Elisabeth Ytteborg defended her PhD thesis on June 25th 2010

Ane Gro Siri Skjelfjord

Morphological and molecular characterization of defeloping vertebral fusions in Atlantic salmon (Salmo salar)

Problems with spinal disorders in Atlantic salmon (Salmo salar) have been increasingly in focus due to the importance of this species in the aquaculture industry. Until recently, the molecular development of spinal deformities in fish has received relatively little attention and most studies have been largely descriptive. Due to economical matters of the industry, previous deformity studies were primarily based on radiographic findings, histological staining and field studies to reveal factors inducing deformities. Consequently, few deformities have been explored beyond the level of association with particular causative factors. However, accumulated studies on intensive production regimes and incidence of deformities have been followed by more advanced studies on vertebral development and bone biology. A better understanding of cellular and molecular events during bone development in teleosts should enable us to better characterize the pathology, define particular requirements and enable us to minimize the occurrence of bone disorders.

To increase the understanding of normal and pathological bone development in Atlantic salmon, fish was exposed to two different temperature regimes from fertilization until 15g size. Fish exposed to high temperature regimes showed a markedly higher growth rate and a significant higher percentage of vertebral fusions than fish reared at low temperatures. The major aim of paper I, II and III was to study the long term effect of hyperthermic conditions upon bone development in Atlantic salmon with focus on pathological development of spinal fusions. Analyzing non-deformed vertebrae from the two temperature regimes (paper I) revealed that the increased risk of developing vertebral fusions was linked to a down-regulated transcription of genes involved in production and mineralization of extracellular matrix components. Furthermore, morphological changes in the arch centra identified chondrocytes with a distorted maturation pattern and an increased zone of hypertrophic chondrocytes. The data presented indicate that production of both bone and cartilage is disrupted when fast growth is promoted. Paper II and III were devoted to characterize developing spinal fusions, hence describe typical hallmarks in the fusion process. An intermediate and a terminal stage of the fusion process were studied at a morphological level using radiography and histology and at a gene expressional level using quantitative real-time PCR, in situ hybridization and immunohistochemistry. In paper II the focus was directed towards bone and cartilage formation in the centra, whereas in paper III the notochord was analyzed. The development of vertebral fusions is a dynamic process, but as suggested in this thesis, the process may be summarized as four major events. First, disorganized and proliferating cells were prominent at the vertebral growth zones and in the notochord. The marked border between the osteoblast growth zones and the chondrocytic arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone (paper II). Second, in situ hybridization visualized that proliferating cells in the intermediate zone co-expressed mixed signals of chondrogenic and osteogenic markers, suggesting a metaplastic shift in these cells (paper II). A similar shift also occurred in the notochord where proliferating chordoblasts changed transcription profile to be more osteogenic (paper II and III). Third, as the pathology progressed, the notochordal sheath stretched and a thinner and more fragmented elastic membrane was detected (paper III). Immunohistochemistry further revealed that the structural organization in the notochordal sheath was altered upon development of vertebral fusions. Fourth, ectopic bone formation was found in the arch centra and in the intervertebral regions (paper II). The formation of ectopic bone indicated that the metaplastic shift in proliferating cells led to cells capable of producing mineralized matrix.

The major aim of paper IV was to develop an in vitro system for Atlantic salmon osteoblasts so that problems related to bone development could be more specifically targeted. Unspecialized primary cells from Atlantic salmon white muscle differentiate in osteogenic medium to osteoblasts-like cells. The cells changed their morphology from elongated to become more cobblestone, started expressing osteoblast specific markers and stained for Alkaline phosphatase during the differentiation period. The differentiated cells were further used to study the effects of two factors that influence bone formation in Atlantic salmon under commercial farming conditions; elevated temperature and polyunsaturated fatty acid composition. The in vitro response showed resemblances with in vivo findings, supporting that we had succeeded in differentiating the precursor cells to become osteoblast like cells.

Through the work presented in this thesis we have added knowledge to both normal and pathological development of the Atlantic salmon vertebrae. Most markers for bone and cartilage development had not previously been described in Atlantic salmon. The defined markers can be used to investigate how the progression of skeletogenesis is modulated by a variety of factors and reveals the potential use of gene transcription profiling as a prognostic approach in aquaculture. Moreover, Atlantic salmon has shown to be comparable to mammalian models used in revealing the complex pathology involved in the development of spinal malformation.

Updated: 25.06.10
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