Limits...
Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

Hannesson KO, Ytteborg E, Takle H, Enersen G, Bæverfjord G, Pedersen ME - Fish Physiol. Biochem. (2015)

Bottom Line: In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared.A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis.Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

View Article: PubMed Central - PubMed

Affiliation: Nofima AS, 1430, Ås, Norway.

ABSTRACT
In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

No MeSH data available.


Related in: MedlinePlus

Immunostaining of different GAG types in longitudinal sections of normal and malformed vertebrae from 15 g salmon. Left panel shows normal vertebrae versus right panel shows malformed vertebrae. a, b KS was found mainly in the lumen of the notochord in normal and malformed, but also appeared strongly in the notochordal sheath in malformed. c, d C-6-S had distinct band with globular structures in the notochordal sheath of normal vertebrae, but these were lost in malformed vertebrae. e, f C-0-S was found closer to the lumen in the notochordal sheath of normal vertebrae in contrast to a more widespread distribution in malformed where this gradient was disturbed. g, h C-4-S/DS showed lamellar structures in the cranio-caudal direction of the notochordal sheath of normal vertebrae, but in malformed, these structures were ruptured and stretched more in the dorsoventral direction. i, j C-4-S showed weak staining in the notochordal sheath of normal vertebrae, but stained more intensively and in a spotted pattern in malformed vertebrae. Scale bar 100 µm; nl notochord lumen, ns notochordal sheath, Norm normal, Malf malformed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection


getmorefigures.php?uid=PMC4495713&req=5

Fig10: Immunostaining of different GAG types in longitudinal sections of normal and malformed vertebrae from 15 g salmon. Left panel shows normal vertebrae versus right panel shows malformed vertebrae. a, b KS was found mainly in the lumen of the notochord in normal and malformed, but also appeared strongly in the notochordal sheath in malformed. c, d C-6-S had distinct band with globular structures in the notochordal sheath of normal vertebrae, but these were lost in malformed vertebrae. e, f C-0-S was found closer to the lumen in the notochordal sheath of normal vertebrae in contrast to a more widespread distribution in malformed where this gradient was disturbed. g, h C-4-S/DS showed lamellar structures in the cranio-caudal direction of the notochordal sheath of normal vertebrae, but in malformed, these structures were ruptured and stretched more in the dorsoventral direction. i, j C-4-S showed weak staining in the notochordal sheath of normal vertebrae, but stained more intensively and in a spotted pattern in malformed vertebrae. Scale bar 100 µm; nl notochord lumen, ns notochordal sheath, Norm normal, Malf malformed

Mentions: HE-added saffron staining of normal and malformed spinal columns showed a weaker external elastic lamina surrounding malformed vertebrae, as indicated by arrows in Fig. 9a, b. The notochordal sheath of malformed vertebrae also had a more elongated appearance containing vertical wavy lamellar structures (arrows, Fig. 9a, b). Alizarin red S visualized changes like thicker compact bone, flattened endplates and reduced IVRs, between normal and malformed vertebrae (Fig. 9c, d), as previous described in the literature (Ytteborg et al. 2010a). Alcian blue visualized a more homogenous GAG distribution in the notochordal sheath of normal vertebrae compared to malformed vertebrae. In the latter, Alcian blue stained the notochordal sheath in different layers, with weaker and intermittent staining in between mainly in the part of the sheath towards the lumen (Fig. 9e, f). Chordoblasts and chordocytes also stained heavier in malformed vertebrae (Fig. 9f), indicating more GAGs in the notochord of these individuals. The distribution of the GAG types and their organization patterns differed markedly between normal and malformed vertebral columns (Fig. 10). The major difference was seen in the distribution of KS epitopes. KS expression was present mainly in the lumen of the notochord of normal vertebral column (Fig. 10a), but also appeared strongly in the vertebral sheath of malformed samples (Fig. 10b). In addition, a more widespread distribution with changes in the organization pattern of CS epitopes in the intervertebral region was observed in the malformed vertebral column (Fig. 10d, f, h, j) compared to normal (Fig. 10c, e, g, i). The entire notochordal sheath in the malformed vertebral column showed intense staining and the staining pattern changed from parallel arranged lamellae to heavily stained, and more wavy irregular structures appeared. C-6-S had distinct band in the notochordal sheath of normal vertebrae; these were lost in malformed vertebrae (Fig. 10d).Fig. 9


Sulphated glycosaminoglycans and proteoglycans in the developing vertebral column of juvenile Atlantic salmon (Salmo salar).

Hannesson KO, Ytteborg E, Takle H, Enersen G, Bæverfjord G, Pedersen ME - Fish Physiol. Biochem. (2015)

Immunostaining of different GAG types in longitudinal sections of normal and malformed vertebrae from 15 g salmon. Left panel shows normal vertebrae versus right panel shows malformed vertebrae. a, b KS was found mainly in the lumen of the notochord in normal and malformed, but also appeared strongly in the notochordal sheath in malformed. c, d C-6-S had distinct band with globular structures in the notochordal sheath of normal vertebrae, but these were lost in malformed vertebrae. e, f C-0-S was found closer to the lumen in the notochordal sheath of normal vertebrae in contrast to a more widespread distribution in malformed where this gradient was disturbed. g, h C-4-S/DS showed lamellar structures in the cranio-caudal direction of the notochordal sheath of normal vertebrae, but in malformed, these structures were ruptured and stretched more in the dorsoventral direction. i, j C-4-S showed weak staining in the notochordal sheath of normal vertebrae, but stained more intensively and in a spotted pattern in malformed vertebrae. Scale bar 100 µm; nl notochord lumen, ns notochordal sheath, Norm normal, Malf malformed
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC4495713&req=5

Fig10: Immunostaining of different GAG types in longitudinal sections of normal and malformed vertebrae from 15 g salmon. Left panel shows normal vertebrae versus right panel shows malformed vertebrae. a, b KS was found mainly in the lumen of the notochord in normal and malformed, but also appeared strongly in the notochordal sheath in malformed. c, d C-6-S had distinct band with globular structures in the notochordal sheath of normal vertebrae, but these were lost in malformed vertebrae. e, f C-0-S was found closer to the lumen in the notochordal sheath of normal vertebrae in contrast to a more widespread distribution in malformed where this gradient was disturbed. g, h C-4-S/DS showed lamellar structures in the cranio-caudal direction of the notochordal sheath of normal vertebrae, but in malformed, these structures were ruptured and stretched more in the dorsoventral direction. i, j C-4-S showed weak staining in the notochordal sheath of normal vertebrae, but stained more intensively and in a spotted pattern in malformed vertebrae. Scale bar 100 µm; nl notochord lumen, ns notochordal sheath, Norm normal, Malf malformed
Mentions: HE-added saffron staining of normal and malformed spinal columns showed a weaker external elastic lamina surrounding malformed vertebrae, as indicated by arrows in Fig. 9a, b. The notochordal sheath of malformed vertebrae also had a more elongated appearance containing vertical wavy lamellar structures (arrows, Fig. 9a, b). Alizarin red S visualized changes like thicker compact bone, flattened endplates and reduced IVRs, between normal and malformed vertebrae (Fig. 9c, d), as previous described in the literature (Ytteborg et al. 2010a). Alcian blue visualized a more homogenous GAG distribution in the notochordal sheath of normal vertebrae compared to malformed vertebrae. In the latter, Alcian blue stained the notochordal sheath in different layers, with weaker and intermittent staining in between mainly in the part of the sheath towards the lumen (Fig. 9e, f). Chordoblasts and chordocytes also stained heavier in malformed vertebrae (Fig. 9f), indicating more GAGs in the notochord of these individuals. The distribution of the GAG types and their organization patterns differed markedly between normal and malformed vertebral columns (Fig. 10). The major difference was seen in the distribution of KS epitopes. KS expression was present mainly in the lumen of the notochord of normal vertebral column (Fig. 10a), but also appeared strongly in the vertebral sheath of malformed samples (Fig. 10b). In addition, a more widespread distribution with changes in the organization pattern of CS epitopes in the intervertebral region was observed in the malformed vertebral column (Fig. 10d, f, h, j) compared to normal (Fig. 10c, e, g, i). The entire notochordal sheath in the malformed vertebral column showed intense staining and the staining pattern changed from parallel arranged lamellae to heavily stained, and more wavy irregular structures appeared. C-6-S had distinct band in the notochordal sheath of normal vertebrae; these were lost in malformed vertebrae (Fig. 10d).Fig. 9

Bottom Line: In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared.A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis.Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

View Article: PubMed Central - PubMed

Affiliation: Nofima AS, 1430, Ås, Norway.

ABSTRACT
In the present study, the distribution of sulphated glycosaminoglycans (GAGs) in the developing vertebral column of Atlantic salmon (Salmo salar) at 700, 900, 1100 and 1400 d° was examined by light microscopy. The mineralization pattern was outlined by Alizarin red S and soft structures by Alcian blue. The temporal and spatial distribution patterns of different types of GAGs: chondroitin-4-sulphate/dermatan sulphate, chondroitin-6-sulphate, chondroitin-0-sulphate and keratan sulphate were addressed by immunohistochemistry using monoclonal antibodies against the different GAGs. The specific pattern obtained with the different antibodies suggests a unique role of the different GAG types in pattern formation and mineralization. In addition, the distribution of the different GAG types in normal and malformed vertebral columns from 15 g salmon was compared. A changed expression pattern of GAGs was found in the malformed vertebrae, indicating the involvement of these molecules during the pathogenesis. The molecular size of proteoglycans (PGs) in the vertebrae carrying GAGs was analysed with western blotting, and mRNA transcription of the PGs aggrecan, decorin, biglycan, fibromodulin and lumican by real-time qPCR. Our study reveals the importance of GAGs in development of vertebral column also in Atlantic salmon and indicates that a more comprehensive approach is necessary to completely understand the processes involved.

No MeSH data available.


Related in: MedlinePlus