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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.

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Presence of IdoA in CS/DS PGs of early embryos. (A) At stage 22, IdoA is present in high molecular mass CS/DS PGs, as demonstrated by the size-fractionation of [35S]-containing PGs. The HS and CS/DS degradation products are produced by nitrous acid and Chase ABC treatment, respectively. CS/DS PGs represent 72% of the high molecular mass PGs (fractions 12-17). (B) SDS-PAGE analysis of [35S]-labeled CS/DS PGs. The same samples analyzed by gel filtration in A were separated using a 4-10% gradient SDS-PAGE following nitrous acid or, alternatively, Chase ABC or Chase B treatments. The fluography indicates CS/DS PGs (brackets) with an apparent molecular mass of 200-300 kDa (Bgn) and ∼1000 kDa (Vcan). The percentages of radioactivity in the framed areas are indicated below each lane. (C,C′) Whole-mount in situ hybridization of Bgn at stage 26. Embryo is shown in the lateral view (C) and transversally sectioned (C′). Arrowheads indicate migrating trunk neural crest cells. The section planes are indicated by the dashed straight line. (D,E) Chase B treatment degrades CS/DS chains in high molecular mass PGs in control-MO-injected embryos (D) but not Dse-MO-injected embryos (E). Bgn, biglycan; Chase, chondroitinase; Vcan, versican.
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DMM024661F3: Presence of IdoA in CS/DS PGs of early embryos. (A) At stage 22, IdoA is present in high molecular mass CS/DS PGs, as demonstrated by the size-fractionation of [35S]-containing PGs. The HS and CS/DS degradation products are produced by nitrous acid and Chase ABC treatment, respectively. CS/DS PGs represent 72% of the high molecular mass PGs (fractions 12-17). (B) SDS-PAGE analysis of [35S]-labeled CS/DS PGs. The same samples analyzed by gel filtration in A were separated using a 4-10% gradient SDS-PAGE following nitrous acid or, alternatively, Chase ABC or Chase B treatments. The fluography indicates CS/DS PGs (brackets) with an apparent molecular mass of 200-300 kDa (Bgn) and ∼1000 kDa (Vcan). The percentages of radioactivity in the framed areas are indicated below each lane. (C,C′) Whole-mount in situ hybridization of Bgn at stage 26. Embryo is shown in the lateral view (C) and transversally sectioned (C′). Arrowheads indicate migrating trunk neural crest cells. The section planes are indicated by the dashed straight line. (D,E) Chase B treatment degrades CS/DS chains in high molecular mass PGs in control-MO-injected embryos (D) but not Dse-MO-injected embryos (E). Bgn, biglycan; Chase, chondroitinase; Vcan, versican.

Mentions: To investigate the PG expression and GAG composition in Xenopus embryos, we microinjected [35S]sulfate into the blastocoel at stage 9 and analyzed the purified PGs at stage 22 after chemical and lyase treatment to degrade distinct GAG chains (Fig. 3). Nitrous acid at low pH was used to degrade HS (Shively and Conrad, 1976), and chondroitinases that are specific for DS (denoted Chase B) or both CS and DS (denoted Chase ABC) were used to degrade the DS or CS/DS chains, respectively. The size separation of the split products indicated that 56% of the total radioactivity corresponded to CS/DS PGs (Fig. 3A). SDS-PAGE analysis demonstrated two broad bands that were resistant to nitrous acid treatment and compatible with being biglycan (Bgn, 200-300 kDa, Moreno et al., 2005) and versican (Vcan, ∼1000 kDa) (Fig. 3B). Both bands disappeared following Chase ABC digestion and decreased following Chase B digestion to 71% (Bgn) and 65% (Vcan). We have previously demonstrated that CS/DS chains are present in Xenopus overexpressing Bgn (Hou et al., 2007). In post-neurula embryos, both Bgn (Fig. 3C,C′) and Vcan (Casini et al., 2008) are expressed in the trunk NC. Chase B treatment demonstrated IdoA in the high molecular mass fractions of control-MO- but not Dse-MO-injected embryos (Fig. 3D,E), which underscores the crucial role of DS-epi1 in DS biosynthesis. The Chase-B-degraded products primarily moved to fractions of 10-20 kDa, which indicates that IdoA is predominately a rare and isolated structure that is surrounded by long stretches of GlcA. We conclude that embryos at the early tailbud stage synthesize high molecular mass CS/DS PGs, such as Bgn and Vcan, with only few IdoA residues per chain.Fig. 3.


Musculocontractural Ehlers – Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin
Presence of IdoA in CS/DS PGs of early embryos. (A) At stage 22, IdoA is present in high molecular mass CS/DS PGs, as demonstrated by the size-fractionation of [35S]-containing PGs. The HS and CS/DS degradation products are produced by nitrous acid and Chase ABC treatment, respectively. CS/DS PGs represent 72% of the high molecular mass PGs (fractions 12-17). (B) SDS-PAGE analysis of [35S]-labeled CS/DS PGs. The same samples analyzed by gel filtration in A were separated using a 4-10% gradient SDS-PAGE following nitrous acid or, alternatively, Chase ABC or Chase B treatments. The fluography indicates CS/DS PGs (brackets) with an apparent molecular mass of 200-300 kDa (Bgn) and ∼1000 kDa (Vcan). The percentages of radioactivity in the framed areas are indicated below each lane. (C,C′) Whole-mount in situ hybridization of Bgn at stage 26. Embryo is shown in the lateral view (C) and transversally sectioned (C′). Arrowheads indicate migrating trunk neural crest cells. The section planes are indicated by the dashed straight line. (D,E) Chase B treatment degrades CS/DS chains in high molecular mass PGs in control-MO-injected embryos (D) but not Dse-MO-injected embryos (E). Bgn, biglycan; Chase, chondroitinase; Vcan, versican.
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DMM024661F3: Presence of IdoA in CS/DS PGs of early embryos. (A) At stage 22, IdoA is present in high molecular mass CS/DS PGs, as demonstrated by the size-fractionation of [35S]-containing PGs. The HS and CS/DS degradation products are produced by nitrous acid and Chase ABC treatment, respectively. CS/DS PGs represent 72% of the high molecular mass PGs (fractions 12-17). (B) SDS-PAGE analysis of [35S]-labeled CS/DS PGs. The same samples analyzed by gel filtration in A were separated using a 4-10% gradient SDS-PAGE following nitrous acid or, alternatively, Chase ABC or Chase B treatments. The fluography indicates CS/DS PGs (brackets) with an apparent molecular mass of 200-300 kDa (Bgn) and ∼1000 kDa (Vcan). The percentages of radioactivity in the framed areas are indicated below each lane. (C,C′) Whole-mount in situ hybridization of Bgn at stage 26. Embryo is shown in the lateral view (C) and transversally sectioned (C′). Arrowheads indicate migrating trunk neural crest cells. The section planes are indicated by the dashed straight line. (D,E) Chase B treatment degrades CS/DS chains in high molecular mass PGs in control-MO-injected embryos (D) but not Dse-MO-injected embryos (E). Bgn, biglycan; Chase, chondroitinase; Vcan, versican.
Mentions: To investigate the PG expression and GAG composition in Xenopus embryos, we microinjected [35S]sulfate into the blastocoel at stage 9 and analyzed the purified PGs at stage 22 after chemical and lyase treatment to degrade distinct GAG chains (Fig. 3). Nitrous acid at low pH was used to degrade HS (Shively and Conrad, 1976), and chondroitinases that are specific for DS (denoted Chase B) or both CS and DS (denoted Chase ABC) were used to degrade the DS or CS/DS chains, respectively. The size separation of the split products indicated that 56% of the total radioactivity corresponded to CS/DS PGs (Fig. 3A). SDS-PAGE analysis demonstrated two broad bands that were resistant to nitrous acid treatment and compatible with being biglycan (Bgn, 200-300 kDa, Moreno et al., 2005) and versican (Vcan, ∼1000 kDa) (Fig. 3B). Both bands disappeared following Chase ABC digestion and decreased following Chase B digestion to 71% (Bgn) and 65% (Vcan). We have previously demonstrated that CS/DS chains are present in Xenopus overexpressing Bgn (Hou et al., 2007). In post-neurula embryos, both Bgn (Fig. 3C,C′) and Vcan (Casini et al., 2008) are expressed in the trunk NC. Chase B treatment demonstrated IdoA in the high molecular mass fractions of control-MO- but not Dse-MO-injected embryos (Fig. 3D,E), which underscores the crucial role of DS-epi1 in DS biosynthesis. The Chase-B-degraded products primarily moved to fractions of 10-20 kDa, which indicates that IdoA is predominately a rare and isolated structure that is surrounded by long stretches of GlcA. We conclude that embryos at the early tailbud stage synthesize high molecular mass CS/DS PGs, such as Bgn and Vcan, with only few IdoA residues per chain.Fig. 3.

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