Development of Teleost Intermuscular Bones Undergoing Intramembranous Ossification Based on Histological-Transcriptomic-Proteomic Data

Abstract

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.

Keywords: genes expression; histological structure; intermuscular bone; ossification process; teleost.

Figure 1

Developmental characteristics and visualization of cross sections from four key Intermuscular bone (IB) developmental stages in M. amblycephala. (A–D) represent ossification process of IBs stained with ARS from S1, S2, S3, and S4, respectively. (A) S1. Body Length ≈ 11 mm (17 dph), the IBs have not emerged in the tail part; (B) S2. Body Length ≈ 16mm (24 dph), several small IBs have ossified in the tail part; (C) S3. Body Length ≈ 21 mm (29 dph), more IBs gradually ossified in the tail part with immature form; (D) S4. Body Length ≈ 32 mm (42 dph), all the IBs ossified in the tail part with mature morphology. Blue arrows indicate IBs; scale bars = 200 μm. (E–H) represent histological structures of the tail part with alizarin red S staining from S1, S2, S3, and S4, respectively. (I–L) represent histological structures of the tail part with alcian blue staining from S1, S2, S3, and S4, respectively. Blue arrows indicate IBs and black arrows indicate notochord sheath. The notochord sheath and vertebra were magnified in the lower left corner in (I–L). Scale bars = 100 μm.

Figure 2

Analysis of bone-regulation pathways from identified proteins. (A) The number of proteins belonging to the pathways associated with IB development. (B) A network of pathways associated with bone. Yellow indicates pathways which are related to osteoclast, blue related to osteoblast, and white related to both osteoclast and osteoblast.

Figure 3

Comparison of proteins identified in six comparison groups. (A) indicates differential expressed proteins. X-axis: names of the comparison groups; Y-axis: the number of differentially expressed proteins; red column: up-regulated proteins; green column: down-regulated proteins. (B) shows a Venn diagram for differentially expressed proteins in three adjacent developmental stages. (C) shows a Venn diagram for differentially expressed proteins in three nonadjacent developmental stages.

Figure 4

The number of bone-related pathways in six comparison groups (A) and the main pathways during IB developmental stages (B) in M. amblycephala based on differentially expressed proteins.

Figure 5

Cluster (A), network (B) and analysis of bone-regulated proteins during IB developmental stages. In the Cluster (A), the color intensity indicates the level of protein expression. Black indicates a low level of protein expression or undetected protein; red indicates a high level of expression.

Figure 6

Verification of gene expression. (A) The relative expression of genes at different stages with qRT-PCR; for each gene, different letters above the bar of each stage indicated significant differences at the level of p < 0.05 among stages. (B) The expression levels of five proteins based on Western blot analysis; GAPDH is used as a loading control. (C) In situ expression analysis of entpd5a and casq1a genes at individual’s tail part from four stages. Blue arrows indicate IBs. Scale bar = 50 μm.

Figure 7

Effects of IWP-L6 (D–F) and K02288 (G–I) inhibitors on IB development in zebrafish larvae (Tg(sp7/GFP)). Control larvae (A–C) were treated with dimethyl sulfoxide (DMSO). Red arrows indicate IBs. (B,E,H) show the tail part; (C,F,I) show the dorsal part.