In this study we present a molecular characterization of mechanisms involved in development of vertebral fusions in salmon. We have previously shown that the non-deformed fish used in this study had indications of soft bone phenotype . They were further characterized by disrupted chondrocytic maturation, increased zones of hypertrophic chondrocytes and delayed endochondral ossification in the arch centra . The number of deformities increased throughout the experiment and an imbalanced bone and cartilage production characterized susceptible fish, predisposed for developing deformities . In this study we wanted to analyze an intermediate and a terminal stage of the fusion process to further characterize developing deformities. Through this experiment, we found that vertebral deformities were developing through a series of events, of which five hallmarks were identified as particularly interesting. First, disorganized and proliferating osteoblasts were prominent in the growth zones of the vertebral body endplates. Second, a metaplastic shift made the borders less distinct between the osteoblastic growth zone and the chondrocytic areas in the arch centra. Third, the arch centra ossified and the endplates became straight, hence giving the vertebral bodies a squared shaped morphology. Fourth, the intervertebral space narrowed down and the notochord was replaced by bone forming cells. Fifth, in a complete fusion all intervertebral tissue was remodeled into bone.
One of the major morphological changes during the fusion process was ossification of the arch centra. Our findings suggest that this ectopic bone formation is a key event in development of vertebral fusions, which involve lack of normal cell differentiation and growth. Immunohistochemistry with PCNA showed that osteoblasts at the growth zone of the vertebral body endplates had a markedly increased cell proliferation during the fusion process. The increased proliferation of osteoblasts was apparently partly counteracted by increased cell death as shown by stronger caspase 3 signaling. Nevertheless, the osteoblasts at the vertebral endplates appeared less organized in intermediate and fused vertebral bodies by toluidine blue staining. In addition, in fused vertebral bodies we observed moderate changes of abaxial translocation of cells from the osteoblast growth zone. Abaxial direction of growth from the borders of vertebral body end-plates and formation of chondroid bone in these areas are also described in previous experiments [14, 27]. The findings of increased proliferation and disorganized osteoblast growth were evident in vertebrae with modest alterations, which may suggest that this is an early event in the fusion process.
During the developing pathology, the marked border between the osteoblast growth zones and the chondrocytic areas connected to the arches became less distinct, as proliferating cells and chondrocytes blended through an intermediate zone. PCNA positive cells further extended along the rims of fusing vertebral bodies. This cell proliferation appeared to be closely linked to fusion of opposing arch centra. During the fusion process a metaplastic shift appeared in the arch centra where cells in the intermediate zone between osteoblasts and chondrocytes co-transcribed col1a, col2a, runx2, osteocalcin and osteonectin, as visualized by ISH. Based on histology, Witten et al.  have previously suggested the involvement of a metaplastic shift in developing fusions. In more progressed fusions, most cells in the arch centra seemed to co-transcribe osteogenic and chondrogenic markers. Our suggestion is therefore that trans-differentiated cells produce the ectopic bone.
Several in vitro studies have demonstrated that chondrocytes associated with calcifying cartilage can acquire properties of osteoblasts  and are able to change their phenotype from a primarily cartilage synthesizing cell type to a bone synthesizing cell type . However, hypertrophic chondrocytes able to trans-differentiate into osteoblasts through a process called trans-chondroid ossification has also been described . Interestingly, this type of growth has been identified during distraction osteogenesis in rats [24, 30], a process where bone is formed rapidly upon stretching. During trans-chondroid ossification, chondrocytes are found to express both col1 and col2 . In a review by Amir et al.  it was speculated if tension stress during distraction inhibited final differentiation of chondrocytes and rather trans-differentiated these cells into osteoblastic cells. At fused stage, early markers for osteoblasts and chondrocytes (runx2, osterix, sox9 and bmp2) were upregulated whereas the osteoblast inhibitor (twist) and genes involved in chondrocyte hypertrophy (bmp4, mef2c, col10a, shh and ihh) were downregulated, results also supported by ISH. Deletion of Ihh has been shown to disrupt the normal pattern of various zones of chondrocyte differentiation in the growth plate , whereas Sox9 accelerate chondrocyte differentiation in proliferating chondrocytes but inhibit hypertrophy . Sustained runx2 expression, as found in our studies, is further associated with trans-differentiation of chondrocytes into bone cells . On the contrary, analyzing the ECM components of both osteoblasts and chondrocytes (col1a, col2a, col10a, osteocalcin and alp) revealed that these transcripts had reduced activity in both intermediate and fused vertebrae. These findings might reflect the reduced radiodensity described in fish reared at elevated temperatures .
To further characterize the pathological bone formation in the chondrocytic areas in the arch centra, we analyzed osteoclast activity. Absence of osteoclasts visualized through TRAP staining was characteristic during the development of vertebral fusions, indicating that normal endochondral ossification was restrained. In addition, cathepsin k had a down-regulated transcription level. In normal developing salmon vertebrae, these areas are modeled through endochondral bone formation, a process requiring invasion of osteoclasts and activity of TRAP, Mmps and Cathepsin K [21, 35]. Transcription of mmp s are up-regulated during IDD  and compression-induced IVD [37, 38] in mammals. Intriguingly, mmp9 and mmp13 were also up-regulated during fusion of vertebral bodies in salmon. Excessive co-activity of mmp9 and mmp13 is linked to development and healing of chronic wounds in rainbow trout  and salmon . Lack of osteoclast activity and reduced activity of genes involved in chondrocyte hypertrophy during development of vertebral fusions may therefore suggest that mmp's were up-regulated in fused vertebral bodies as a response to chronic injury rather than bone resorption.
Our results suggest that the ossification type during development of spinal fusions and fast growth could be trans-chondroid ossification. A mixed type of intramembraneous and endochondral ossification, as suggested by Yasui et al.  and demonstrated by Okafuji et al.  may also occur, however the lack of osteoclast activity makes this less likely. Our findings indicate that chondrocytes had not only differentiated towards osteoblast-like cells, but also completed the differentiation to cells that were capable of producing mineralized bone matrix. Whether the suggested trans-chondroid ossification is trans-differentiation as a sudden switch from the chondrogenic to the osteogenic phenotype or a continuous differentiation was not assessed in this experiment. However, based on our results, a pathway to bone formation through chondrocytes might be possible during development of vertebral fusions.
The completing step in the fusion process is transformation of notochordal tissue into bone . As intervertebral space narrowed down, proliferating chordoblasts and denser packet chordocytes were revealed through toluidine blue staining and PCNA antibody binding, respectively. The structured chordoblast layer increased and more of these cells stained for col2a. As the pathology progressed, proliferating chordoblasts seemed to occupy most of the intervertebral space and vacuolated chordocytes disappeared. Moreover, cells in the notochord had a transcription profile resembling the trans-differentiating cell at the borders between the osteoblast growth zones and the chondrocytic areas connected to the arches. Transcription of marker genes changed from chondrogenic to also include osteogenic, as mRNA of osteocalcin, runx2, osteonectin and col1a were detected. QPCR further showed up-regulated transcription of both runx2 and sox9 throughout the developing deformity. Comparative to our findings, disc cell proliferation and a switch in the synthesis of ECM components are associated with disc degeneration [41, 42]. However, ISH revealed that whereas sox9 and col2a was present in chordoblasts from the non-deformed stage, runx2 and col1a was only detected in fused samples, when intervertebral space was severely narrowed. This co-transcription of chondrocytic and osteogenic markers in the notochord supports the hypothesis of a metaplastic shift during vertebral fusions in salmon .
The metaplastic shift in the notochord and arch centra may be induced to produce more robust cells, able to withstand increased mechanical load. However, as bone replaced chondrocytic areas throughout the pathology, notochordal tissue did not calcify until the deformity developed into severe fusion. We therefore suggest that metaplasia leads to cell types more suited to the new environment but that changes are related to a threshold of the stimuli, in this case, grade of fusion. A shift in NP cell population coincides with spinal disorders like IDD and changes in the synthesis of matrix molecules differ with the degree of degeneration . A comparative pathological process to our findings is mammalian "Bamboo spine", describing a condition where vertebral bodies have fused and reshaped through ectopic bone formation [44, 45]. Similar rescue processes have also been found in the mammalian AF, where it is strengthened through cartilage formation upon elevated mechanical load [46, 47]. Overall, the vertebral fusion process seen in salmon might reflect an effort to restore and strengthen a vertebral area of a weakened vertebral column.