The Intermuscular Bones And The Sclerostin Gene(SOST) In Carassius Auratus In Qihe River

Intermuscular bones in vertebral side muscle interval of membranous bony small thorn, originate from mesenchyme cell in muscle connective tissue and ossify directly without cartilage phase. They only exist in lower teleost fish. This way of ossification is called intramembranous ossification or ossification of connective tissue, also known as fibrous ossification or membranous hard bone ossification. Osteoblast(OB) is generated by the calcinosis of bone tissue in this way. In the body of fish, intermuscular bone is generally divided into epineural bones, epicentral bones and epipleural bones. In this research, the possible physiological mechanism of intermuscular bone was analyzed, the ossification, quantity and morphogenetic of intermuscular bone was investigated, intermuscular bone occurrence related protein, SOST gene was cloned and the expression pattern was analyzed. The results were showed bellow,Some of the tendons in muscle in teleosts ossify via intramembranous ossification.Some or all of these tendons can ossify along the corresponding parts of the body, and eventually become intermuscular bone. Variation in the number of ossified tendons has been demonstrated to be partly under genetic control, bothid fishes and the common carp e.g. Intermuscular bones in Hemibarbus labeo, Hypophthalmichthys molitrix and Danio rerio ossified after the others’ ossification had been over. Compared with the other bones which developed by same mode, the regulation of intermuscular bone formation may involve particular molecules. With the generation, simple ossification and subsequent mineralization of connective tissue between myomeres, the intermuscular bone formed, and then its shape changed with new branch or unchanged, and grew continually. When the connective tissue membrane between myomeres appears, blood vessels adhere and proliferate here, to supply oxygen and nutrients, and then bone begin to form in these parts with mesenchymal cells concentrating gradually and differentiating into osteoprogenitor cells firstly. A few of the new cells grow into osteoblasts and then secrete osteoid encasing themselves, resulting in that the osteoblasts become osteocytes. Then, osteoid is mineralized into bone matrix, and the ossification center, also known as the ossification point appears.The new bone surface always have osteoprogenitor cells or osteoblasts adhesion which continue the above physiological activities. The ossification goes on along with the growth of the fish body. At the same time, the shape changes from point to line, and then to "y" or "卜", etc. Up to now, the available reference shows that related researches are limited on tissue anatomy and morphology analysis, tiny study involved in molecules. At the cellular level, the main part of the procession focused on the mechanisms of mesenchymal stem cells to osteoblast directional differentiation and the latter mature, proliferation, secretion of bone matrix and mineralization. In vivo environment, osteoblasts are well connected with osteocytes and vascular endothelial cells, and regulated by autocrine and paracrine factors at the same time. The appearance sequence of intermuscular bones in silver carp and zebrafish from tail firstly to head lastly, this may be caused by systemic regulation factors such as hormone regulates. The directional differentiation of osteoblasts to osteocytes are regulated by the Core binding factor 1 A, Cbfα1, small related transcription factor 2, Runx2, Osterix, Osx, Indian Hedgehog, Ihh.The appearance and the form change of intermuscular bone in larval Carassius auratus were viewed and concluded, with the shape and quantity in mature bodies after they were wholly dyed or dissected respectively. Results showed that there were epineural bone and epipleural bone, no epicentral bone, and the number was between 83 and 86 totally in Carassius auratus in Qihe river. When it was 25 th day past fertilization, epineural bones appeared firstly in 21 th, 22 th, 23 th myoseptaes. When it was 28 th day past fertilization, epipleural bones arised in 22 th, 23 th, 24 th myoseptaes. At 45 th day past fertilization, each intermuscular bone could be seen, and the epipleural bone existed after cloaca, while epineural bone distributed among back muscle evenly. Their simple shape likes “I” or “y”, except for 8 thick intermuscular bones in tail muscle.The cDNA was synthesized with total RNA as template extracted from muscle in Qihe crucian carp. The corresponding primers, RT-R and RT-F, were designed based on the conservative homologous sequence in zebra fish. By RACE(rapid amplification of cDNA ends), the precursor cDNA with 2190 bases encoding precursor peptide of 211 amino acids was obtained. Multiple sequence alignment suggested that SOST C-terminal cystine knot-like domain(CTCK) was highly conserved among vertebrates. Taking it by and large, SOST was conservatively grouped with teleosts in the phylogenetic analysis. Tissue expression analysis showed that SOST mRNA exhibited extensive tissue distribution including brain, liver, spleen, gill, kidney and intestine, except for heart and back muscle, and highest in brain. These results implicated that SOST may participate to negatively regulate the formation of the intermuscular bones in Qihe crucian carp.Above all, Qihe crucian carp has a large number of intermuscular bones, and their formation procession is complicated, while the intramembranous ossification mechanism in higher vertebrates is useful for the research but limited. Beside, SOST may negatively regulate the formation of the intermuscular bones in Qihe crucian carp.