Intermuscular bones (IBs) specially exist in lower teleost fish and the molecular mechanism for its development remains to be clarified. In this study, different staining methods and comparative proteomics were conducted to investigate the histological structure and proteome of IB development in Megalobrama amblycephala, including four key IB developmental stages (S1—IBs have not emerged in the tail part; S2—several small IBs started to ossify in the tail part; S3—IBs appeared rapidly; S4—all the IBs appeared with mature morphology). Alcian blue and alizarin red S stained results indicated that IBs were gradually formed from S2 to S4, undergoing intramembranous ossification without a cartilaginous phase. A total of 3368 proteins were identified by using the isobaric tags for relative and absolute quantitation (iTRAQ) approach. Functional annotation showed that proteins which were differentially expressed among stages were involved in calcium, MAPK, Wnt, TGF-β, and osteoclast pathways which played a critical role in bone formation and differentiation. Three proteins (collagen9α1, stat1, tnc) associated with chondrocytes did not exhibit significant changes through S2 to S4; however, proteins (entpd5, casq1a, pvalb, anxa2a, anxa5) which associated with osteoblasts and bone formation and differentiation showed significantly a higher expression level from S1 to S2, as well as to S3 and S4. These further demonstrated that development of IBs did not go through a cartilaginous phase. The inhibitors of TGF-β and Wnt pathways were tested on zebrafish (sp7/eGFP) and the results indicated that both inhibitors significantly delayed IB development. This study provides a comprehensive understanding of the IB ossification pattern, which will help further elucidate the molecular mechanisms for IB development in teleosts.
Intermuscular bones (IBs) and ribs both are a part of skeletal system in teleosts, but with different developing process. The chemical composition of fish IBs and ribs as well as the underlying mechanism about their development have not been investigated. In the present study, histological structures showed that one bone cavity containing osteoclasts were existed in ribs, but not in IBs of Megalobrama amblycephala. We constructed the first proteomics map for fish bones including IBs and ribs, and identified the differentially expressed proteins between IBs and ribs through iTRAQ LC-MS/MS proteomic analysis. The proteins extracted from IBs and ribs at 1- to 2-year old M. amblycephala were quantified 2,342 proteins, with 1,451 proteins annotated with GO annotation in biological processes, molecular function and cellular component. A number of bone related proteins as well as pathways were identified in the study. A total of 93 and 154 differently expressed proteins were identified in comparison groups of 1-IB-vs-1-Rib and 2-IB-vs-2-Rib, which indicated the obvious differences of chemical composition between these two bone tissues. The two proteins (vitronectin b precursor and matrix metalloproteinase-2) related to osteoclasts differentiation were significantly up-regulated in ribs compared with IBs (P < 0.05), which was in accordance with the results from histological structures. In comparison groups of 1-IB-vs-2-IB and 1-Rib-vs-2-Rib, 33 and 51 differently expressed proteins were identified and the function annotation results showed that these proteins were involved in regulating bone development and differentiation. Subsequently, 11 and 13 candidate proteins in comparison group of 1-IB-vs-1-Rib and 1-IB-vs-2-IB related to bone development were validated by MRM assays. Our present study suggested the different key proteins involved in the composition of fish ribs and IBs as well as their growth development. These findings could provide important clues towards further understanding of fish skeletal system and the roles of proteins playing in regulating diverse biological processes in fish.
Coilia nasus is a threatened migratory species in the Yangtze River Basin. To reveal the genetic diversity of natural and farmed populations of C. nasus and the status of germplasm resources in the Yangtze River, the genetic diversity and structure of two wild populations (Yezhi Lake: YZ; Poyang Lake: PY) and two farmed populations (Zhenjiang: ZJ; Wuhan: WH) of C. nasus were analyzed using 44,718 SNPs obtained via 2b-RAD sequencing. The results indicate that both the wild and farmed populations had low genetic diversity, and germplasm resources have undergone varying degrees of degradation. Population genetic structure analyses indicated that the four populations may have come from two ancestral groups. Different amounts of gene flow were identified among WH, ZJ, and PY populations, but gene flow among YZ and other populations was low. It is speculated that the river–lake isolation of Yezhi Lake is the main cause of this phenomenon. In conclusion, this study revealed that genetic diversity reduction and germplasm resource degradation had occurred in both wild and farmed C. nasus, suggesting that conservation of its resources is of great urgency. This study provides a theoretical basis for the conservation and rational exploitation of germplasm resources for C. nasus.