spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Yip, G. W.
Right arrow Articles by Copp, A. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Yip, G. W.
Right arrow Articles by Copp, A. J.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Heparan sulphate proteoglycans and spinal neurulation in the mouse embryo

George W. Yip1,*, Patrizia Ferretti1 and Andrew J. Copp2,{dagger}

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



View larger version (23K):

[in a new window]
  		Fig. 1. Effect of chlorate on embryonic growth parameters. (A) Crown-rump length, (B) head length and (C) Brown and Fabro morphological score of E9.5 embryos following culture for 14 hours in the presence of 0, 10, 20 and 30 mM chlorate. Statistical comparison using one-way ANOVA shows no significant effect of chlorate concentration on any of the growth parameters (P=0.307, 0.178, 0.308 in A,B,C, respectively). (D) Total embryonic protein content does not differ between cultures containing 0 and 30 mM chlorate (Student’s t-test; p=0.854). Values represent mean±s.e.m. of at least 9 embryos in each group in A-C, and at least five embryos per group in D.

 


View larger version (16K):

[in a new window]
  		Fig. 2. Effect of chlorate on developmental progression and closure of the spinal neural tube. E9.5 embryos following culture for 14 hours in the presence of 0, 10, 20 or 30 mM chlorate. Comparison of somite number (A) and posterior neuropore length (B) between the four groups using one-way ANOVA shows no significant effect of chlorate concentration on somite number (P=0.641) but a significant effect on neuropore length (P<0.0001) which is significantly smaller in embryos treated with 30 mM chlorate than in the 0 mM group (Dunnett’s test; P<0.01). Treatment with 10 mM and 20 mM chlorate did not significantly reduce the posterior neuropore length compared with the 0 mM group (P>0.05 in each case). Values represent mean±s.e.m. of at least nine embryos in each group. PNP, posterior neuropore.

 


View larger version (20K):

[in a new window]
  		Fig. 3. Specific requirement for heparan sulphate in spinal neurulation. Embryos were cultured in the presence of PBS, 30 mM chlorate alone or 30 mM chlorate plus (A) 10 mM sulphate, (B) 100 ng/ml heparan sulphate, (C) 100 ng/ml heparan sulphate pre-treated with heparitinase or (D) 100 ng/ml chondroitin-6-sulphate. Posterior neuropore length varies significantly between treatment groups (one-way ANOVA; P<0.0001) in all panels with a significantly shorter posterior neuropore length in the chlorate alone group compared with the PBS-treated group in each case (Dunnett’s test: P<0.01). Embryos treated with chlorate plus exogenous sulphate (A) or heparan sulphate (B) do not differ in posterior neuropore length significantly from PBS-treated embryos (P>0.05), while embryos treated with chlorate plus heparitinase-treated heparan sulphate (C) or chondroitin-6-sulphate (D) do not differ from embryos treated with chlorate alone (P>0.05). Values represent mean±s.e.m. of at least 12 embryos per group in A-C and at least nine embryos per group in D. CS, chondroitin-6-sulphate; HS, heparan sulphate; htx-HS, heparitinase-treated heparan sulphate.

 


View larger version (26K):

[in a new window]
  		Fig. 4. Chlorate inhibits sulphation of GAGs in cultured mouse embryos. (A) [35S]sulphate-labelled GAGs from PBS-treated embryos were separated by anion exchange chromatography, yielding two peaks (red line), representing heparan sulphate (HS) and chondroitin sulphate (CS). Culture of embryos in chlorate (green line) abolishes sulphation of chondroitin sulphate and dramatically reduces the sulphation of heparan sulphate, resulting in the formation of Peak HS'. There is no effect on the elution profile when exogenous heparan sulphate (100 ng/ml) is added to the chlorate-treated embryos (blue line). (B) The single 35S-labelled GAG peak (HS') from chlorate-treated embryos (red line) is unaffected by pre-treatment with chondroitinase (green line), whereas it is significantly reduced in height by heparitinase treatment, giving rise to Peak HS'' (blue line). Note the expanded y-axis used for plotting the level of radioactivity in B compared with A. Brown line represents elution gradient of sodium chloride (right axis). NNC, non-negatively charged 35S-labelled material eluting in the buffer wash.

 


View larger version (15K):

[in a new window]
  		Fig. 5. Importance of N- and O-sulphate groups in heparan sulphate. Embryos were cultured in the presence of PBS, 30 mM chlorate alone or 30 mM chlorate plus (A) 100 ng/ml de-N-sulphated or (B) 100 ng/ml de-O-sulphated heparan sulphate. Posterior neuropore length varies significantly between treatment groups (one-way ANOVA; P<0.0001) in both panels. Although the chlorate-induced reduction in posterior neuropore length was statistically significant in both experiments (Dunnett’s test; P<0.01 in both cases), there was no significant difference between embryos treated with chlorate alone and those treated additionally with de-N- or de-O-sulphated heparan sulphate (P>0.05). Values represent mean±s.e.m. of at least nine embryos per group. de-N-HS, de-N-sulphated heparan sulphate; de-O-HS, de-O-sulphated heparan sulphate.

 


View larger version (107K):

[in a new window]
  		Fig. 6. Heparan sulphate affects neural plate bending during spinal neurulation. (A-C) E9.5 embryos following culture showing the normal appearance of the posterior neuropore (between black arrows) in embryos treated with PBS (A) and chlorate plus heparan sulphate (C). By contrast, embryos treated with chlorate alone exhibit a convex neural plate within a shortened neuropore (blue arrow in B). (E-I) H&E-stained transverse sections through the rostral end of the posterior neuropore of embryos cultured in the presence of PBS (E), chlorate (F), chlorate plus sulphate (G), chlorate plus heparan sulphate (H) or chlorate plus chondroitin-6-sulphate (I). Note the loss of median hinge point, accentuation of dorsolateral bending and convex neural plate (blue arrows) in F,I compared with the normal appearance in E,G,H. Level of sections is shown in D. Posterior neuropore is shorter in embryos in F,I, compared with those in E,G,H, so sections at the level of the rostral end of the neuropore are more distal: hence, the reduced section diameter in F,I. Reagent concentrations as in Fig. 3. Red arrows, paired dorsolateral hinge points; star, median hinge point; ClO3, chlorate; CS, chondroitin sulphate; HS, heparan sulphate; SO4, sulphate. Scale bars: 0.5 mm in A-C; 100 µm in E-I.

 


View larger version (73K):

[in a new window]
  		Fig. 7. Chlorate reduces the sulphation of GAGs in the posterior neuropore region. Immunohistochemical localisation of heparan sulphate (A-C) and chondroitin sulphate (D,E) on transverse sections through the posterior neuropore region prepared from embryos cultured in the presence of PBS (A,D), chlorate alone (B,E) or chlorate plus heparan sulphate (C). Heparan sulphate staining is reduced dramatically after chlorate treatment (B), whereas chlorate plus heparan sulphate-treated embryos show increased heparan sulphate staining, particularly in the ectodermal basement membrane (C, blue arrows), indicating access of exogenous heparan sulphate to embryonic tissues. Chondroitin sulphate exhibits specific loss of staining in the chlorate-treated neuroepithelium (E), whereas mesodermal staining is maintained at low intensity. Black arrows, paired dorsolateral hinge points; star, median hinge point; ClO3, chlorate; CS, chondroitin sulphate; HS, heparan sulphate; neb, neuroepithelial basement membrane. Scale bars: 100 µm.

 


View larger version (63K):

[in a new window]
  		Fig. 8. Altered expression of Ptch, but not Shh, in embryos cultured in the presence of chlorate. Embryos cultured in the presence of either PBS (A,C) or 30 mM chlorate (B,D,E) were processed for whole-mount in situ hybridisation analysis of Shh (A,B) and Ptch (C-E), and then sectioned transversely through the rostral end of the posterior neuropore. The pattern of Shh expression does not differ between PBS-treated and chlorate-treated embryos (compare A with B), whereas the distribution of Ptch transcripts is markedly altered (compare C with D,E). In particular, notochordal expression of Ptch is strongly upregulated, as seen in two different chlorate-treated embryos (D,E), while midline neuroepithelial Ptch expression is less intense and exhibits less marked ventrodorsal variation. Arrows, paired dorsolateral hinge points; star, median hinge point; ClO3, chlorate; nc, notochord. Scale bars: 100 µm.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2002