Dynamic mRNA and miRNA expression analysis in response to intermuscular bone development of blunt snout bream (Megalobrama amblycephala)

Shi-Ming Wan,Ze-Xia Gao,Ning-Nan Guan,Jia Zhong,Shao-Kui Yi,Chun-Hong Nie,Wei-Zhuo Zhang

doi:10.1038/srep31050

Abstract

Intermuscular bone (IB), which occurs only in the myosepta of lower teleosts, is attracting more attention because they are difficult to remove and make the fish unpleasant to eat. By gaining a better understanding of the genetic regulation of IB development, an integrated analysis of miRNAs and mRNAs expression profiling was performed on Megalobrama amblycephala. Four key development stages were selected for transcriptome and small RNA sequencing. A number of significantly differentially expressed miRNAs/genes associated with bone formation and differentiation were identified and the functional characteristics of these miRNAs/genes were revealed by GO function and KEGG pathway analysis. These were involved in TGF-β, ERK and osteoclast differentiation pathways known in the literature to affect bone formation and differentiation. MiRNA-mRNA interaction pairs were detected from comparison of expression between different stages. The function annotation results also showed that many miRNA-mRNA interaction pairs were likely to be involved in regulating bone development and differentiation. A negative regulation effect of two miRNAs was verified through dual luciferase reporter assay. As a unique public resource for gene expression and regulation during the IB development, this study is expected to provide forwards ideas and resources for further biological researches to understand the IBs’ development.

Highlights

It is well known that most of the freshwater aquaculture fishes around the world, especially Cyprinidae species, possessed a certain amount of intermuscular bones (IBs), which are hard-boned spicules located in the muscle tissue on both sides of the vertebrae[1]
The size distribution of these contigs and unigenes is shown in Supplementary Fig. S1-4 and 5
Venn diagrams displayed no overlapping DEGs for the three adjacent pairwise comparisons, while 57 overlapping DEGs were identified with nonadjacent pairwise developmental stage comparisons (Fig. 3B). These results indicated that comparisons of nonadjacent developmental stages can obtain more key DEGs, which may play an important role during long-term development of IBs

Summary

Introduction

It is well known that most of the freshwater aquaculture fishes around the world, especially Cyprinidae species, possessed a certain amount of intermuscular bones (IBs), which are hard-boned spicules located in the muscle tissue on both sides of the vertebrae[1]. Xu et al.[23] has identified an intermuscular bone-deficient grass carp (Ctenopharyngodon idellus) mutant in an artificial gynogenetic group Based on these two reports, it has been speculated that there maybe one or several key genes that regulate the development of IBs. In the present study, we used one typical Cyprinidae species, blunt snout bream (Megalobrama amblycephala), for which the morphological characteristics, emergent periods and morphogenesis of IBs are known from our previous research[24]. With the aim to generate the fundamental molecular resources across the different developmental stages from emerging to complete formation of IBs and try to find the putative key genes or miRNAs for regulating IBs’ development, the transcriptome property, mRNA and miRNA expression profiling were investigated in the four key stages related to IBs development of M. amblycephala in this study. Some interaction networks and regulatory modes of mRNAs and miRNAs were revealed based on the integrated analysis of miRNA and mRNA expression profiles

Methods

Results

Conclusion