Comparative Biochemistry and Physiology Part D: Genomics and Proteomics
Comparative microRNAs expression profiles analysis during embryonic development of common carp, Cyprinus carpio
Graphical abstract
Introduction
MicroRNAs (miRNAs) are highly-conserved, small (19–24 nucleotides), non-coding RNA that play important roles in regulating specific messenger RNA target genes expression in many biological processes (Bartel, 2004; Krol et al., 2010; Luo et al., 2018). Embryonic development is a continuous and complex morphogenetic process involving proliferation and differentiation of embryonic cells (Kocamis et al., 2013). The miRNAs regulate the developmental timing, cell division, differentiation, migration, apoptosis, morphogenesis, and organogenesis processes in the period of embryonic development (Hwang and Mendell, 2007; Williams et al., 2009).
MiRNAs have been suggested to be important during embryonic development of animals. For instance, the human follicular fluid miRNA-320 affected mouse embryonic development (Feng et al., 2015). MiRNA-450a-3p repressed cell proliferation and regulated embryonic development in mouse (Luo et al., 2012). The miRNA-51 family was broadly expressed from mid-embryogenesis onward in Caenorhabditis elegans (Shaw et al., 2010). MiRNA-138 modulated cardiac patterning during embryonic development and miRNA-140-5p regulated embryonic bone development of zebrafish (Danio rerio) (Morton et al., 2008; Gan et al., 2016). The miRNA-10 family directly regulated the Hox gene family during Oreochromis niloticus embryogenesis (Giusti et al., 2016). Although microRNA expressions in embryonic development of model zebrafish have been investigated (Wienholds et al., 2005), research on the molecular mechanism of miRNAs regulating embryo development of fish, especially non-model fish, is still in its infancy.
The common carp (Cyprinus carpio L.) is an important food fish with over hundred strains and varieties in the world. It is one of the dominant aquaculture species and shows some morphological and genetic variations after selection breeding in china (Dong et al., 2015). The genome sequence and genetic diversity of C. carpio has been reported (Xu et al., 2014), but the miRNAs information during embryonic development is relatively limited. MiRNAs in the fertilized oocytes, 72 hour post-fertilization (hpf) and 1 day post-hatching (dph) embryonic development stages were identified without common carp genome as reference sequences (Zhu et al., 2012). And identification of miRNAs of neurula (13 hpf) and complete yolk sac absorption (2 dph) embryonic development stages were finished in common carp (Wang et al., 2017). However, systemic research on the miRNAs during embryonic development of C. carpio has not been reported yet. Here in, we constructed the small RNA (sRNA) libraries from six main embryonic development stages (cleavage (2 hpf), blastocyst (6 hpf), gastrulation (12 hpf), organ formation (20 hpf), hatching stage (64 hpf) and 1 dph larva) (Fig. 1) to investigate the miRNAs expression profiles during embryonic development of C. carpio. The results would provide new insight into the roles of miRNAs in embryo development, leading to assist the exploitation of the genetic mechanisms of the occurrence of excellent features in fish.
Section snippets
Animal and embryo collection
The experimental fish used in the experiment is a new aquaculture variety called FFRC (Freshwater Fisheries Research Center) No. 2 strain common carp. Embryos were obtained by natural spawning in the Qiting Pilot Research Station (Yixing, China), which is affiliated to the FFRC, CAFS (Chinese Academy of Fishery Sciences). The stages of embryonic development were observed and photographed under a microscope. The test samples consisted of six embryonic stages: cleavage stage (2 hpf) (P1),
Overview of sRNA-sequencing
To obtain the miRNA expression signature in different embryonic stages of common carp, we constructed totally 18 small RNA sequencing libraries in six stages (P1, P2, P3, P4, P5 and P6), with three biological replicates in each stage. A mean of 12,744,989 raw reads were obtained from each library, corresponded to 9,888,123 clean reads after removing disordered reads (Table 1). To realize distribution of sRNAs on genome, the clean reads mentioned above were mapped to the C. carpio reference
Discussion
In this study, the length distribution of all samples in P1 to P4 stages showed a peak length at 24 nt, while 22 nt class being the most abundant in P5 to P6. The identification of miRNAs of 13 hpf and 2 dph displayed similar read-length distributions of 22 and 23 nt in common carp (Wang et al., 2017). The length distribution (24 nt) increased in the first four embryonic stages of common carp, possibly indicating the abundant expression of Piwi-interacting RNAs (piRNAs) from 26 to 31 nt.
Conclusions
The study provides the first miRNAs expression profiles of six important embryonic development stages in common carp. We screened the stage specific miRNAs and significant DEMs by pairwise comparison. The repertoire of 11 out of 15 developmental stage-specific DEMs played important roles in the embryonic development via its target genes. KEGG analysis identified numerous signaling pathways including the phototransduction, hippo signaling pathway, Wnt, melanogenesis, histidine metabolism and
CRediT authorship contribution statement
W.L. generated and wrote the manuscript, D.Z. and X.P. designed and supervised the study, S.F. gained the embryo samples of different developmental stages, Y.H. performed qRT-PCR, F.J. and Z.W. analyzed the results. All authors reviewed and approved the manuscript.
Ethics statement
All animal experiments in this study were approved by the Bioethical Committee of FFRC of CAFS (BC 2013863, 9/2013), guidelines for the Care and Use of Experimental Animals of China.
Declaration of competing interest
The authors declare that they have no conflict of interests.
Acknowledgements
This research was funded by the project supported by the Jiangsu Provincial Postdoctoral Research program in 2018 (2018K208C), the Central Public-interest Scientific Institution Basal Research Fund, Chinese Academy of Fishery Sciences (2020TD37) and the Chinese Earmarked Fund for Modern Agro-industry Technology Research System (CARS-45-05).
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