Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

doi:10.1186/s12864-017-3530-z

Comparative Study

. 2017 Feb 10;18(1):147.

doi: 10.1186/s12864-017-3530-z.

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

Chun-Hong Nie^{1

2}, Shi-Ming Wan^{1

2}, Tea Tomljanovic³, Tomislav Treer³, Chung-Der Hsiao⁴, Wei-Min Wang¹, Ze-Xia Gao^{5

6

7}

Affiliations

¹ College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
² Collaborative Innovation Center for Healthy Freshwater Aquaculture of Hubei Province, Wuhan, 430070, China.
³ Department for Fisheries, Beekeeping, Game management and Special Zoology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia.
⁴ Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan.
⁵ College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, 430070, China. gaozexia@hotmail.com.
⁶ Collaborative Innovation Center for Healthy Freshwater Aquaculture of Hubei Province, Wuhan, 430070, China. gaozexia@hotmail.com.
⁷ Hubei Provincial Engineering Laboratory for Pond Aquaculture, Wuhan, 430070, Hubei, China. gaozexia@hotmail.com.

PMID: 28183283
PMCID: PMC5301324
DOI: 10.1186/s12864-017-3530-z

Comparative Study

Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

Chun-Hong Nie et al. BMC Genomics. 2017.

. 2017 Feb 10;18(1):147.

doi: 10.1186/s12864-017-3530-z.

Authors

Chun-Hong Nie^{1

2}, Shi-Ming Wan^{1

2}, Tea Tomljanovic³, Tomislav Treer³, Chung-Der Hsiao⁴, Wei-Min Wang¹, Ze-Xia Gao^{5

6

7}

Affiliations

¹ College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, 430070, China.
² Collaborative Innovation Center for Healthy Freshwater Aquaculture of Hubei Province, Wuhan, 430070, China.
³ Department for Fisheries, Beekeeping, Game management and Special Zoology, Faculty of Agriculture, University of Zagreb, Zagreb, Croatia.
⁴ Department of Bioscience Technology, Chung Yuan Christian University, Chung-Li, Taiwan.
⁵ College of Fisheries, Key Lab of Agricultural Animal Genetics, Breeding and Reproduction of Ministry of Education/Key Lab of Freshwater Animal Breeding, Ministry of Agriculture, Huazhong Agricultural University, Wuhan, Hubei, 430070, China. gaozexia@hotmail.com.
⁶ Collaborative Innovation Center for Healthy Freshwater Aquaculture of Hubei Province, Wuhan, 430070, China. gaozexia@hotmail.com.
⁷ Hubei Provincial Engineering Laboratory for Pond Aquaculture, Wuhan, 430070, Hubei, China. gaozexia@hotmail.com.

PMID: 28183283
PMCID: PMC5301324
DOI: 10.1186/s12864-017-3530-z

Abstract

Background: 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.

Results: 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.

Conclusions: 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.

Keywords: Growth and differentiation; Intermuscular bones; MRM validation; Megalobrama amblycephala; Ribs; iTRAQ proteomics.

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Figures

**Fig. 1**
HE staining results for IBs and ribs of *M. amblycephala*, a-ribs of fish at 1-year old; b-IBs of fish at 1-year old; c-ribs of fish at 2-year old; d-IBs of fish at 2-year old. a showed osteoblast; b showed osteoclast; c showed osteocyte

**Fig. 2**
Proteins were functionally annotated for (a) biological process, (b) molecular function, as well as (c) cellular component and Venn diagram of proteins annotated for three processes (d)

**Fig. 3**
Comparison of proteins identified in 1-IB-vs-1-Rib and 2-IB-vs-2-Rib groups. a indicates differential expressed proteins, *X-axis*: names of the comparison groups; *Y-axis*: the number of differentially expressed proteins; *Red column*: up-regulation proteins; *Green column*: down-regulation proteins. b shows COG functional classification of differentially expressed proteins in two comparison groups. c shows Venn diagram for differentially expressed proteins between 1-IB-vs-1-Rib and 2-IB-vs-2-Rib

**Fig. 4**
Comparison of proteins identified in 1-IB-vs-2-IB and 1-Rib-vs-2-Rib groups, a indicates differential expressed proteins, *X-axis*: names of the comparison groups; *Y-axis*: the number of differentially expressed proteins; *Red column*: up-regulation proteins; *Green column*: down-regulation proteins. b shows COG functional classification of differentially expressed proteins from iTRAQ data. c shows Venn diagram for 1-IB-vs-1-Rib and 2-IB-vs-2-Rib

**Fig. 5**
Distribution of the differentially expressed proteins in 1-IB-vs-2-IB and 1-Rib-vs-2-Rib by their functions. Using marked proteins were associated with calcium or calcium-related process. Using marked proteins related to bone formation and growth. Using marked proteins related to osteoblast formation. The *black* type of proteins represented co-expressed differentially proteins in both 1-IB-vs-2-IB and 1-Rib-vs-2-Rib. The *red* type of proteins represented specific differentially expressed proteins in 1-Rib-vs-2-Rib. The *green* type of proteins represented specific differentially expressed proteins in 1-IB-vs-2-IB

formula image — **Fig. 5**
Distribution of the differentially expressed proteins in 1-IB-vs-2-IB and 1-Rib-vs-2-Rib by their functions. Using marked proteins were associated with calcium or calcium-related process. Using marked proteins related to bone formation and growth. Using marked proteins related to osteoblast formation. The *black* type of proteins represented co-expressed differentially proteins in both 1-IB-vs-2-IB and 1-Rib-vs-2-Rib. The *red* type of proteins represented specific differentially expressed proteins in 1-Rib-vs-2-Rib. The *green* type of proteins represented specific differentially expressed proteins in 1-IB-vs-2-IB

**Fig. 6**
Cluster analysis of four pathway proteins in the 1-IB, 2-IB, 1-Rib and 2-Rib tissues. 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. 1-IB and 2-IB were IBs at 1 and 2 years old of *M. amblycephala*. 1-Rib and 2-Rib were ribs at 1 and 2 years old of *M. amblycephala*

**Fig. 7**
The correlation between iTRAQ quantified log₂ (protein ratio) and MRM quantified log₂ (protein ratio) for target proteins of the two comparison groups. a is for 1-IB-vs-1-Rib and b is for 1-IB-vs-2-IB

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References

1. Patterson C, Johnson GD. The intermuscular bones and ligaments of teleostean fishes. 1995. pp. 1–85.
1. Ma LR, et al. The research progress on intermuscular bones of teleosts. Jiangsu Agric Sci. 2012;40:234–5.
1. Hensley DA. Larval development of Engyophrys senta (Bothidae), with comments on intermuscular bones in flatfishes. Bull Mar Sci. 1977;27:681–703.
1. Johnson GD, Patterson C. The intermuscular system of acanthomorph fish: a commentary. Am Mus Novit. 2001;3312:1–24. doi: 10.1206/0003-0082(2001)312<0001:TISOAF>2.0.CO;2. - DOI
1. Bing Z. On the myoseptal spines of the carp (Cyprinus carpio L) Acta Zoolog Sin. 1962;14:175–8.

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[1] Patterson C, Johnson GD. The intermuscular bones and ligaments of teleostean fishes. 1995. pp. 1–85.

[2] Patterson C, Johnson GD. The intermuscular bones and ligaments of teleostean fishes. 1995. pp. 1–85.

[3] Ma LR, et al. The research progress on intermuscular bones of teleosts. Jiangsu Agric Sci. 2012;40:234–5.

[4] Ma LR, et al. The research progress on intermuscular bones of teleosts. Jiangsu Agric Sci. 2012;40:234–5.

[5] Hensley DA. Larval development of Engyophrys senta (Bothidae), with comments on intermuscular bones in flatfishes. Bull Mar Sci. 1977;27:681–703.

[6] Hensley DA. Larval development of Engyophrys senta (Bothidae), with comments on intermuscular bones in flatfishes. Bull Mar Sci. 1977;27:681–703.

[7] Johnson GD, Patterson C. The intermuscular system of acanthomorph fish: a commentary. Am Mus Novit. 2001;3312:1–24. doi: 10.1206/0003-0082(2001)312<0001:TISOAF>2.0.CO;2. - DOI

[8] Johnson GD, Patterson C. The intermuscular system of acanthomorph fish: a commentary. Am Mus Novit. 2001;3312:1–24. doi: 10.1206/0003-0082(2001)312<0001:TISOAF>2.0.CO;2. - DOI

[9] Bing Z. On the myoseptal spines of the carp (Cyprinus carpio L) Acta Zoolog Sin. 1962;14:175–8.

[10] Bing Z. On the myoseptal spines of the carp (Cyprinus carpio L) Acta Zoolog Sin. 1962;14:175–8.

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Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

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Comparative proteomics analysis of teleost intermuscular bones and ribs provides insight into their development

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