Comparative microRNAs expression profiles analysis during embryonic development of common carp, Cyprinus carpio

https://doi.org/10.1016/j.cbd.2020.100754Get rights and content

Highlights

  • It provides the first miRNAs expression profile of six embryonic stages in C. carpio.

  • We screened the stage specific miRNAs and differentially expressed miRNAs (DEMs).

  • 15 known DEMs seemed to play important roles in embryogenesis.

  • The specifically expressed miRNAs-mRNA interaction is crucial for mechanisms.

  • Many pathways related to embryonic development were identified.

Abstract

MicroRNAs (miRNAs) play important roles in biological processes by regulating specific gene expression. Limited miRNAs information is available on embryonic development in common carp (Cyprinus carpio) so far. In this study, six important embryonic development stages of C. carpio were collected to perform a times-series of small RNA-seq experiments from cleavage, blastocyst, gastrulation, organ formation, hatching stage to 1 day post-hatching larva. The expression profiles of miRNAs were identified and differentially expressed miRNAs (DEMs) were screened out based on pairwise comparison. A mean of 12,744,989 raw reads and 9,888,123 clean reads were obtained from each library. A total of 2565 miRNAs were identified. 68 of 204 DEMs were overlapped with stage-specific miRNAs, in which 15 were known miRNAs and seemed to play a key role in embryogenesis. Additionally, time-course expression reveals several intriguing fluctuations during embryogenesis. Numerous signaling pathways were identified in embryonic development, including the phototransduction, hippo signaling pathway, Wnt, melanogenesis, histidine metabolism and fatty acid biosynthesis. The results would provide new insight into the roles of miRNAs in embryonic development, and would help us to advance the understanding of miRNA-mediated mechanisms in embryonic development of fish.

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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