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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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DS-epi1 has a tissue-autonomous role in CNC cell migration, adherence to fibronectin and cell polarization. (A,A′) Schemes for transplantation experiments. A CNC explant from an embryo injected with 300 pg GFP mRNA was homotypically grafted at stage 17. MOs were injected into the donor (A) or host embryo (A′). (B-E) Lateral view of embryos at stage 26. Grafted GFP+ CNC cells migrate ventrally when derived from control-MO-injected embryos (B); however, they do not properly migrate when derived from Dse-MO-injected embryos (C). br, branchial segment; hy, hyoid segment; ma, mandibular segment. The CNC cell migration was normal when the host embryo was injected with control-MO or Dse-MO (D,E). Three independent experiments were performed (n=3). (F) Scheme illustrating the culture of stage 17 morphant CNC explants on fibronectin-coated plates. (G,G′) At 2 h after plating (G), the control-MO-injected CNC explant exhibits collective cell migration in one direction (arrow). The inset shows a magnification of spread cells. After 4 h (G′), the cells migrate in distinct streams (asterisks). (H,H′) Cells of Dse-MO-injected CNC explants detach from each other and fail to adhere to the fibronectin substrate. The inset depicts a magnification of the spherical cells. (I-K) Confocal microscopy of fixed CNC cells after 5 h of explant culture on fibronectin. Phalloidin–Alexa-Fluor-488 and DAPI label F-actin and cell nuclei, respectively. The Dse-5MM-MO-injected control cell (I) exhibits lamellipodia at the leading edge (arrowhead) and stress fibers in the inner regions of the cell (arrow in inset). Dse-morphant cells (J) exhibit cortical networks of stress fibers and lack polarized protrusions. Co-injection of Dse-MO and 1 ng Dse* mRNA per embryo (K) restores the normal cytoskeleton and cell shape. (L,M) Quantification of cell spreading (L) and formation of polarized cell protrusions (M) in dissociated phalloidin-stained single cells from CNC explants following 5 h of culture on fibronectin. Cell spreading and polarized protrusions were quantified by calculating the cell size as the square number of pixels (ImageJ) and determining the percentage of cells with lamellipodia or filopodia, respectively. Uninjected and Dse-5MM-MO-injected explants exhibit a similar extent of cell spreading and formation of polarized protrusions. The reduction in the cell size and the lack of lamellipodia and filopodia are rescued by the co-injection of Dse* mRNA in Dse-morphant explants. A minimum of 100 cells per sample were evaluated in each experiment. Number of independent experiments (n≥3). Results are mean±s.d. (N) Cell–matrix adhesion of dissociated single CNC cells on fibronectin- or BSA-coated plates. Following the co-injection of MO and 300 pg GFP mRNA, CNC explants from stage 17 embryos were dissociated in Ca2+- and Mg2+-free medium and cultured for 45 min on fibronectin or BSA. The Dse-morphant cells exhibit decreased adhesion to fibronectin compared with the control and Dse-5MM-MO-injected cells. None of the analyzed cell samples exhibited significant cell adhesion to BSA. At least three independent experiments were performed for each sample (n≥3). Results are mean±s.d. The proportion of examined explants or cells with the indicated phenotype was as follows: B, 10/12; C, 11/13; D, 7/7; E, 9/9; G, 30/34; H, 26/28. Scale bars: 100 µm (G-H′); 10 µm (I-K). **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA multiple comparisons test with Tukey correction).
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DMM024661F6: DS-epi1 has a tissue-autonomous role in CNC cell migration, adherence to fibronectin and cell polarization. (A,A′) Schemes for transplantation experiments. A CNC explant from an embryo injected with 300 pg GFP mRNA was homotypically grafted at stage 17. MOs were injected into the donor (A) or host embryo (A′). (B-E) Lateral view of embryos at stage 26. Grafted GFP+ CNC cells migrate ventrally when derived from control-MO-injected embryos (B); however, they do not properly migrate when derived from Dse-MO-injected embryos (C). br, branchial segment; hy, hyoid segment; ma, mandibular segment. The CNC cell migration was normal when the host embryo was injected with control-MO or Dse-MO (D,E). Three independent experiments were performed (n=3). (F) Scheme illustrating the culture of stage 17 morphant CNC explants on fibronectin-coated plates. (G,G′) At 2 h after plating (G), the control-MO-injected CNC explant exhibits collective cell migration in one direction (arrow). The inset shows a magnification of spread cells. After 4 h (G′), the cells migrate in distinct streams (asterisks). (H,H′) Cells of Dse-MO-injected CNC explants detach from each other and fail to adhere to the fibronectin substrate. The inset depicts a magnification of the spherical cells. (I-K) Confocal microscopy of fixed CNC cells after 5 h of explant culture on fibronectin. Phalloidin–Alexa-Fluor-488 and DAPI label F-actin and cell nuclei, respectively. The Dse-5MM-MO-injected control cell (I) exhibits lamellipodia at the leading edge (arrowhead) and stress fibers in the inner regions of the cell (arrow in inset). Dse-morphant cells (J) exhibit cortical networks of stress fibers and lack polarized protrusions. Co-injection of Dse-MO and 1 ng Dse* mRNA per embryo (K) restores the normal cytoskeleton and cell shape. (L,M) Quantification of cell spreading (L) and formation of polarized cell protrusions (M) in dissociated phalloidin-stained single cells from CNC explants following 5 h of culture on fibronectin. Cell spreading and polarized protrusions were quantified by calculating the cell size as the square number of pixels (ImageJ) and determining the percentage of cells with lamellipodia or filopodia, respectively. Uninjected and Dse-5MM-MO-injected explants exhibit a similar extent of cell spreading and formation of polarized protrusions. The reduction in the cell size and the lack of lamellipodia and filopodia are rescued by the co-injection of Dse* mRNA in Dse-morphant explants. A minimum of 100 cells per sample were evaluated in each experiment. Number of independent experiments (n≥3). Results are mean±s.d. (N) Cell–matrix adhesion of dissociated single CNC cells on fibronectin- or BSA-coated plates. Following the co-injection of MO and 300 pg GFP mRNA, CNC explants from stage 17 embryos were dissociated in Ca2+- and Mg2+-free medium and cultured for 45 min on fibronectin or BSA. The Dse-morphant cells exhibit decreased adhesion to fibronectin compared with the control and Dse-5MM-MO-injected cells. None of the analyzed cell samples exhibited significant cell adhesion to BSA. At least three independent experiments were performed for each sample (n≥3). Results are mean±s.d. The proportion of examined explants or cells with the indicated phenotype was as follows: B, 10/12; C, 11/13; D, 7/7; E, 9/9; G, 30/34; H, 26/28. Scale bars: 100 µm (G-H′); 10 µm (I-K). **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA multiple comparisons test with Tukey correction).

Mentions: To investigate where the CS/DS chains affect cell migration, we performed transplantation experiments with green fluorescent protein (GFP) as a lineage tracer. First, we homotypically grafted CNC explants from embryos that were co-injected with a morpholino oligonucleotide and GFP mRNA into uninjected sibling hosts at stage 17 (Fig. 6A). Cells from the control-MO-injected CNC grafts migrated ventrally and formed well-orchestrated mandibular, hyoid and branchial streams (Fig. 6B). In contrast, the migration of the Dse-MO-injected CNC cells was disturbed and only a few labeled cells reached their correct destination (Fig. 6C). When GFP+ CNC cells were transplanted into control- and Dse-morphant host embryos (Fig. 6A′), their cell migration was normal (Fig. 6D,E). These findings suggest that DS-epi1 is needed in the CNC graft, but apparently not in surrounding tissue, which indicates a tissue-autonomous function of CS/DS PGs in CNC cell migration.Fig. 6.


Musculocontractural Ehlers – Danlos syndrome and neurocristopathies: dermatan sulfate is required for Xenopus neural crest cells to migrate and adhere to fibronectin
DS-epi1 has a tissue-autonomous role in CNC cell migration, adherence to fibronectin and cell polarization. (A,A′) Schemes for transplantation experiments. A CNC explant from an embryo injected with 300 pg GFP mRNA was homotypically grafted at stage 17. MOs were injected into the donor (A) or host embryo (A′). (B-E) Lateral view of embryos at stage 26. Grafted GFP+ CNC cells migrate ventrally when derived from control-MO-injected embryos (B); however, they do not properly migrate when derived from Dse-MO-injected embryos (C). br, branchial segment; hy, hyoid segment; ma, mandibular segment. The CNC cell migration was normal when the host embryo was injected with control-MO or Dse-MO (D,E). Three independent experiments were performed (n=3). (F) Scheme illustrating the culture of stage 17 morphant CNC explants on fibronectin-coated plates. (G,G′) At 2 h after plating (G), the control-MO-injected CNC explant exhibits collective cell migration in one direction (arrow). The inset shows a magnification of spread cells. After 4 h (G′), the cells migrate in distinct streams (asterisks). (H,H′) Cells of Dse-MO-injected CNC explants detach from each other and fail to adhere to the fibronectin substrate. The inset depicts a magnification of the spherical cells. (I-K) Confocal microscopy of fixed CNC cells after 5 h of explant culture on fibronectin. Phalloidin–Alexa-Fluor-488 and DAPI label F-actin and cell nuclei, respectively. The Dse-5MM-MO-injected control cell (I) exhibits lamellipodia at the leading edge (arrowhead) and stress fibers in the inner regions of the cell (arrow in inset). Dse-morphant cells (J) exhibit cortical networks of stress fibers and lack polarized protrusions. Co-injection of Dse-MO and 1 ng Dse* mRNA per embryo (K) restores the normal cytoskeleton and cell shape. (L,M) Quantification of cell spreading (L) and formation of polarized cell protrusions (M) in dissociated phalloidin-stained single cells from CNC explants following 5 h of culture on fibronectin. Cell spreading and polarized protrusions were quantified by calculating the cell size as the square number of pixels (ImageJ) and determining the percentage of cells with lamellipodia or filopodia, respectively. Uninjected and Dse-5MM-MO-injected explants exhibit a similar extent of cell spreading and formation of polarized protrusions. The reduction in the cell size and the lack of lamellipodia and filopodia are rescued by the co-injection of Dse* mRNA in Dse-morphant explants. A minimum of 100 cells per sample were evaluated in each experiment. Number of independent experiments (n≥3). Results are mean±s.d. (N) Cell–matrix adhesion of dissociated single CNC cells on fibronectin- or BSA-coated plates. Following the co-injection of MO and 300 pg GFP mRNA, CNC explants from stage 17 embryos were dissociated in Ca2+- and Mg2+-free medium and cultured for 45 min on fibronectin or BSA. The Dse-morphant cells exhibit decreased adhesion to fibronectin compared with the control and Dse-5MM-MO-injected cells. None of the analyzed cell samples exhibited significant cell adhesion to BSA. At least three independent experiments were performed for each sample (n≥3). Results are mean±s.d. The proportion of examined explants or cells with the indicated phenotype was as follows: B, 10/12; C, 11/13; D, 7/7; E, 9/9; G, 30/34; H, 26/28. Scale bars: 100 µm (G-H′); 10 µm (I-K). **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA multiple comparisons test with Tukey correction).
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DMM024661F6: DS-epi1 has a tissue-autonomous role in CNC cell migration, adherence to fibronectin and cell polarization. (A,A′) Schemes for transplantation experiments. A CNC explant from an embryo injected with 300 pg GFP mRNA was homotypically grafted at stage 17. MOs were injected into the donor (A) or host embryo (A′). (B-E) Lateral view of embryos at stage 26. Grafted GFP+ CNC cells migrate ventrally when derived from control-MO-injected embryos (B); however, they do not properly migrate when derived from Dse-MO-injected embryos (C). br, branchial segment; hy, hyoid segment; ma, mandibular segment. The CNC cell migration was normal when the host embryo was injected with control-MO or Dse-MO (D,E). Three independent experiments were performed (n=3). (F) Scheme illustrating the culture of stage 17 morphant CNC explants on fibronectin-coated plates. (G,G′) At 2 h after plating (G), the control-MO-injected CNC explant exhibits collective cell migration in one direction (arrow). The inset shows a magnification of spread cells. After 4 h (G′), the cells migrate in distinct streams (asterisks). (H,H′) Cells of Dse-MO-injected CNC explants detach from each other and fail to adhere to the fibronectin substrate. The inset depicts a magnification of the spherical cells. (I-K) Confocal microscopy of fixed CNC cells after 5 h of explant culture on fibronectin. Phalloidin–Alexa-Fluor-488 and DAPI label F-actin and cell nuclei, respectively. The Dse-5MM-MO-injected control cell (I) exhibits lamellipodia at the leading edge (arrowhead) and stress fibers in the inner regions of the cell (arrow in inset). Dse-morphant cells (J) exhibit cortical networks of stress fibers and lack polarized protrusions. Co-injection of Dse-MO and 1 ng Dse* mRNA per embryo (K) restores the normal cytoskeleton and cell shape. (L,M) Quantification of cell spreading (L) and formation of polarized cell protrusions (M) in dissociated phalloidin-stained single cells from CNC explants following 5 h of culture on fibronectin. Cell spreading and polarized protrusions were quantified by calculating the cell size as the square number of pixels (ImageJ) and determining the percentage of cells with lamellipodia or filopodia, respectively. Uninjected and Dse-5MM-MO-injected explants exhibit a similar extent of cell spreading and formation of polarized protrusions. The reduction in the cell size and the lack of lamellipodia and filopodia are rescued by the co-injection of Dse* mRNA in Dse-morphant explants. A minimum of 100 cells per sample were evaluated in each experiment. Number of independent experiments (n≥3). Results are mean±s.d. (N) Cell–matrix adhesion of dissociated single CNC cells on fibronectin- or BSA-coated plates. Following the co-injection of MO and 300 pg GFP mRNA, CNC explants from stage 17 embryos were dissociated in Ca2+- and Mg2+-free medium and cultured for 45 min on fibronectin or BSA. The Dse-morphant cells exhibit decreased adhesion to fibronectin compared with the control and Dse-5MM-MO-injected cells. None of the analyzed cell samples exhibited significant cell adhesion to BSA. At least three independent experiments were performed for each sample (n≥3). Results are mean±s.d. The proportion of examined explants or cells with the indicated phenotype was as follows: B, 10/12; C, 11/13; D, 7/7; E, 9/9; G, 30/34; H, 26/28. Scale bars: 100 µm (G-H′); 10 µm (I-K). **P<0.01, ***P<0.001, ****P<0.0001 (one-way ANOVA multiple comparisons test with Tukey correction).
Mentions: To investigate where the CS/DS chains affect cell migration, we performed transplantation experiments with green fluorescent protein (GFP) as a lineage tracer. First, we homotypically grafted CNC explants from embryos that were co-injected with a morpholino oligonucleotide and GFP mRNA into uninjected sibling hosts at stage 17 (Fig. 6A). Cells from the control-MO-injected CNC grafts migrated ventrally and formed well-orchestrated mandibular, hyoid and branchial streams (Fig. 6B). In contrast, the migration of the Dse-MO-injected CNC cells was disturbed and only a few labeled cells reached their correct destination (Fig. 6C). When GFP+ CNC cells were transplanted into control- and Dse-morphant host embryos (Fig. 6A′), their cell migration was normal (Fig. 6D,E). These findings suggest that DS-epi1 is needed in the CNC graft, but apparently not in surrounding tissue, which indicates a tissue-autonomous function of CS/DS PGs in CNC cell migration.Fig. 6.

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&ndash;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&ndash;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