Transcriptome sequencing of Atlantic salmon (Salmo salar L.) notochord prior to development of the vertebrae provides clues to regulation of positional fate, chordoblast lineage and mineralisation.

BACKGROUND: In teleosts such as Atlantic salmon (Salmo salar L.), segmentation and subsequent mineralisation of the notochord during embryonic stages are essential for normal vertebrae formation. However, the molecular mechanisms leading to segmentation and mineralisation of the notochord are poorly understood. The aim of this study was to identify genes/pathways acting in gradients over time and along the anterior-posterior axis during notochord segmentation and immediately prior to mineralisation of the vertebral bodies in Atlantic salmon. RESULTS: Notochord samples were collected from unsegmented, pre-segmented and segmented developmental stages. In each stage, the cellular core of the notochord was cut into three pieces along the longitudinal axis (anterior, mid, posterior). RNA was sequenced (22 million pair-end 100 bp/ library) and mapped to the salmon genome. 66569 transcripts were predicted and 55775 were annotated. In order to identify possible gradients leading to segmentation of the notochord, all 71 notochord-expressed hox genes were investigated, most of them displaying a typical anterior-posterior expression pattern along the notochord axis. The clustering of hox genes revealed a pattern that could be related to notochord segmentation. We further investigated how mineralisation is initiated in the notochord, and several factors related to chondrogenic lineage were identified (sox9, sox5, sox6, tgfb3, ihhb and col2a1), suggesting a cartilage-like character of the notochord. KEGG analysis of differentially expressed genes between stages revealed down-regulation of pathways associated with ECM, cell division, metabolism and development at onset of notochord segmentation. This implies that inhibitory signals produce segmentation of the notochord. One such potential inhibitory signal was identified, col11a2, which was detected in segments of non-mineralising notochord. CONCLUSIONS: An incomplete salmon genome was successfully used to analyse RNA-seq data from the cellular core of the Atlantic salmon notochord. In transcriptome we found; hox gene patterns possibly linked to segmentation; down-regulation of pathways in the notochord at onset of segmentation; segmented expression of col11a2 in non-mineralised segments of the notochord; and a chondroblast-like footprint in the notochord.

Mineralization of the vertebral bodies in Atlantic salmon (Salmo salar L.) is initiated segmentally in the form of hydroxyapatite crystal accretions in the notochord sheath.

We performed a sequential morphological and molecular biological study of the development of the vertebral bodies in Atlantic salmon (Salmo salar L.). Mineralization starts in separate bony elements which fuse to form complete segmental rings within the notochord sheath. The nucleation and growth of hydroxyapatite crystals in both the lamellar type II collagen matrix of the notochord sheath and the lamellar type I collagen matrix derived from the sclerotome, were highly similar. In both matrices the hydroxyapatite crystals nucleate and accrete on the surface of the collagen fibrils rather than inside the fibrils, a process that may be controlled by a template imposed by the collagen fibrils. Apatite crystal growth starts with the formation of small plate-like structures, about 5 nm thick, that gradually grow and aggregate to form extensive multi-branched crystal arborizations, resembling dendritic growth. The hydroxyapatite crystals are always oriented parallel to the long axis of the collagen fibrils, and the lamellar collagen matrices provide oriented support for crystal growth. We demonstrate here for the first time by means of synchroton radiation based on X-ray diffraction that the chordacentra contain hydroxyapatite. We employed quantitative real-time PCR to study the expression of key signalling molecule transcripts expressed in the cellular core of the notochord. The results indicate that the notochord not only produces and maintains the notochord sheath but also expresses factors known to regulate skeletogenesis: sonic hedgehog (shh), indian hedgehog homolog b (ihhb), parathyroid hormone 1 receptor (pth1r) and transforming growth factor beta 1 (tgfb1). In conclusion, our study provides evidence for the process of vertebral body development in teleost fishes, which is initially orchestrated by the notochord.

Identification of vimentin- and elastin-like transcripts specifically expressed in developing notochord of Atlantic salmon (Salmo salar L.).

The notochord functions as the midline structural element of all vertebrate embryos, and allows movement and growth at early developmental stages. Moreover, during embryonic development, notochord cells produce secreted factors that provide positional and fate information to a broad variety of cells within adjacent tissues, for instance those of the vertebrae, central nervous system and somites. Due to the large size of the embryo, the salmon notochord is useful to study as a model for exploring notochord development. To investigate factors that might be involved in notochord development, a normalized cDNA library was constructed from a mix of notochords from ∼500 to ∼800 day°. From the 1968 Sanger-sequenced transcripts, 22 genes were identified to be predominantly expressed in the notochord compared to other organs of salmon. Twelve of these genes were found to show expressional regulation around mineralization of the notochord sheath; 11 genes were up-regulated and one gene was down-regulated. Two genes were found to be specifically expressed in the notochord; these genes showed similarity to vimentin (acc. no GT297094) and elastin (acc. no GT297478). In-situ results showed that the vimentin- like transcript was expressed in both chordocytes and chordoblasts, whereas the elastin- like transcript was uniquely expressed in the chordoblasts lining the notochordal sheath. In salmon aquaculture, vertebral deformities are a common problem, and some malformations have been linked to the notochord. The expression of identified transcripts provides further insight into processes taking place in the developing notochord, prior to and during the early mineralization period.

Vitamin K-dependent gamma-glutamylcarboxylase in Atlantic salmon (Salmo salar L.).

Due to problems with bone deformities in farmed Atlantic salmon, there is a growing interest in the possible involvement of vitamin K in normal bone development, and sensitive biomarkers for evaluating vitamin K status are therefore needed. The vitamin K-dependent (VKD) enzyme gamma-glutamylcarboxylase (GGCX, EC 6.4.x.x) requires vitamin K as a cofactor for its post-translational modification of glutamic acid (Glu) residues to gamma-carboxyglutamic acid (Gla) residues in VKD proteins, and is required for their function in haemostasis and bone metabolism. The present study was designed to evaluate the enzyme assay for GGCX activity in isolated liver microsomes and its distribution in the tissues of Atlantic salmon. The effect of KH(2) and menadione on the GGCX activity in salmon liver was also compared. Results from the present study show a widespread tissue distribution and expression of GGCX in Atlantic salmon. The GGCX activity and ggcx expression in all bony tissues examined imply the presence of vitamin K, and suggest the involvement of vitamin K in bone metabolism of Atlantic salmon. We propose the GGCX assay as a sensitive marker for vitamin K status, and confirm that menadione does not work as a cofactor for GGCX in Atlantic salmon liver.

Differential gene expression of bgp and mgp in trabecular and compact bone of Atlantic salmon (Salmo salar L.) vertebrae.

The tissue-specific gene expression of the vitamin K-dependent proteins bone gamma-carboxyglutamate-protein (BGP) and matrix gamma-carboxyglutamate-protein (MGP) in Atlantic salmon (Salmo salar L.) was investigated. In previous studies, BGP, the most abundant non-collagenous protein of bone, was almost exclusively associated with bone, whereas the non-structural protein MGP has a more widespread tissue distribution. In-situ hybridization of juvenile Atlantic salmon ( approximately 40 g, fresh water) vertebrae demonstrated expression of bgp and mgp mRNA in osteoblasts lining the trabecular bone, whereas no staining was observed in the compact bone. By separating the trabecular and compact bone of both juvenile ( approximately 40 g, fresh water) and adult ( approximately 1000 g, sea water) Atlantic salmon, we observed that the two vertebral bone compartments displayed different levels of bgp, whereas no such differences were seen for mgp. Measurements of the mineral content and Ca/P molar ratio in adult salmon revealed no significant differences between trabecular and compact bone. In conclusion, the osteoblasts covering the salmon vertebrae have unique gene expression patterns and levels of bgp and mgp. Further, the study confirms the presence of mRNA from the vitamin K-dependent proteins BGP and MGP in the vertebrae, fin and gills of Atlantic salmon.

Stepwise enforcement of the notochord and its intersection with the myoseptum: an evolutionary path leading to development of the vertebra?

The notochord constitutes the main axial support during the embryonic and larval stages, and the arrangement of collagen fibrils within the notochord sheath is assumed to play a decisive role in determining its functional properties as a fibre-wound hydrostatic skeleton. We have found that during early ontogeny in Atlantic salmon stepwise changes occur in the configuration of the collagen fibre-winding of the notochord sheath. The sheath consists of a basal lamina, a layer of type II collagen, and an elastica externa that delimits the notochord; and these constituents are secreted in a specific order. Initially, the collagen fibrils are circumferentially arranged perpendicular to the longitudinal axis, and this specific spatial fibril configuration is maintained until hatching when the collagen becomes reorganized into distinct layers or lamellae. Within each lamella, fibrils are parallel to each other, forming helices around the longitudinal axis of the notochord, with a tangent angle of 75-80 degrees to the cranio-caudal axis. The helical geometry shifts between adjacent lamellae, forming enantiomorphous left- and right-handed coils, respectively, thus enforcing the sheath. The observed changes in the fibre-winding configuration may reflect adaptation of the notochord to functional demands related to stage in ontogeny. When the vertebral bodies initially form as chordacentra, the collagen lamellae of the sheath in the vertebral region are fixed by the deposition of minerals; in the intervertebral region, however, they represent a pre-adaptation providing torsional stability to the intervertebral joint. Hence, these modifications of the sheath transform the notochord per se into a functional vertebral column. The elastica externa, encasing the notochord, has serrated surfaces, connected inward to the type II collagen of the sheath, and outward to type I collagen of the mesenchymal connective tissue surrounding the notochord. In a similar manner, the collagen matrix of the neural and haemal arch cartilages is tightly anchored to the outward surface of the elastic membrane. Hence, the elastic membrane may serve as an interface between the notochord and the adjacent structures, with an essential function related to transmission of tensile forces from the musculature. The interconnection between the notochord and the myosepta is discussed in relation to function and to evolution of the arches and the vertebra. Contrary to current understanding, this study also shows that notochord vacuolization does not result in an increased elongation of the embryo, which agrees with the circular arrangement of type II collagen that probably only enables a restricted increase in girth upon vacuolization, not aiding elongation. As the vacuolization occurs during the egg stage, this type of collagen disposition, in combination with an elastica externa, also probably facilitates flexibility and curling of the embryo.