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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

Distribution of chondroitin 4-sulphate (C-4-S) and dermatan sulphate (DS). a Longitudinal section at 700 d° showing strong staining of the notochordal sheath and the perichordal cartilage. The staining of notochordal sheath appeared granular, as clearly outlined in b. c The transverse section showing a more continuous staining of the sheath and staining of the cartilaginous arches. d Longitudinal section at 900 d° showing the staining of the notochordal sheath become weaker in areas of developing chordacentra, enlarged in e. f Transverse section showing stronger staining towards the lumen. g Longitudinal section at 1100 d°, the strong staining persisted in developing IVRs, higher magnification in h, showing a granular staining pattern in the external region of the IVR in contrast to the layered appearance in the central part. i Transverse section showing stronger staining towards the lumen. j, k Longitudinal sections at 1400 d°, showing a strong staining of the IVR in the central region and distinct, labelled wings extending towards the mineralizing zones (arrow), enlarged in k. Some staining was also seen in the chordoblast layer (arrow). l Transverse sections showing strong, lamellar staining of the notochordal sheath. Scale bar 100 µm; nl notochord lumen, nc neural cord, ac arch centra, ns notochordal sheath
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Fig4: Distribution of chondroitin 4-sulphate (C-4-S) and dermatan sulphate (DS). a Longitudinal section at 700 d° showing strong staining of the notochordal sheath and the perichordal cartilage. The staining of notochordal sheath appeared granular, as clearly outlined in b. c The transverse section showing a more continuous staining of the sheath and staining of the cartilaginous arches. d Longitudinal section at 900 d° showing the staining of the notochordal sheath become weaker in areas of developing chordacentra, enlarged in e. f Transverse section showing stronger staining towards the lumen. g Longitudinal section at 1100 d°, the strong staining persisted in developing IVRs, higher magnification in h, showing a granular staining pattern in the external region of the IVR in contrast to the layered appearance in the central part. i Transverse section showing stronger staining towards the lumen. j, k Longitudinal sections at 1400 d°, showing a strong staining of the IVR in the central region and distinct, labelled wings extending towards the mineralizing zones (arrow), enlarged in k. Some staining was also seen in the chordoblast layer (arrow). l Transverse sections showing strong, lamellar staining of the notochordal sheath. Scale bar 100 µm; nl notochord lumen, nc neural cord, ac arch centra, ns notochordal sheath

Mentions: The mAb 2B6 binds to both C-4-S and DS when the sections are treated with chondroitinase ABC but only to C-4-S when the sections are treated with chondroitinase ACII, as described in materials and methods. Subsequently, C-4-S was identified by direct observations of stained structures after treatment with the latter, whereas DS containing structures were identified as those that were stained with the former and not the latter. A different expression pattern of C-4-S and DS epitopes during development of the salmon spine was detected (Fig. 4). Using mAb 2B6 + cABC lyase for detection of both C-4-S and DS containing structures, strong staining of both the notochordal sheath and the perichordal cartilage was found in sections at 700 d° (Fig. 4a–c). In the longitudinal section (Fig. 4b), staining of the notochordal sheath appeared granular, whereas in the transverse section (Fig. 4c), heavily stained notochordal sheath showed a more uniform appearance, with traces of concentric lamellae. At 900 d°, the staining of the sheath became weak in the mineralized areas and the granular staining pattern became more uniform (Fig. 4d, e). A strong label persisted in developing IVRs that appeared at 1100 d° in parallel layers (Fig. 4g, h). However, in the outer rims of these regions, more granular staining could be seen (Fig. 4h), thus indicating a different organization pattern of C-4-S/DS (Fig. 4h). A faint staining of the mineralized part of the sheath was also evident and the chondrogenic arches stained strongly at all developmental stages. At 1400 d°, a well-organized staining pattern appeared in the IVR, showing a strong and uniform staining of the central region with distinct, labelled wings extending towards the mineralizing zones (arrow, Fig. 4j) was visualized. Furthermore, staining was seen in the chordoblast area (arrow, Fig. 4k). Strong staining was seen in the sheath and in the arches in the transverse sections (Fig. 4l).Fig. 4


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)

Distribution of chondroitin 4-sulphate (C-4-S) and dermatan sulphate (DS). a Longitudinal section at 700 d° showing strong staining of the notochordal sheath and the perichordal cartilage. The staining of notochordal sheath appeared granular, as clearly outlined in b. c The transverse section showing a more continuous staining of the sheath and staining of the cartilaginous arches. d Longitudinal section at 900 d° showing the staining of the notochordal sheath become weaker in areas of developing chordacentra, enlarged in e. f Transverse section showing stronger staining towards the lumen. g Longitudinal section at 1100 d°, the strong staining persisted in developing IVRs, higher magnification in h, showing a granular staining pattern in the external region of the IVR in contrast to the layered appearance in the central part. i Transverse section showing stronger staining towards the lumen. j, k Longitudinal sections at 1400 d°, showing a strong staining of the IVR in the central region and distinct, labelled wings extending towards the mineralizing zones (arrow), enlarged in k. Some staining was also seen in the chordoblast layer (arrow). l Transverse sections showing strong, lamellar staining of the notochordal sheath. Scale bar 100 µm; nl notochord lumen, nc neural cord, ac arch centra, ns notochordal sheath
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Related In: Results  -  Collection

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Fig4: Distribution of chondroitin 4-sulphate (C-4-S) and dermatan sulphate (DS). a Longitudinal section at 700 d° showing strong staining of the notochordal sheath and the perichordal cartilage. The staining of notochordal sheath appeared granular, as clearly outlined in b. c The transverse section showing a more continuous staining of the sheath and staining of the cartilaginous arches. d Longitudinal section at 900 d° showing the staining of the notochordal sheath become weaker in areas of developing chordacentra, enlarged in e. f Transverse section showing stronger staining towards the lumen. g Longitudinal section at 1100 d°, the strong staining persisted in developing IVRs, higher magnification in h, showing a granular staining pattern in the external region of the IVR in contrast to the layered appearance in the central part. i Transverse section showing stronger staining towards the lumen. j, k Longitudinal sections at 1400 d°, showing a strong staining of the IVR in the central region and distinct, labelled wings extending towards the mineralizing zones (arrow), enlarged in k. Some staining was also seen in the chordoblast layer (arrow). l Transverse sections showing strong, lamellar staining of the notochordal sheath. Scale bar 100 µm; nl notochord lumen, nc neural cord, ac arch centra, ns notochordal sheath
Mentions: The mAb 2B6 binds to both C-4-S and DS when the sections are treated with chondroitinase ABC but only to C-4-S when the sections are treated with chondroitinase ACII, as described in materials and methods. Subsequently, C-4-S was identified by direct observations of stained structures after treatment with the latter, whereas DS containing structures were identified as those that were stained with the former and not the latter. A different expression pattern of C-4-S and DS epitopes during development of the salmon spine was detected (Fig. 4). Using mAb 2B6 + cABC lyase for detection of both C-4-S and DS containing structures, strong staining of both the notochordal sheath and the perichordal cartilage was found in sections at 700 d° (Fig. 4a–c). In the longitudinal section (Fig. 4b), staining of the notochordal sheath appeared granular, whereas in the transverse section (Fig. 4c), heavily stained notochordal sheath showed a more uniform appearance, with traces of concentric lamellae. At 900 d°, the staining of the sheath became weak in the mineralized areas and the granular staining pattern became more uniform (Fig. 4d, e). A strong label persisted in developing IVRs that appeared at 1100 d° in parallel layers (Fig. 4g, h). However, in the outer rims of these regions, more granular staining could be seen (Fig. 4h), thus indicating a different organization pattern of C-4-S/DS (Fig. 4h). A faint staining of the mineralized part of the sheath was also evident and the chondrogenic arches stained strongly at all developmental stages. At 1400 d°, a well-organized staining pattern appeared in the IVR, showing a strong and uniform staining of the central region with distinct, labelled wings extending towards the mineralizing zones (arrow, Fig. 4j) was visualized. Furthermore, staining was seen in the chordoblast area (arrow, Fig. 4k). Strong staining was seen in the sheath and in the arches in the transverse sections (Fig. 4l).Fig. 4

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