Evidence for the conservation of miR-223 in zebrafish (Danio rerio): Implications for function.

GENE-40428; No. of pages: 9; 4C: Gene xxx (2015) xxx–xxx

Contents lists available at ScienceDirect

Gene journal homepage: www.elsevier.com/locate/gene

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V.P. Roberto a,b,1, D.M. Tiago a,1,⁎, K. Gautvik d,e, M.L. Cancela a,c,⁎⁎ a

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Article history: Received 14 October 2014 Received in revised form 5 April 2015 Accepted 9 April 2015 Available online xxxx

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Keywords: miR-223 Conservation miRNA expression miRNA targets Haematopoiesis Osteoclastogenesis

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Evidence for the conservation of miR-223 in zebrafish (Danio rerio): Implications for function☆ Centre of Marine Sciences, University of Algarve, Faro, Portugal PhD Program in Biomedical Sciences, University of Algarve, Faro, Portugal Department of Biomedical Sciences and Medicine, University of Algarve, Faro, Portugal d Oslo University Hospital, Department of Medical Biochemistry, Lovisenberg Deacon Hospital, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo, Norway e Oslo University Hospital, Institute of Medical Biochemistry, Lovisenberg Deacon Hospital, University of Oslo, P.O. Box 1112 Blindern, 0317 Oslo, Norway b

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MicroRNAs (miRNAs) are an abundant and conserved class of small RNAs, which play important regulatory functions by interacting with the 3′ untranslated region (UTR) of target mRNAs. Through this mechanism, miR-223 was shown to regulate genes involved in mammalian haematopoiesis, both in physiological and pathological contexts. MiR-223 is essential for normal myelopoiesis in mammals, promoting granulocyte, osteoclast and megakaryocyte differentiation and suppressing erythropoiesis. However, there is a general lack of knowledge regarding miR-223 function in other vertebrates, which could help to clarify its role in other processes, such as development. In this work, we explored the functional conservation of miR-223 using zebrafish as a model. We show that miR-223 gene structure and genomic context have been maintained between human and zebrafish. In addition, we identified 22 novel sequences of miR-223 precursor and demonstrate that it contains domains highly conserved among vertebrates, suggesting function preservation throughout evolution. Furthermore, collected evidences show that miR-223 expression is highly correlated with haematopoietic events and osteoclastogenesis throughout zebrafish development. In adults, expression of miR-223 in zebrafish tissues mimics the distribution in mice, with high levels found in the major fish haematopoietic organ, the head kidney. These results suggest a conservation of miR-223 role in haematopoiesis, and osteoclastogenesis between zebrafish and human. Accordingly, validated targets of miR-223 in mammalian models were investigated and defined as putative targets in zebrafish, by in silico and gene expression analysis. Our data compiles critical evidence showing that miR-223, a highly conserved miRNA, appears to have kept similar regulatory functions throughout evolution. © 2015 Published by Elsevier B.V.

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1. Introduction

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MicroRNAs (miRNAs), are a conserved class of noncoding small RNAs located at diverse genomic locations and transcribed by RNA polymerase II as long primary transcripts (pri-miRNA) (Lee et al., 2004), which are then processed by the nuclear RNase III Drosha generating a miRNA precursor (pre-miRNA). Once exported to the cytoplasm, cleavage of the pre-miRNA is catalysed by Dicer resulting in a doublestranded 20–23 nt product (Davis and Hata, 2009). After loading into

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Abbreviations: miRNA, microRNA; dpf, days post-fertilization; hpf, hours postfertilization; bp, base pairs; kb, kilobase pairs; nt, nucleotide; RACE, rapid amplification of cDNA ends; qPCR, real time-PCR; UTR, untranslated region; U6, U6 small nuclear RNA. ☆ Disclosures: The authors declare that there is no conflict of interests regarding the publication of this paper and that all authors agree with the content of this paper. ⁎ Corresponding author. ⁎⁎ Correspondence to: M.L. Cancela, Centre of Marine Sciences, University of Algarve, Faro, Portugal. E-mail addresses: [email protected] (D.M. Tiago), [email protected] (M.L. Cancela). 1 Both authors equally contributed to this study.

the RNA induced silencing complex (RISC), mature miRNAs guide this complex to target mRNAs and bind by complementarity to their 3′ UTRs, leading to translation inhibition or cleavage of mRNA transcripts (Davis and Hata, 2009; Eulalio et al., 2008). As regulators of gene expression, miRNAs have been implicated in a variety of physiological and pathological processes, including cell proliferation, apoptosis, differentiation and cell fate decisions (Erson and Petty, 2008; Huang et al., 2011). One of these is the multi-stage process of blood cell formation and development from haematopoietic stem cells (HSCs), named haematopoiesis (Vasilatou et al., 2010). Several studies have linked miRNAs to normal and malignant haematopoiesis and the particular miRNA focused in this study, miR-223, has been shown to affect both processes (Vasilatou et al., 2010; Haneklaus et al., 2013). Essential for normal myeloid cell differentiation, miR-223 was shown to participate in granulocyte differentiation and maturation (Fazi et al., 2005; Lu et al., 2013; Johnnidis et al., 2008), as well as in osteoclast and erythocyte differentiation (Sugatani and Hruska, 2009; Felli et al., 2009). Despite the recent progress towards elucidation of miR-223 function in mammals, there is a general lack of knowledge regarding the role of this miRNA in other vertebrates.

http://dx.doi.org/10.1016/j.gene.2015.04.022 0378-1119/© 2015 Published by Elsevier B.V.

Please cite this article as: Roberto, V.P., et al., Evidence for the conservation of miR-223 in zebrafish (Danio rerio): Implications for function, Gene (2015), http://dx.doi.org/10.1016/j.gene.2015.04.022

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achieved by rapid amplification of cDNA ends (RACE) using Advantage cDNA polymerase mix (Clontech) according to the manufacturer's conditions. Specific forward and reverse primers (listed in Supplementary Table S1) were designed according to expressed sequence tags (ESTs) available in NCBI database (http://www.ncbi.nlm.nih.gov) and combined with universal adapter primers (Table S1). Amplified PCR products were inserted into pCRII-TOPO (Invitrogen), cloned and further analysed by standard DNA sequencing.

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2.4. Quantitative real-time PCR (qPCR) analysis

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Total RNA was extracted from adult tissues of zebrafish (pool of 4 specimens) and mouse (one specimen) and from zebrafish larvae and juveniles (pool of up to twenty individuals), from 1k-cell (approximately 3 hpf) to 81 dpf, as previously described. Then, RNA was treated with RQ1 RNase-free DNase (Promega), re-purified in phenol/chloroform/ isoamyl alcohol (25:24:1) mixture, quantified by UV spectrophotometry (NanoDropND-1000) and its quality assessed by agarose gel electrophoresis. For qPCR analysis of mature miRNAs, total RNA (1 μg) was polyadenylated and reverse-transcribed using the NCode miRNA FirstStrand cDNA Synthesis kit (Invitrogen), according to the manufacturer's instructions. For PCR amplifications 1.6 ng of reverse transcribed RNA, miRNA-specific primers (Table S1) and the NCODE qPCR kit (Invitrogen) were used. For qPCR analysis of mRNAs, 1 μg of total RNA was treated with RQ1 RNase-free DNase and reverse-transcribed using MMLV-RT (Invitrogen) and oligo-d(T)-adapter primer (Table S1). For mRNA amplification we used 10 ng of cDNA, specific primers (Table S1) and SsoFast EvaGreen Supermix (Bio-Rad), according to the manufacturer's instructions. QPCR analysis was performed using the StepOnePlus system (Applied Biosystems) and relative expression of mRNAs and miRNAs was determined through the ΔΔCt method (Livak and Schmittgen, 2001), using EF1α and U6 small nuclear RNA transcript for normalization, respectively. Specificity of each qPCR reaction was verified by melt curve analysis and electrophoresis.

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2.5. In silico analysis of miR-223 gene and transcripts

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Precursor and mature miR-223 sequences from different vertebrate species were obtained from miRbase release 20 (http://www.mirbase. org). EST and whole genome shotgun (WGS) sequences were obtained from NCBI database. Hairpin precursor sequences were inferred based on: i) nucleotide, size and position similarity, when compared with species from the same taxa; and ii) hairpin minimum free energy. Precursor sequences were aligned using Clustal Omega tool (http:// www.ebi.ac.uk/Tools/msa/clustalo/), displayed as a logo using Weblogo (http://weblogo.berkeley.edu/logo.cgi), and used to generate consensus secondary structures using RNAalifold (http://rna.tbi.univie.ac.at/ cgi-bin/RNAalifold.cgi). Pairwise sequence identities among miR-223 sequences were determined using Sequence Manipulation Suite (http://www.bioinformatics.org/sms2/ident_sim.html). Individual structural analysis of miR-223 precursors was performed using Sfold (http://sfold.wadsworth.org/cgi-bin/srna.pl). Finally, Ensembl (http:// www.ensembl.org/index.html) was used to identify loci and flanking regions of miR-223 genes, and synteny was determined by comparing miR-223 flanking genes. Genes were designated syntenic if one or more genes were conserved between zebrafish and human chromosomes, irrespective of orientation or order.

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2.1. Ethics statement

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All animal manipulations were performed in compliance with the Guidelines of the European Union Council (86/609/EU) and transposed to the Portuguese laws for the use of laboratory animals by “DL n° 129/ 92 de 06 de Julho, Portaria n° 1005/92 de 23 de Outubro de 1992” and on the directive of the European parliament and of the council on the protection of animals used for scientific purposes (2010/63/EU). All animal protocols were performed under a “Group-I” license from the DirecçãoGeral de Veterinária, Ministério da Agricultura, do Desenvolvimento Rural e das Pescas, Portugal.

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Wild-type zebrafish were reared at 28.5 °C on a 14:10 h light:dark cycle and zebrafish eggs were obtained by natural spawning. Larvae were maintained and raised at standard conditions, as previously described (Westerfield, 2000). Individuals were collected randomly at regular intervals, from hatching to adult stages. Fish were euthanized with a lethal dose of MS-222 (Sigma) and either frozen in liquid nitrogen and preserved at −80 °C, or fixed in 4% buffered paraformaldehyde (PFA) at 4 °C for further processing. Adult mice tissue samples were obtained from Mus musculus specimens maintained in our animal facilities as previously described (Roberto et al., 2014).

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2.3. Marathon library and molecular cloning of zebrafish miR-223 primary transcript

2.6. Prediction of miR-223 target transcripts

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Zebrafish specimens (48 h post-fertilization (hpf), 20 days post-fertilization (dpf), 25 dpf, 40 dpf, two adult males and two adult females) were used to extract total RNA following the Chomczynski and Sacchi method (Chomczynski and Sacchi, 1987). RNA was then used to construct a Marathon™ cDNA library (Clontech), following the manufacturers' protocol. The 5′ and 3′ ends of zebrafish miR-223 were

The 3′-UTR of zebrafish orthologs of known miR-223 targets were fed to three algorithms: i) TargetScanFish Release 6.2 (http://www. targetscan.org/fish_62/), which relies on conservation of miRNA binding site (seed) and also on number of sites, type and context (Grimson et al., 2007; Lewis et al., 2005; Ulitsky et al., 2012; Garcia et al., 2011); ii) PITA algorithm (http://genie.weizmann.ac.il/pubs/

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Such understanding will contribute to further elucidate miR-223 role in haematopoietic and osteoclastogenic events, for instance during development. In that sense, zebrafish, a widely used organism for studies of vertebrate development, gene function and human disease (Haffter et al., 1996; Lieschke and Currie, 2007), could be a valuable model to improve the current knowledge on miR-223 functions. Advantages of using zebrafish to study haematopoiesis include a rapid external embryonic development and transparency, survival for several days without circulating blood cells (Weinstein et al., 1996), and high conservation of the haematopoietic process compared with mammals (De Jong and Zon, 2005). In fact, both mammals and zebrafish experience two waves of haematopoiesis: 1) primitive haematopoiesis, involving initial migration of primitive macrophages from cephalic mesoderm to yolk ball, and later development of erythrocyte in the intermediate cell mass (ICM) (De Jong and Zon, 2005; Bertrand and Traver, 2009; Davidson and Zon, 2004); and 2) definitive haematopoiesis, involving initial generation of erythroid-myeloid progenitors (EMPs) in the posterior blood island (PBI) (Bertrand et al., 2007), and later production of haematopoietic stem cells (HSCs) in the AGM (aorta-gonad-mesonephros), which migrate to the caudal haematopoietic tissue (CHT), thymus and kidney (Bertrand and Traver, 2009; Paik and Zon, 2010). Despite the potential advantages of using zebrafish to study miR-223, no data is available in this species. In this work we have investigated miR-223 conservation between zebrafish and mammals, regarding its gene organization, genomic context, miRNA structure, pattern of expression (development and adults) and predicted targets. We show a general conservation of miR-223 features, indicating that zebrafish is a valuable model to study miR-223 functions with particular emphasis in haematopoiesis and osteoclastogenesis.


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3.1. MiR-223 is conserved throughout evolution: insights from gene and precursor analysis

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In order to investigate the putative miR-223 conservation in vertebrates, human and zebrafish pre-miR-223 sequences were collected from miRBase and used as template to search for full length miR-223 transcripts (pri-miR-223), using BLAST tool from NCBI. While for human sequences two complete mRNA sequences (accession numbers DQ680071 and DQ680072) were retrieved, for zebrafish only a single EST (accession number EB991492) corresponding to a partial pri-miR223 was identified (Fig. 1). This sequence was used as template to design specific primers and obtain a complete cDNA by RACE-PCR amplification. A sequence spanning 1146 bp and matching zebrafish miR-223 was identified and submitted to NCBI database as pri-miR-223 (accession number KJ634046). Finally, in order to infer miR-223 gene structures, both human and zebrafish pri-miR-223 were mapped to their specific locations in the corresponding genomes, using Ensembl database. Zebrafish miR-223 is located in Chr 5 (Zv9 5: 24017735–24017833 [−]) while human miR-223 is located in Chr X (GRCh37 chrXq21.1: 65238712–65238821 [+]) (Table S2). In both species miR-223 is organized in three exons with the precursor sequence being contained in the third exon (Fig. 1), demonstrating a structural genomic conservation

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Fig. 2. Schematic representations of genomic contexts of human and zebrafish miR-223 genes. The physical localization of syntenic genes present in the vicinity of miR-223 is indicated for both human and zebrafish. Chr — chromosome.

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This work focused on exploring the structural and functional conservation of miR-223 throughout evolution, from zebrafish to mammals. The conservation of miR-223 was explored in different perspectives: from sequence to structure, from gene organization to genomic context, and from levels of expression to mRNA targets. The main goal was to infer about a possible use of zebrafish as a model to investigate miR-223 role in haematopoiesis and osteoclastogenesis.

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mir07/index.html), which computes the difference between free energy associated with miRNA-target duplex and free energy associated with target secondary structure (Kertesz et al., 2007); and iii) RNAhybrid (http://bibiserv.techfak.uni-bielefeld.de/rnahybrid/submission.html), based on the minimum free energy of hybridization of a long and a short RNA (Rehmsmeier et al., 2004). Putative targets were considered whenever the same binding sites were predicted by at least two algorithms.

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Fig. 1. Structural organization of miR-223 gene. Schematic representations of human and zebrafish miR-223 genes located, respectively, in Chr X and 5, are presented. Below the respective genes are represented 2 annotated human miR-223 transcript variants, and the identified zebrafish primary transcript and zebrafish EST sequence. Boxes represent exons 1, 2 and 3 indicated in white, black and grey respectively. Pre-miR-223 is indicated in a light grey box, within exon 3.


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Fig. 3. Conserved features of miR-223 hairpin among vertebrate species. a) Weblogo representation of pre-miR-223 sequence alignment. Sequence logos are presented as a graphical display where the height of each letter is proportional to its frequency in each position and black letters indicate nucleotides that are 100% conserved. b) MiR-223 hairpin alignment using Clustal Omega is displayed together with taxonomic tree (left) and consensus secondary structure in dot-bracket notation (bottom, where dots and brackets represent unpaired and paired bases, respectively). Mature miR-223 sequence is highlighted in dark grey 100% conserved nucleotides enclosed in a box. Other conserved domains among species are highlighted in soft grey. A domain only conserved within the same group of species, preceding star strand, is indicated with a medium grey box. Consensus boundaries of pre-miR-223 are indicated as dashed lines at the bottom. Species corresponding to sequences retrieved from miRbase are indicated in bold. Taxonomic groups investigated were: Actinopterygii (A), Acanthopterygii (Ac), Amniota (Am), Amphibia (Ap), Artiodactyla (Ar), Aves (Av), Beliniformes (Be), Coelacanthimorpha (C), Carnívora (Ca), Cetacea (Ce), Cypriniformes (Ci), Columbiformes (Co), Cyprinodontiformes (Cy), Diapsida (D), Eutheria (E), Euteleostei (Eu), Falconiformes (Fa), Galliformes (G), Gasterosteiformes (Ga), Holacanthopterygii (H), Lepidosauria (L), Marsupialia (M), Monotremata (Mo), Osteichthyes (O), Ostariophysi (Os), Protacanthopterygii (P), Passeriformes (Pa), Perciformes (Pe), Perissodactyla (Pi), Primates (Pr), Psittaciformes (Ps), Rodentia (Ro), Sarcopterygii (S), Siluriformes (Si), Synapsida (Sy), Testudines (T), and Tetraodontiformes (Te).


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mammalian osteoclastogenic events (Sugatani and Hruska, 2009; Sugatani and Hruska, 2007). To shed some light into the putative functions of miR-223 in other vertebrates, the expression pattern of this miRNA was investigated in zebrafish. Since in zebrafish crucial events of haematopoiesis (Bertrand et al., 2007; Paik and Zon, 2010; Bertrand et al., 2008; Sood and Liu, 2012) and osteoclastogenesis (Witten et al., 2001) were previously described to occur between 10 hpf and 6 dpf, and from 20 dpf until adulthood, respectively, we analysed zebrafish miR-223 expression from blastula and larval stages to adulthood. In addition, expression in adult zebrafish and mouse tissues were analysed for comparison. During zebrafish development (Fig. 4), miR-223 is up-regulated between 31 and 36 hpf, which could be related with two important haematopoietic events: i) formation of committed erythromyeloid progenitors in the PBI, known to occur between 24 and 48 hpf (Bertrand et al., 2007); and ii) formation of haematopoietic precursors in the AGM, occurring from 30 to 36 hpf (Bertrand et al., 2008). Furthermore, miR-223 was also up-regulated between 4 and 6 dpf, and from 30 dpf to adulthood, which could be associated, respectively, to the seeding of the head kidney by HSCs and to definitive haematopoiesis support (De Jong and Zon, 2005; Bertrand and Traver, 2009; Paik and Zon, 2010). Analysis of miR-223 transcripts in tissues from adult zebrafish and mouse showed expression in all analysed tissues, with the lowest levels observed in brain and cartilaginous tissues (branchial arches and ear; Fig. 5a, b). Both species presented relatively high levels of expression in the vertebrae, muscle and heart. Since miR-223 has been associated with differentiation of haematopoietic lineages, the higher levels of expression observed in the heart and muscle could be associated with their high degree of vascularization (probably due to circulation of immature cells). Alternatively, miR-223 could be associated with specific functions in these organs. Most importantly, the highest levels of miR-223 expression were observed in major sites of haematopoiesis, i.e. head kidney in zebrafish and bone marrow in mouse (Fig. 5). The head kidney is a well-described haematopoietic organ in zebrafish and is considered the equivalent to the bone marrow of mammals (Paik and Zon, 2010; Sood and Liu, 2012). Our finding that miR-223 is highly expressed in this organ is definetely in favour of a putative role of miR223 in zebrafish haematopoiesis and further emphasizes the usefulness of using zebrafish as model to elucidate miR-223 function. Another molecular process that could be related to miR-223 expression in zebrafish, specially during development (10-fold induction of miR-223 expression after 30 dpf; Fig. 4) is osteoclastogenesis/bone remodelling, which normally occurs from 20 to 30 dpf onwards (Witten et al., 2001; Witten and Huysseune, 2009). Although osteoclasts derive from the haematopoietic lineage, the molecular pathway(s) involved in this process are still not completely understood in mammals or zebrafish. However, miR-223 was previously shown to be a key factor in osteoclast differentiation in mammals, being differentially expressed during different phases of osteoclast differentiation (Sugatani and Hruska, 2009; Sugatani and Hruska, 2007; Kagiya and Nakamura, 2013; Shibuya et al., 2013). Because of its similarities with mammals in terms of gene structure and targets, zebrafish might be an important model to clarify the exact function of miR-223 microRNA in osteoclastogenesis. In that sense, transgenic lines that allow in vivo imaging of osteoclast maturation and migration (as already available in medaka (Chatani et al., 2011)) could be crossed with other lines, in which miR-223 promoter could drive expression of a fluorescent reporter gene thus providing relevant in vivo tools to further understand miR-223 function in vertebrate osteoclastogenesis.

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3.2. MiR-223 expression is correlated with haematopoiesis and osteoclastogenesis in zebrafish

3.3. Evidences for conservation of miR-223 functions in zebrafish

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In mammalian models, miR-223 is known to play critical roles in haematopoiesis, promoting granulocyte differentiation (Fazi et al., 2005; Fukao et al., 2007) and suppressing erythrocyte differentiation (Felli et al., 2009). Moreover, miR-223 was also associated with

In general, the identification of miRNA target transcripts is essential to fully elucidate miRNA functions. In that sense, the identification of known miR-223 targets was performed through extensive search of Pubmed database, using the following keywords: miR-223 and target

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among vertebrates, which is in agreement with previous data (Fukao et al., 2007). However, miR-223 genes are more compact in mammals than in zebrafish (5 kb in human vs. 11.8 kb in zebrafish, Table S2), and present two alternative splicing forms (already described) (Fukao et al., 2007), while in our study only one transcript was identified in zebrafish. In order to confirm that this transcript is an ortholog of the human, a more detailed analysis of miR-223 human and zebrafish genomic contexts was performed. Gene synteny analysis revealed a block of 8 genes upstream miR-223 locus that were preserved, and in the same order, between the two species. Downstream the miR-223 locus, the order of syntenic genes was less preserved, although several genes were still found to be conserved between the two species (Figs. 2 and S1). Therefore our data confirmed that, besides gene structure, the genomic contexts have also been maintained in the two species, indicating that zebrafish miR-223 is most likely the ortholog of the corresponding human gene. To explore conservation of miR-223 primary and secondary structures (Auyeung et al., 2013; Zeng and Cullen, 2005; Zeng et al., 2005; Han et al., 2006; Zhang and Zeng, 2010), a broader group of species were compared. First, pre-miR-223 and mature miR-223 sequences from mammalian, amphibian, birds, reptiles and teleost fish species were collected from miRbase and their pairwise identities determined. Because of the poor availability of sequences from certain taxonomic groups (e.g. birds and teleost fish), additional sequences were collected from EST and WGS databases available in NCBI. In total, 46 miR-223 precursor sequences were collected and analysed (24 sequences previously annotated in miRbase, and 22 new sequences identified in the present study). While the identity of pre-miR-223 sequences between zebrafish and other vertebrate species was less than 70% (Fig. S2a), the same analysis using mature miR-223 sequences showed a homology higher than 85% (Fig. S2b), indicating a remarkable conservation. Finally, after inferring hairpin precursor sequences, conservation of primary structure was determined using Clustal Omega alignments (Fig. 3) (represented by logos using WebLogo; Fig. 3a) and conservation of secondary structure was identified by analysis of individual and consensus hairpin structures (created using Sfold and RNAalifold, respectively, Figs. S3 and 3b). Data confirmed that miR-223 mature sequence is remarkably conserved among vertebrates. Furthermore, it provided evidence that a key element in target recognition and translation inhibition (Pillai, 2005), named the seed region, was 100% preserved, suggesting that both miR-223 targets and its function are likely to have been conserved among vertebrates. Other important features of miR-223 were also preserved, including the star strand and flanking sequences of both mature and star miR-223 (Fig. 3b), suggesting a conservation in miR-223 processing. In particular, the following regions were maintained at hairpins folding (Figs. 3 and S3): i) a terminal loop, which is critical for Drosha and Dicer optimal processing, and contributes to determine the cleavage site based on the distance to the loop (Zeng et al., 2005; Zhang and Zeng, 2010); ii) single-stranded extensions on the pri-miRNA hairpin in the basal segment, which are vital for DGCR8 binding and distance counting for Drosha cleavage (Zeng and Cullen, 2005; Zeng et al., 2005; Han et al., 2006); iii) 2 helix turns (~22 nt) that encode the miRNA:miRNA* duplex; iv) 1 helix turn (~11 nt) of the lower stem, which is also important for processing (Zeng et al., 2005; Han et al., 2006); and v) the mature miR-223, maintained in the 3p arm of stem-loops in all analysed species. In general, structural information pointed towards a notable conservation of miR-223 throughout evolution, raising the hypothesis that its function could also be maintained.


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in mammals). The remaining 36 studies corresponded to a total of 9 miR-223 mammalian targets, listed in Table 1. Then, corresponding zebrafish orthologs were retrieved from NCBI database and analysed for putative miR-223 binding sites using three different bioinformatic tools: i) TargetScanFish, ii) PITA algorithm and iii) RNAhybrid. Potential binding sites were considered if identified by at least two algorithms (also listed in Table 1). This survey revealed that all the identified mammalian miR-223 targets had at least one corresponding zebrafish

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(last checked in November 18, 2013). Our search retrieved a total of 92 reports confirming the general idea that each miRNA can regulate hundreds of genes (Pillai, 2005). From that initial list, 56 publications were excluded from this analysis due to the lack of one or more of the following inclusion criteria: i) presence of known ortholog in zebrafish; ii) knowledge of full-length cDNA sequence in zebrafish; iii) association of targets to mammalian haematopoiesis or osteoclastogenesis (since miR-223 role was shown to be mainly associated with those processes

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Fig. 4. Analysis of mature miR-223 expression during zebrafish development. MiR-223 expression was determined by qPCR analysis using RNA samples from different stages of zebrafish development, and normalized to levels of zebrafish U6 small RNA. Values are the mean of at least 3 independent replicates; hpf indicates hours post-fertilization; dpf indicates days postfertilization, N.D. indicates non-detected. Gap in the y axis separates two different scales.

Fig. 5. Analysis of mature miR-223 expression in zebrafish (a) and mouse (b) adult tissues. MiR-223 expression, determined by qPCR analysis in zebrafish and mouse tissues, was normalized to levels of U6 small RNA. Values are the mean of at least 3 independent replicates.


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References

Observations

Aces. num.

Seed match

Fazi et al. (2005) Sugatani and Hruska (2009) Jia et al. (2011)

miR-223 represses translation of NFI-A favouring granulocytic differentiator Through regulation of NFI-A, miR-223 stimulates osteoclast differentiation by favouring M-CSFR expression. Although Rasa1 was not the functional target in the system studied, Rasa1 is a direct target of miR-223.

XM_005161709.1

1922–1928 TargetScan, PITA, RNAhybrid

t1:7

NFIA

t1:8

RASA1

t1:9 Q2

E2F1

t1:10 Q3

FBXW7 Kurashige et al. (2012)

miR-223 represses FBXW7 and has no adverse impact on ESCC patients survival.

t1:11

FOXO1

Wu et al. (2012)

Overexpression of miR-223 leads to down-regulation foxo1a of FOXO1 and inhibition of cell proliferation. (BX649258.11)

t1:12

IGF1R

t1:13

MEF2C

Johnnidis et al. (2008) Jia et al. (2011) Lu et al. (2013) Johnnidis et al. (2008) Rangrez et al. (2012)

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STMN1

Wong et al. (2008) Kang et al. (2012)

miR-223 controls granulocyte function potentially by modulation of IGF1R. miR-223 suppressed HeLa cell proliferation by targeting IGF-1R. miR-223 regulates eosinophil differentiation probably through IGF-1R. miR-223 targets Mef2c in myeloid progenitors inhibiting their proliferation and granulocyte function. miR-223 overexpression promotes VSMC proliferation and downregulates Mef2c and RhoB. STMN1 is a downstream target of miR-223 in hepatocellular carcinoma. STMN1 is a putative target of miR-223 in gastric cancer cells.

t1:15

LMO2

Yuan et al. (2009) Felli et al. (2009)

igf1ra (BX470160.9)

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3450–3458 3468–3474

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mef2cb (BX465834.20)

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miR-223 reversibly regulates erythroid and megakaryocytic differentiation of K562 cells by targeting LMO2. Enforced expression of miR-223 impairs differentiation of erythroid cells by repressing LMO2.

XM_005170948.1

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Pulikkan et al. (2012)

264–270 rasa1a (XM_001341973.5) 329–335 rasa1b (XM_001921687.4) miR-223 regulates myeloid cell proliferation through XM_005174533.1 39–46 a mutual negative feedback loop involving E2F1. 1705–1713

Biological process DNA replication; DNA-templated, regulation of transcription, DNA-dependent transcription

TargetScan, PITA, RNAhybrid

Regulation of small GTPase mediated signal transduction

TargetScan, PITA, RNAhybrid TargetScan, PITA, RNAhybrid PITA, RNAhybrid TargetScan, PITA, RNAhybrid PITA, RNAhybrid TargetScan, PITA PITA, RNAhybrid

Unknown

Negative regulation of Notch signalling pathway Regulation of transcription, DNA-dependent transcription, DNA-templated

PITA, RNAhybrid

Anterior/posterior pattern specification; heart morphogenesis and development; embryo development; IGF receptor signalling pathway Cardiac muscle cell development and differentiation heart formation and development regulation of transcription, DNA-dependent Regulation of microtubule polymerization

PITA, RNAhybrid

Blood vessel development; embryonic hemopoiesis erythrocyte differentiation

375 376 377 378 379 380 381 382 383 384 385 386 387 388 389 390 391 392 393 394 395 396

R

373 374

ortholog that was also predicted by our in silico analysis to be a miR-223 target, further supporting our hypothesis of a conservation of miR-223 function. However, to the best of our knowledge, the majority of the identified targets had no haematopoietic function assigned in zebrafish (with the exception of lmo2) and their levels of expression were poorly described. To overcome this issue, and although an inverse correlation between mRNA target and miRNA levels is not always observed, we investigated the expression pattern of six putative target genes in zebrafish (Fig. 6 and Fig. S4). Interestingly, higher levels of lmo2, igf1ra, rasa1b and fbxw7 expression were observed at 1k-cell stage (Fig. 6), a stage where we could not detect miR-223 (Fig. 4). Furthermore, the expression levels of these 4 genes were predominantly lower than at 24 hpf, both throughout zebrafish development and in juvenile stages, when miR-223 levels were found to be higher. Regarding mef2cb and rasa1a, we could not find an evident correlation with the levels of miR-223 expression in any of the developmental stages analysed (Fig. S4): mef2cb showed 3 peaks of expression at 24 hpf, 6 dpf and 60 dpf (Fig. S4a), while rasa1 expression was predominantly higher after 3 dpf (Fig. S4b). This lack of correlation could be related to a specific inability of miR-223 to promote mRNA degradation of these putative targets. Nevertheless, our analysis showed a good inverse correlation between miR-223 and the majority of analysed putative targets, providing additional evidences for specific post-transcriptional regulation of these genes in zebrafish. In mammals, the majority of these targets have known functions in haematopoiesis or osteoclastogenesis, and at least one target, i.e. LMO2, has a conserved function in both zebrafish and mammals. LMO2

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370

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Q1

Predicted by

F

t1:6

Mammals

O

Gene

R O

t1:5

Table 1 Target genes of miR-223 analysed in this study. Position of seed match refers to counting from first nucleotide after stop codon. E2F1, E2F transcription factor 1; FBW7, F-box and WD repeat domain-containing 7; FOXO1, forkhead box O1; IGF1R, insulin-like growth factor 1 receptor; LMO2, LIM domain only 2 (rhombotin-like 1); MEF2C, myocyte enhancer factor 2C; NFIA, nuclear factor I/A; RASA1, RAS p21 protein activator 1; STMN1, stathmin 1.

P

t1:1 t1:2 t1:3 t1:4

7

is required for primitive and definitive haematopoiesis and for angiogenesis both in mammals and zebrafish (Patterson et al., 2007; Yamada et al., 1998; Yamada et al., 2000; Zhu et al., 2005); also, it was shown to be down-regulated by miR-223 in humans, thus suppressing differentiation of erythroid cells (Felli et al., 2009; Yuan et al., 2009) and increasing megakaryocytic differentiation (Yuan et al., 2009). In mammals, NFIA was shown to be down-regulated by miR-223, promoting granulocytic (Fazi et al., 2005), and osteoclastic differentiation (Sugatani and Hruska, 2009). Ablation of MEF2C, a modulator of HSCs cell-fate decision in mammals (Schüler et al., 2008), was shown to suppress granulocyte progenitor proliferation, thus correcting mice phenotype promoted by miR-223 knockout (Johnnidis et al., 2008). Also, in miR-223 knockout, IGF1R was shown to be increased, which resulted in higher proliferation of eosinophil progenitors (Lu et al., 2013). In both cases, MEF2C and IGF1R genes were validated as direct miR-223 targets (Johnnidis et al., 2008; Jia et al., 2011). In general, our in silico and gene expression analysis evidenced that the genes here studied are most likely miR-223 targets also in zebrafish, and although further validation is still required, we present additional clues towards a functional conservation of miR-223 in vertebrates. Finally, it is worthy to mention that miR-223 and its predicted targets are also associated with other processes, such as malignant haematopoiesis, by the regulation of E2F1, STMN1, FOXO1, FBXW7 (Pulikkan et al., 2010; Kurashige et al., 2012; Mansour et al., 2013; Wu et al., 2012; Wong et al., 2008; Kang et al., 2012), and vascular development, by the modulation of MEF2C (Rangrez et al., 2012). In addition, RASA1 (Kranenburg et al., 2004; Ren et al., 2013), LMO2 (Yamada


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Fig. 6. Analysis of lmo2, igf1ra, rasa1b and fbxw7 expression during zebrafish development. Gene expression was determined by qPCR analysis using RNA samples from different stages of zebrafish development, and normalized to levels of zebrafish EF1α. Values are the mean of at least 3 independent replicates; hpf indicates hours post-fertilization; dpf indicates days postfertilization. Gap in the y axis separates two different scales.

430

4. Conclusions

431

Our data revealed that miR-223 structural and functional features have been conserved throughout evolution. Its genomic organization and context are maintained between human and zebrafish, and the conservation of primary and secondary structures of miR-223 precursors suggests that processing and function of miR-223 were maintained across vertebrate evolution. We provide additional evidence supporting the use of zebrafish as model to study miR-223 function by showing that its expression pattern during development is consistent with a role in primitive and definitive haematopoiesis and consequently also in osteoclastogenesis. We also predicted, as putative targets for zebrafish miR-223, those genes for which the corresponding mammalian orthologs are established miR-223 targets and already shown to be involved in haematopoiesis and/or osteoclastogenesis. The data presented here contributes decisively to define a pool of miR-223 target genes and physiological processes including haematopoiesis, osteoclastogenesis, malignancy and cardiovascular development, which should be further investigated in order to clarify the role of miR-223. Furthermore, we provide strong evidence supporting the use of zebrafish as model to study the involvement of miR-223 in those functions. Supplementary data to this article can be found online at http://dx. doi.org/10.1016/j.gene.2015.04.022.

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et al., 2000; Zhu et al., 2005), FBXW7 (Izumi et al., 2012), MEF2C (Edmondson et al., 1994; Hinits et al., 2012) and IGF1R (Galer et al., 2011; Huang et al., 2013) also play roles in angiogenesis and cardiovascular development, both in mammals and zebrafish, suggesting the involvement of miR-223 in physiological and/or pathological events associated with those processes.

Funding

452

This work was supported by grants from the Calouste Gulbenkian Foundation (program “Na Fronteira das Ciências da Vida”; to D.M.T.), by the European Regional Development Fund (ERDF) through COMPETE Program and by national funds through the FCT — Foundation for Science and Technology, under the project “PEst-C/MAR/LA0015/2011” and by Helse SørØst, Norway. V.P.R. and D.M.T. were the recipients of doctoral (SFRH/BD/38607/ 2007) and post-doctoral (SFRH/BPD/45034/2008) fellowships respectively, from the Portuguese Foundation for Science and Technology (FCT).

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Uncited references

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Shen et al., 2013 Teittinen et al., 2012

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A physiologically based toxicokinetic model for the zebrafish Danio rerio.

Manipulating galectin expression in zebrafish (Danio rerio).

Functional characterization of zebrafish (Danio rerio) Bcl10.

Short-term memory in zebrafish (Danio rerio).

The Control of Calcium Metabolism in Zebrafish (Danio rerio).

Genetic Analysis of the Touch Response in Zebrafish (Danio rerio).

Characterization of glutathione-S-transferases in zebrafish (Danio rerio).

Transformation of tributyltin in zebrafish eleutheroembryos (Danio rerio).

Evaluation of visible implant elastomer tags in zebrafish (Danio rerio).

dark preference in zebrafish (Danio rerio).

Primary intestinal and vertebral chordomas in laboratory zebrafish (Danio rerio).

Effectiveness of recommended euthanasia methods in larval zebrafish (Danio rerio).

Mechanism of TiO2 nanoparticle-induced neurotoxicity in zebrafish (Danio rerio).

Cardiotoxicity evaluation of anthracyclines in zebrafish (Danio rerio).

Identification of novel antigen candidates for a tuberculosis vaccine in the adult zebrafish (Danio rerio).

Evidence of common cadmium and copper uptake routes in zebrafish Danio rerio.

Vascular toxicity of silver nanoparticles to developing zebrafish (Danio rerio).

Brain Transcriptomic Response to Social Eavesdropping in Zebrafish (Danio rerio).

Chronic bisphenol A exposure alters behaviors of zebrafish (Danio rerio).

Long-term (30 days) toxicity of NiO nanoparticles for adult zebrafish Danio rerio.

Distribution of vitellogenin in zebrafish (Danio rerio) tissues for biomarker analysis.

A role for transcription factor glial cell missing 2 in Ca2+ homeostasis in zebrafish, Danio rerio.

The common neural parasite Pseudoloma neurophilia is associated with altered startle response habituation in adult zebrafish (Danio rerio): Implications for the zebrafish as a model organism.

Are zebrafish Danio rerio males better swimmers than females?