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Musculocontractural Ehlers – Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin

View Article: PubMed Central - PubMed

ABSTRACT

Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers–Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial–mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.

No MeSH data available.


Related in: MedlinePlus

CS/DS-PGs in CNC cells. (A) qPCR analysis in uninjected CNC explants at stage 18. Note abundant expression of Itga5, Itgb1 and Sdc4. Results are mean±s.d. from triplicates (n≥4 biological replicates). (B) Dse-MO does not differentially affect the mRNA levels of Itga5, Itgb1 and Sdc4 compared with Dse-5MM-MO. Results are mean±s.d. (n≥4 biological replicates). (C) Dse-MO does not reduce the protein amount of integrin β1 in explants enriched in neural crest and epidermis of stage 18 embryos. Western blotting was performed on a 7.5% Mini-Protean TGX Stain-free gel (Bio-Rad). The loading control was ascertained prior to blotting using the ChemiDoc Touch Imaging System. Resuts is representative of two independent experiments (n=2). (D) Metabolic labeling of PGs in stage 18 CNC explants. Note that Chase B partially degrades CS/DS PGs >18 kDa. The IdoA is a rare modification because the split chains are ∼10 kDa.
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DMM024661F7: CS/DS-PGs in CNC cells. (A) qPCR analysis in uninjected CNC explants at stage 18. Note abundant expression of Itga5, Itgb1 and Sdc4. Results are mean±s.d. from triplicates (n≥4 biological replicates). (B) Dse-MO does not differentially affect the mRNA levels of Itga5, Itgb1 and Sdc4 compared with Dse-5MM-MO. Results are mean±s.d. (n≥4 biological replicates). (C) Dse-MO does not reduce the protein amount of integrin β1 in explants enriched in neural crest and epidermis of stage 18 embryos. Western blotting was performed on a 7.5% Mini-Protean TGX Stain-free gel (Bio-Rad). The loading control was ascertained prior to blotting using the ChemiDoc Touch Imaging System. Resuts is representative of two independent experiments (n=2). (D) Metabolic labeling of PGs in stage 18 CNC explants. Note that Chase B partially degrades CS/DS PGs >18 kDa. The IdoA is a rare modification because the split chains are ∼10 kDa.

Mentions: Based on the tissue-autonomous role of IdoA in CS/DS, we propose that DS-epi1 knockdown affects PGs that are synthesized in the CNC. The integrin α5β1 heterodimer (encoded by the Itga5 and Itgb1 genes), members of the syndecan (Sdc) family, Bgn, Vcan and CD44 have been presented as CS or CS/DS PGs, some of which have functions in cell adhesion (Thelin et al., 2013). In CNC explants of stage 18 embryos, qPCR indicated abundant mRNA levels of Itga5, Itgb1 and Sdc4, whereas Sdc1, Sdc3, Bgn, Vcan and CD44 were not expressed or were only expressed at very low levels (Fig. 7A). We did not identify changes in the Itga5, Itgb1 or Sdc4 mRNA levels in the Dse-morphant compared with the Dse-5MM-MO-injected control CNC cells (Fig. 7B). Moreover, western blot analysis demonstrated similar levels of integrin β1 protein in the DS-epi1+ and DS-epi1-deficient NC- and epidermis-enriched explants (Fig. 7C), which suggests that reduced IdoA levels do not alter the expression of integrins. In some mammalian cell types, it has been reported that the α5 and β1 integrin subunits (Veiga et al., 1997; Franco et al., 2009) and Sdc4 (Shworak et al., 1994; Deepa et al., 2004; Holmborn et al., 2012) are hybrid PGs that carry both CS and HS chains; however, it is not known whether they contain CS/DS chains. To investigate whether migratory CNC cells also synthesize IdoA in CS/DS chains, we isolated CNC explants at stage 18 and cultivated the cells in vitro in [35S]-containing medium for 24 h to label the PGs. Chase B degraded 47% of the labeled CS/DS PGs, which covered a wide range of molecular mass of over 18 kDa (Fig. 7D), and the size distribution of the degraded products indicated that isolated IdoA moieties are present in the native chains. The molecular mass of the IdoA-containing PGs is compatible with integrin α5β1 subunits (120-150 kDa) (Alfandari et al., 2003) and Sdc4 (40-250 kDa) (Gopal et al., 2010). However, we could not identify CS, CS/DS and HS chains on endogenous integrin α5 and β1 in Xenopus CNC tissue, as well as explants enriched in epidermis and neural crest (Fig. S8A,B). Moreover, we demonstrated that HS, but not CS and CS/DS, chains in Flag-tagged Sdc4 were produced in the mRNA-injected explants (Fig. S8C). In summary, integrin α5β1 and Sdc4 are expressed in CNC cells and do not appear to be decorated with CS or CS/DS chains in Xenopus embryos. These findings suggest that other not yet identified CS/DS PGs might mediate the DS-epi1-dependent CNC cell adhesion on fibronectin.Fig. 7.


Musculocontractural Ehlers – Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin
CS/DS-PGs in CNC cells. (A) qPCR analysis in uninjected CNC explants at stage 18. Note abundant expression of Itga5, Itgb1 and Sdc4. Results are mean±s.d. from triplicates (n≥4 biological replicates). (B) Dse-MO does not differentially affect the mRNA levels of Itga5, Itgb1 and Sdc4 compared with Dse-5MM-MO. Results are mean±s.d. (n≥4 biological replicates). (C) Dse-MO does not reduce the protein amount of integrin β1 in explants enriched in neural crest and epidermis of stage 18 embryos. Western blotting was performed on a 7.5% Mini-Protean TGX Stain-free gel (Bio-Rad). The loading control was ascertained prior to blotting using the ChemiDoc Touch Imaging System. Resuts is representative of two independent experiments (n=2). (D) Metabolic labeling of PGs in stage 18 CNC explants. Note that Chase B partially degrades CS/DS PGs >18 kDa. The IdoA is a rare modification because the split chains are ∼10 kDa.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920151&req=5

DMM024661F7: CS/DS-PGs in CNC cells. (A) qPCR analysis in uninjected CNC explants at stage 18. Note abundant expression of Itga5, Itgb1 and Sdc4. Results are mean±s.d. from triplicates (n≥4 biological replicates). (B) Dse-MO does not differentially affect the mRNA levels of Itga5, Itgb1 and Sdc4 compared with Dse-5MM-MO. Results are mean±s.d. (n≥4 biological replicates). (C) Dse-MO does not reduce the protein amount of integrin β1 in explants enriched in neural crest and epidermis of stage 18 embryos. Western blotting was performed on a 7.5% Mini-Protean TGX Stain-free gel (Bio-Rad). The loading control was ascertained prior to blotting using the ChemiDoc Touch Imaging System. Resuts is representative of two independent experiments (n=2). (D) Metabolic labeling of PGs in stage 18 CNC explants. Note that Chase B partially degrades CS/DS PGs >18 kDa. The IdoA is a rare modification because the split chains are ∼10 kDa.
Mentions: Based on the tissue-autonomous role of IdoA in CS/DS, we propose that DS-epi1 knockdown affects PGs that are synthesized in the CNC. The integrin α5β1 heterodimer (encoded by the Itga5 and Itgb1 genes), members of the syndecan (Sdc) family, Bgn, Vcan and CD44 have been presented as CS or CS/DS PGs, some of which have functions in cell adhesion (Thelin et al., 2013). In CNC explants of stage 18 embryos, qPCR indicated abundant mRNA levels of Itga5, Itgb1 and Sdc4, whereas Sdc1, Sdc3, Bgn, Vcan and CD44 were not expressed or were only expressed at very low levels (Fig. 7A). We did not identify changes in the Itga5, Itgb1 or Sdc4 mRNA levels in the Dse-morphant compared with the Dse-5MM-MO-injected control CNC cells (Fig. 7B). Moreover, western blot analysis demonstrated similar levels of integrin β1 protein in the DS-epi1+ and DS-epi1-deficient NC- and epidermis-enriched explants (Fig. 7C), which suggests that reduced IdoA levels do not alter the expression of integrins. In some mammalian cell types, it has been reported that the α5 and β1 integrin subunits (Veiga et al., 1997; Franco et al., 2009) and Sdc4 (Shworak et al., 1994; Deepa et al., 2004; Holmborn et al., 2012) are hybrid PGs that carry both CS and HS chains; however, it is not known whether they contain CS/DS chains. To investigate whether migratory CNC cells also synthesize IdoA in CS/DS chains, we isolated CNC explants at stage 18 and cultivated the cells in vitro in [35S]-containing medium for 24 h to label the PGs. Chase B degraded 47% of the labeled CS/DS PGs, which covered a wide range of molecular mass of over 18 kDa (Fig. 7D), and the size distribution of the degraded products indicated that isolated IdoA moieties are present in the native chains. The molecular mass of the IdoA-containing PGs is compatible with integrin α5β1 subunits (120-150 kDa) (Alfandari et al., 2003) and Sdc4 (40-250 kDa) (Gopal et al., 2010). However, we could not identify CS, CS/DS and HS chains on endogenous integrin α5 and β1 in Xenopus CNC tissue, as well as explants enriched in epidermis and neural crest (Fig. S8A,B). Moreover, we demonstrated that HS, but not CS and CS/DS, chains in Flag-tagged Sdc4 were produced in the mRNA-injected explants (Fig. S8C). In summary, integrin α5β1 and Sdc4 are expressed in CNC cells and do not appear to be decorated with CS or CS/DS chains in Xenopus embryos. These findings suggest that other not yet identified CS/DS PGs might mediate the DS-epi1-dependent CNC cell adhesion on fibronectin.Fig. 7.

View Article: PubMed Central - PubMed

ABSTRACT

Of all live births with congenital anomalies, approximately one-third exhibit deformities of the head and face. Most craniofacial disorders are associated with defects in a migratory stem and progenitor cell population, which is designated the neural crest (NC). Musculocontractural Ehlers–Danlos syndrome (MCEDS) is a heritable connective tissue disorder with distinct craniofacial features; this syndrome comprises multiple congenital malformations that are caused by dysfunction of dermatan sulfate (DS) biosynthetic enzymes, including DS epimerase-1 (DS-epi1; also known as DSE). Studies in mice have extended our understanding of DS-epi1 in connective tissue maintenance; however, its role in fetal development is not understood. We demonstrate that DS-epi1 is important for the generation of isolated iduronic acid residues in chondroitin sulfate (CS)/DS proteoglycans in early Xenopus embryos. The knockdown of DS-epi1 does not affect the formation of early NC progenitors; however, it impairs the correct activation of transcription factors involved in the epithelial–mesenchymal transition (EMT) and reduces the extent of NC cell migration, which leads to a decrease in NC-derived craniofacial skeleton, melanocytes and dorsal fin structures. Transplantation experiments demonstrate a tissue-autonomous role for DS-epi1 in cranial NC cell migration in vivo. Cranial NC explant and single-cell cultures indicate a requirement of DS-epi1 in cell adhesion, spreading and extension of polarized cell processes on fibronectin. Thus, our work indicates a functional link between DS and NC cell migration. We conclude that NC defects in the EMT and cell migration might account for the craniofacial anomalies and other congenital malformations in MCEDS, which might facilitate the diagnosis and development of therapies for this distressing condition. Moreover, the presented correlations between human DS-epi1 expression and gene sets of mesenchymal character, invasion and metastasis in neuroblastoma and malignant melanoma suggest an association between DS and NC-derived cancers.

No MeSH data available.


Related in: MedlinePlus