking sulphation of heparan sulphate by chlorate enables these cells to respond to FGF2 and proliferate. Heparan sulphate that inhibits a mitogenic response to FGF1 and FGF7 has also been described (Bonneh-Barkay et al., 1997
; Pye et al., 2000
), and this inhibitory activity has been correlated with the sulphation pattern of heparan sulphate. Thus, cells in the median hinge point could have a prolonged cell cycle because of their inability to respond to growth factors (such as FGFs), owing to the inhibitory action of heparan sulphate. Blocking sulphation of heparan sulphate by chlorate treatment releases the cells from this inhibition, enabling their increased proliferation, and abolishing median hinge point formation.
Heparan sulphate and sonic hedgehog signalling
Heparan sulphate is required for localisation and propagation of the hedgehog signal (Bellaiche et al., 1998; Lin et al., 2000
; The et al., 1999
) through its interaction with the transmembrane protein Dispatched. This promotes the release of hedgehog from producing cells and helps to propagate the signal through the tissues to the receiving cells (Burke et al., 1999
). In addition, a specific requirement for sulphate groups on heparan sulphate is indicated by the disruption of hedgehog signalling where sulphation of heparan sulphate is perturbed, as in the Drosophila sulfateless mutant (The et al., 1999
). Thus, it could be postulated that the absence of a median hinge point and the increased bending at the paired dorsolateral hinge points in the chlorate-treated embryos are caused by disruption of Shh signalling. Indeed, dorsolateral bending of the neural plate in the posterior neuropore has been shown to be negatively regulated by Shh (Ybot-Gonzalez et al., 2002
). However, induction of midline bending by the notochord (Smith and Schoenwolf, 1989
; Van Straaten et al., 1985
) does not appear to depend primarily on Shh action. For example, median hinge point formation is not abolished in embryos lacking Shh function (Ybot-Gonzalez et al., 2002
). We conclude that modulation of Shh function may be responsible for some, but not all, of the abnormalities of posterior neuropore closure observed in chlorate-treated embryos.
We have shown that chlorate alters the expression pattern of Ptch mRNA, whereas Shh expression appears unaffected. We cannot exclude the possibility that Ptch expression is affected directly by chlorate, or indirectly affected by altered neural plate bending. Nevertheless, our results are consistent with a reduced propagation of the Shh signal in the presence of chlorate. It is well established that the influence of Shh from the notochord and floor plate induces a ventrodorsal gradient of Ptch expression in the neuroepithelium (Marigo and Tabin, 1996). We suggest that during normal development, Shh signalling from the notochord is propagated towards the overlying neuroepithelium through the action of heparan sulphate in the intervening extracellular matrix. This suggestion is supported by the immunohistochemical studies of Martí et al. (Martí et al., 1995
), which show that Shh peptide is cleared from notochordal cells (despite continuing Shh transcription in these cells) and accumulates in the adjacent floor plate region of E9.5 mouse embryos [see Figure 5F,G by Martí et al. (Martí et al., 1995
)]. We suggest, moreover, that inhibition of heparan sulphation by chlorate treatment abolishes this propagation of the Shh signal. Shh peptide is now able to induce strong expression of Ptch within the notochord, at the site of Shh production, as well as in adjacent paraxial mesodermal cells that normally receive only low concentrations of Shh peptide, whereas Ptch is induced to a lesser degree in the overlying neuroepithelium, and the dorsoventral gradient of neuroepithelial Ptch expression is diminished.
The situation we observe in chlorate-treated mouse embryos differs from that in the Drosophila tout-velu mutant, where Ptc protein is absent, rather than ectopically expressed (Bellaiche et al., 1998). This difference could be explained by a quantitative and qualitative difference in heparan sulphate. In the tout-velu mutant, heparan sulphate is almost undetectable, owing to a lack of heparan sulphate co-polymerase, which is required for chain elongation (Toyoda et al., 2000
). By contrast, chlorate treatment merely inhibits O-sulphation and, to a lesser extent N-sulphation, and does not have a significant effect on chain elongation or other structural modifications of heparan sulphate (Conrad, 1998
; Greve et al., 1988
; Safaiyan et al., 1999
).
In conclusion, we have demonstrated a role for HSPGs in regulating mouse spinal neurulation. This role seems likely to be mediated via a combination of the effects of heparan sulphate on the cytoskeleton, cell cycle and the propagation of extracellular signals.
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ACKNOWLEDGMENTS |
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