Physiol Genomics 47: 158169, 2015.
First published March 3, 2015; doi:10.1152/physiolgenomics.00001.2015.
Dynamics of gene expression patterns during early development of the
European seabass (Dicentrarchus labrax)
E. Kaitetzidou,1,2 J. Xiang,3 E. Antonopoulou,2 C. S. Tsigenopoulos,1 and E. Sarropoulou1
1
Institute of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for Marine Research, Greece; 2School of
Biology, Faculty of Science, Aristotle University of Thessaloniki, Greece; and 3Genomics Resources Core Facility, Weill
Cornell Medical College, New York, New York
Submitted 9 January 2015; accepted in final form 23 February 2015
Kaitetzidou E, Xiang J, Antonopoulou E, Tsigenopoulos CS,
Sarropoulou E. Dynamics of gene expression patterns during early
development of the European seabass (Dicentrarchus labrax). Physiol
Genomics 47: 158 169, 2015. First published March 3, 2015;
doi:10.1152/physiolgenomics.00001.2015.Larval and embryonic
stages are the most critical period in the life cycle of marine fish. Key
developmental events occur early in development and are influenced
by external parameters like stress, temperature, salinity, and photope-
riodism. Any failure may cause malformations, developmental delays,
poor growth, and massive mortalities. Advanced understanding of
molecular processes underlying marine larval development may lead
to superior larval rearing conditions. Today, the new sequencing and
bioinformatic methods allow transcriptome screens comprising mes-
senger (mRNA) and microRNA (miRNA) with the scope of detecting
differential expression for any species of interest. In the present study,
we applied Illumina technology to investigate the transcriptome of
early developmental stages of the European seabass (Dicentrarchus
labrax). The European seabass, in its natural environment, is a
euryhaline species and has shown high adaptation processes in early
life phases. During its embryonic and larval phases the European
seabass lives in a marine environment and as a juvenile it migrates to
coastal zones, estuaries, and lagoons. Investigating the dynamics of
gene expression in its early development may shed light on factors
promoting phenotypic plasticity and may also contribute to the im-
provement and advancement of rearing methods of the European
seabass, a species of high economic importance in European and
Mediterranean aquaculture. We present the identification, character-
ization, and expression of mRNA and miRNA, comprising paralogous
genes and differentially spliced transcripts from early developmental
stages of the European seabass. We further investigated the detection
of possible interactions of miRNA with mRNA.
RNA-Seq; transcriptomics; aquaculture; European seabass; Dicen-
trarchus labrax; miRNA; mRNA; development
FISH DEVELOPMENT, LIKE IN all vertebrates, is determined during
the development of an embryo (embryogenesis) and can be
considered as a continuum of morphological changes. To
enable the analysis of embryogenesis this continuum has been
broken down into distinct developmental stages, and much
effort has been invested to define those developmental stages
along with their critical aspects. It is known that embryonic and
larval development in vertebrates share main developmental
processes, like the determination of the overall body plan and
organogenesis (18, 82). On the other hand, studies also have
shown high phenotypic plasticity during development due to
species particularities (36, 43). Phenotypic plasticity in teleost
Address for reprint requests and other correspondence: E. Sarropoulou, Inst.
of Marine Biology, Biotechnology and Aquaculture, Hellenic Centre for
Marine Research, Thalassocosmos, Gournes Pediados, PO Box 2214, 71003
Heraklion, Crete, Greece (e-mail: sarris@hcmr.gr).
158 1094-8341/15 Copyright 2015 the American Physiological Society
may be explained by fish-specific genome duplication (FSGD)
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showing different retention rates among species as well as
different expression levels. This is important especially con-
cerning transcription factors and signaling genes responsible
for the regulatory control of development. Furthermore, regu-
latory processes by microRNA (miRNA), chromatin remodel-
ing (DNA methylation and histone modification), transcription
factors, or mRNA processing are important factors promoting
phenotypic plasticity (65). Transcriptomic studies in embryo-
genesis may shed light on the underlining mechanisms of
morphological developmental changes and larval plasticity.
Here, we have applied Illumina HiSeq sequencing technology
to investigate the transcriptome of the early embryogenesis of
the European seabass (Dicentrarchus labrax). The European
seabass has shown high adaptation processes in early life
phases (15), and it is of high commercial value in the Medi-
terranean (FAO, 2012, http://www.fao.org/fishery/species/
2291/en). During its embryonic and larval phases the European
seabass lives in a marine environment, and as a juvenile it
migrates to coastal zones, estuaries, and lagoons. The genomic
toolbox of the European seabass has been significantly en-
riched in the recent years, facilitating the identification and
characterization of genes (9, 13, 29, 81). In addition, some
high-throughput gene expression studies have been performed
in the European seabass to better understand the role of genes
implicated in several physiological mechanisms such as growth
rate and immune response (e.g., Ref. 71), nutrition (27), salin-
ity tolerance (9), as well as late larval development and
metamorphosis (16). Several high-throughput transcriptomic
studies have also be performed in other species of commercial
interest, like, e.g., the gilt-head sea bream (Sparus aurata),
Atlantic cod (Gadus morhua), and catfish (Ictalurus punctatus)
(10, 46, 55). However, little or no attention has been paid to
paralogous genes despite the FSGD event that also gave rise to
changes in regulatory control (22, 40, 80, 84). It is also known
that paralogous genes may have altered regulation (subfunc-
tionalization) and/or novel function (neofunctionalization). In-
terestingly, studies also have shown that for some genes the
expression of a paralog gradually decreases, whereas that of
the other paralog increases (70, 73, 74). In addition, Garcia de
la Serrana et al. (26) recently highlighted the systematic dif-
ferences of paralogous gene retainment between Acanthoptery-
gii and Ostariophysi. Most of the studies looking at the role of
various paralogous genes have been performed in the model
fish zebrafish (Danio rerio) (40, 91, 98). Zebrafish belongs to
the superorder Ostariophysi, and hence a high amount of data
is available, whereas less information has been produced for
the superorder Acanthopterygii as it comprises mainly non-
model teleost species.
 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax
To better understand the mechanisms underlying develop-
mental processes, besides the exploration of differentially ex-
pressed transcripts during early development, we must also
address the effect of regulative elements such as miRNAs or
small RNAs. It has been shown that miRNAs are largely
expressed in animals, plants, and viruses (62, 79) and that they
are involved in a wide range of gene regulation of various
biological processes. In humans miRNA are extensively stud-
ied, and it is known that altered miRNA expression plays an
important role in human diseases and especially in cancer (2,
38). Next-generation sequencing technologies have enabled the
high-throughput research of miRNA in model as well as in
nonmodel species. Thus, during the last decade some studies
were undertaken to investigate tissue-specific miRNAs as well
as different developmental stages (2, 6, 7, 12, 89). Most studies
of teleosts have been performed in zebrafish (12, 21, 86, 88,
97). In other teleosts, e.g., in Atlantic salmon (5), rainbow trout
(69), medaka (Oryzias latipes) (52), and stickleback (Gaster-
osteus aculeatus) (44), miRNAs have been described indepen-
dently of tissue. Tissue-specific, i.e., muscle-specific, miRNA
expression is shown for, e.g., tilapia and zebra finch (57, 93).
The present study uses Illumina technology to investigate
the transcriptome, comprising mRNA and miRNA transcripts,
of the European seabass during seven early developmental
stages (from morula to 24 h after hatching). We assessed
paralogous genes as well as differentially spliced transcripts.
Expression profiles of mRNA in each developmental stage of the
European seabass are depicted. The study of miRNA focuses on
the identification of miRNAs in the European seabass that have
already been described in other species (new miRNA) and their
expression. Finally first approaches of regulatory control during
development via miRNAs are carried out.
MATERIALS AND METHODS
All procedures involving the handling and treatment of fish used
during this study were approved by the Hellenic Centre for Marine
Research Institutional Animal Care and Use Committee following the
three Rs (Replacement, Reduction, Refinement) guiding principles for
more ethical use of animals in testing, first described by Russell and
Burch in 1959 (EU Directive 2010/63). These principles are now
followed in many testing establishments worldwide prior to initiation
of experiments. The larvae were anesthetized with 100 200 mg/l
MS222 (tricaine methanesulfonate, Sigma-Aldrich) depending on fish
size.
Sampling. Eggs and larvae of seabass were collected in tanks of
the Institute of Marine Biology, Biotechnology and Aquaculture at
the Hellenic Centre for Marine Research in Crete, Greece. In total
we collected samples from seven developmental stages, compris-
ing the morula, 50% epiboly, late gastrula-organogenesis, organo-
genesis-embryo ( 23 of meridian), late organogenesis, hatching, and
24 h after hatching. Staging was performed following gilt-head sea
breams developmental stages (17). All samples were immersed in the
RNAlater (Ambion, Austin, TX) and transferred to a 80C ultralow
freezer until preparation of RNA.
mRNA and miRNA extraction. mRNA and miRNA were extracted
from all developmental stages sampled, with a Nucleospin miRNA kit
for isolation of small and mRNA (Macherey-Nagel, Duren, Germany)
according to the manufacturers instructions. We disrupted pools of
eggs and larvae with mortar and pestle in liquid nitrogen and homog-
enized them in lysis buffer by passing lysate through a 23-gauge (0.64
mm) needle five times. Quantity of RNA was estimated with Nano-
Drop ND-1000 spectrophotometer (NanoDrop Technologies, Wil-
mington, DE), and the quality was further evaluated by agarose (1%)
Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org
159
gel electrophoresis and Agilent 2100 Bioanalyzer using RNA Nano
Bioanalysis chip. All samples had an RNA integrity number value
between 8.9 and 9.9.
RNA sequencing. mRNA and miRNA of all developmental stages
was sequenced at Cornell University Core Laboratories Center using
Illumina HiSeq vs 2000. For mRNA paired-end libraries and for
miRNA single-end libraries were prepared and sequenced over two
Illumina lanes (38 Gb). Use of multiplex identifier tags allowed us
to distinguish among RNA of different stages.
Quality control and de novo assembly. Raw data were sorted by
developmental stage. Quality control was assessed with FastQC (ver-
sion 0.10.0; http://www.bioinformatics.babraham.ac.uk/projects/
fastqc). Low-quality reads were discarded by Trimmomatic software
(8). Sequences of all stages were assembled into a reference transcrip-
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tome using Trinity version 2012-06-08 (28).
Differential expression analysis. Reads of each developmental
stage were mapped to the reconstructed transcriptome with Bowtie
(47, 48), allowing a maximum of three mismatches for each read.
RSEM (version 1.2.3; Ref. 50) was used for quantification of read
abundances, and transcripts represented less than once per million
mappable reads were excluded from the following analysis. Differen-
tial expression between different stages was assessed with the R
Bioconductor package DESeq (3), and transcripts P 0.05 in their
expression were assigned as differentially expressed.
BLAST, annotation, and classification. The filtered sequences were
compared using BLAST tools (version 2.2.25; Ref. 1) to a nonredun-
dant protein database and a nonredundant nucleotide database using.
The results were analyzed with Blast2GO software (14). First, se-
quences were mapped to Gene Ontology (GO) annotations according
to the GO database. Thereafter, sequences that were successfully
mapped were annotated according to the following GO terms: cellular
component, molecular function, and biological process. To determine
putative paralogous genes, we undertook manual evaluation of the
alignment analysis to exclude the classification to different clusters
due to alternative splicing. Subsequently all putative paralogous genes
were mapped onto the published genome of the European seabass
(81), and BLAT searches (41) were performed against five teleost
species (zebrafish medaka, stickleback, tetraodon, and fugu).
Cluster analysis. Significantly differentially expressed transcripts
were partitioned according to their expression pattern by the k-means
clustering method in the R statistical package (version 3.0.2), with the
Hartigan-Wong algorithm (iteration number 200). The number of
centers was determined by the plot of the within-groups sum of
squares by number of clusters (32).
Venn diagram. A Venn diagram was constructed with the web
application jvenn provided for free and nonprofit use from Geno-
Tool Bioinformatics (http://bioinfo.genotoul.fr/jvenn) (4) from all the
differentially expressed genes in six stages and comparing them with
the first (morula) stage.
Enrichment analysis. Fischers exact test tool in Blast2GO was
used with term filter modes FDR and pval, term filter values: false
discovery rate (FDR) 0.05, FDR 0.01, and P 0.005, as well as
two-tailed test. Transcripts grouped into the first and the fourth cluster
after k-means partitioning were used as test datasets that were com-
pared with the annotated transcriptome (reference dataset).
miRNA analysis. All reads were trimmed with Trimmomatic soft-
ware (8), and a quality check was performed with FastQC (version
0.10.0; http://www.bioinformatics.babraham.ac.uk/projects/fastqc).
Trimmed sequences were clustered with the cdHIT (53) cluster program,
and poorly supported transcripts were filtered out for downstream anal-
ysis. Differential expression analysis was carried out with the R Biocon-
ductor package DESeq (3), and transcripts with P value 0.005 were
used for miRNA identification by National Center for Biotechnology
Information (NCBI) database search as well as miRBase search.
Putative targets were identified with the online version of RNAhybrid
(45) and the miRanda web tool (19). Prediction was proceeded with
the default parameters and an energy threshold of mfe 20 Kcal.
160 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax
Network analysis. A module network was created by the LeMoNe
software (61) from highly significant (P value 0.005) differentially
expressed transcripts detected between all stages under study and
expression data from the 42 known miRNA. The normalized log2-fold
values were used as input of transcript expression, and the 42 known
miRNAs were used as potential regulators. The output of the software
presents a set of clusters of coexpressed transcripts and one or more
assigned regulator(s) of particular weight. Cytoscape (version 2.7.0)
(76) was used for the visualization of the clusters and their correlation
with particular regulators and with each other.
RESULTS
Transcriptome sequencing of early developmental stages of
seabass. RNA Illumina sequencing of European seabass seven
developmental stages resulted in 149,642,793 of 100 bp paired-
end reads in total. Raw sequence data have been deposited in
NCBIs Short Read Archive database under the accession
numbers SRX796243 (morula), SRX796245 (50% epiboly),
SRX796246 (late gastrula-organogenesis), SRX796264 (or-
ganogenesis-embryo 23 of meridian), SRX796265 (late
organogenesis), SRX796276 (hatching), and SRX796587 (24 h
after hatching). Trimming of low-quality reads and adaptors
yielded 129,467,381 paired-end reads of high quality, which
were used for the assembly analysis (Table 1). De novo
assembly of the 129,467,381 reads resulted in 201,130 tran-
scripts of an average length of 1,472 bp. The filtering process
led to a final reference transcriptome of 69,794 transcripts,
which was used for downstream analysis (Table 2).
Gene identification and annotation. More than half of the
transcripts, 40,898 (58.6%), had a significant BLASTX simi-
larity matches with sequences deposited in the NCBI nonre-
dundant database (nonredundant GenBank CDS translations
RefSeq a PDP SwitProt PIR PRF). Almost 28,000 clusters plot (Fig. 1A). Further discriminant analysis showed
transcripts had significant similarity with sequences of two fish
species belonging to Perciformes: Maylandia zebra and Oreo-
chromis niloticus. Among the retrieved BLAST matches a set
of transcripts showed similar or even the same BLAST
matches indicating either different splice forms or paralogous
genes. A subset was further examined by a comparative map-
ping approach. Table 3 shows putative paralogous genes, their
genome location, as well as their homolog group in five teleost
species.
Blast2GO successfully mapped 20,991 sequences and gene
ontology (GO) terms were assigned to 16,459 transcripts.
Under the category biological process 3,858 transcripts
(7.24%) were classified to the GO term developmental pro-
cesses. Other GO child terms of biological process are cellular
process (20.21%), metabolic process (15.93%), single-organ-
ism process (14.74%), biological regulation (9.57%), multicel-
Table 1. Sequencing and quality filtering summary
Stage Input Read Pairs Both Ends Survived
Morula 19,436,879 17,416,068 (89.60%)
50% epiboly 21,404,651 17,614,326 (82.29%)
Late gastrulation-organogenesis 21,101,942 17,831,063 (84.50%)
Organogenesis-embryo 3/4 of
the meridian 17,234,236 14,913,368 (86.53%)
Late organogenesis 22,929,410 21,179,558 (92.37%)
Hatching 20,954,714 18,412,965 (87.87%)
24 h after hatching 26,562,961 22,100,033 (83.20%)
Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org
Table 2. Summary of reads used for de novo assembly
before and after filtering
Assembly Statistics
Before Filtering After Filtering
Transcripts 201,130 69,794
Contig N50 3,015 2,769
Total bases 296,031,440 119,954,223
Min. sequence length, bp 201 201
Max. sequence length, bp 27,326 27,326
Average sequence length, bp 1,471 1,718
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lular organismal process (7.38%), response to stimulus
(6.48%), localization (5.08%), signaling (4.60%), cellular com-
ponent organization or biogenesis (4.42%), locomotion
(1.21%), immune system process (0.97%), growth (0.70%),
biological adhesion (0.65%), reproduction (0.56%) and multi-
organism process (0.27%).
Assessment of differential gene expression. Transcripts with
P value 0.05 were assigned as differentially expressed.
The number of differentially expressed genes comparing
each pair of condition is given in Table 4. Thus after
pairwise comparison, 18,140 unique transcripts were found
to be significantly differentially expressed and were used for
downstream analysis. Subsequently those transcripts found
to be differentially expressed at least in one of the stages
were subjected to k-means clustering. Cluster methods are
widely used for categorizing genes according to their ex-
pression patterns. k-means clustering partitioned signifi-
cantly differentially expressed transcripts into four clusters
determined from the within sum of squares by number of
that 98.8% of original grouped cases are correctly classified.
Gene expression profiles of each cluster are illustrated as
heat maps in Fig. 1B. Each row of the heat map represents
the pattern of one transcripts expression, while columns
represent the developmental stage. Cluster 1 contained
2,939 transcripts, while cluster 2, cluster 3, and cluster 4
were composed of 842; 12,968; and 1,391 transcripts, respec-
tively. Enrichment analysis of cluster 1 and cluster 4 is shown in
Fig. 2. The majority of GO terms in cluster 1 (FDR 0.05) are
related to developmental process. In cluster 4 the analysis with
term mode FDR 0.05 and FDR 0.1 resulted in four and
five GO terms, respectively. To retrieve a broader range of GO
terms in this cluster we applied a term mode value of P
0.005, resulting in GO terms mainly involved in regulation of
gene expression and transcription. Additionally, differentially
expressed genes of all seven stages studied were submitted to
principal component analysis (PCA) shown in Fig. 3. The gene
expression values of the stages as shown in the plot reflect the
successiveness of the developmental stages. The first studied st-
age morula is noticeably separated from the remaining six stages.
The last two stages, hatching and 24 h after hatching, are also
grouped more closely together and further away from the other
four stages. To extract stage-specific transcripts for the later
stages, we constructed a Venn diagram using the morula stage as
the reference stage. Therefore, differential expressed tran-
scripts between morula and one of the remaining six stages
were used, resulting in a total of 11,486 transcripts and repre-
senting 4,930 unique transcripts. The number of transcripts that
 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax 161
Table 3. Transcripts considered candidate paralogs in seabass after mapping to different chromosomes or gene groups of
other fish species
TranscriptID BLAST Match Accession No. D. labrax D. rerio O. latipes G. aculeatus T. nigroviridis T. rubripes

comp48446_c1_seq3, myozenin-2 like XP_008284262 LG3 n/a 22 VII Un 8

comp48446_c1_seq5
comp50487_c0_seq1, myozenin-2-like XP_008284938 LG7 1 scf461 IX Un 17
comp50487_c0_seq2
comp44838_c0_seq2 junctional adhesion molecule XP_003452956 LG15 n/a scf2132 XVI 2 1
b-like
comp50898_c0_seq3 junctional adhesion molecule XP_003440998 LG24 n/a 21 I 3 8
b-like
comp53857_c0_seq1 hepatocyte nuclear factor 4 ACO56245 LG2225 23 7 XII 9 HE591758
alpha
comp51024_c0_seq1 hepatocyte nuclear factor XP_004543170 LG5 n/a 7 II n/a n/a

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4-beta-like isoform x2
comp57791_c14_seq5 orthodenticle 2 (otx2) XP_005916803.1 LG12 17 22 XV 10 2
protein
comp50517_c0_seq3 orthodenticle 2 (otx2) XP_003453097 LG17 17 22 XVIII 14 16
protein
comp55416_c0_seq10 homeobox protein XP_003456262 LG15 1 Uc 256 XVI Un 1
engrailed-1-like
comp55416_c0_seq3 homeobox protein XP_003440904 LG24 1 2 I Un 8
engrailed-1-like
comp55416_c0_seq7 homeobox protein engrailed XP_006795130 LG1821 7 20 XXI 6 10
2a like
comp55416_c0_seq5 homeobox protein engrailed XP_003459215 LG10 7 17 III Un 22
2b like
comp50528_c0_seq2, homeobox protein XP_005929900 LG19 21 9 XIV 4 6
comp50528_c0_seq4 orthopedia B-like
comp50528_c0_seq3 homeobox protein XP_003451102 LG20 21 9 XIII 12 21
orthopedia B-like
comp57506_c7_seq1, transcritpion factor SOX-6- XP_004564551 LG6 7 6 XIX 13 9
comp57506_c7_seq4 like isoform
comp57506_c7_seq2, Transcritpion factor SOX-6- XP_004564551 LG6 7 6 XIX 13 9
comp57506_c7_seq7 like isoform
comp26500_c0_seq1, transcritpion factor SOX-6- XP_004543251 LG5 7 3 II 5 13
comp49142_c0_seq2 like isoform
comp47884_c0_seq2 diencephalon mesencephalon XP_003975776 LG10 2 17 III 15 22
homeobox protein 1-a-like
comp47884_c2_seq1, diencephalon mesencephalon XP_004570628 LG10 6 17 III 15 22
comp47884_c2_seq2 homeobox protein 1-a-like
comp56751_c9_seq2 diencephalon mesencephalon XP_004553852 LG4 n/a 4 VIII 1 HE591782
homeobox protein 1-like
comp51561_c0_seq5, serpin h1-like XP_003441717 LG13 15 13 I n/a 11
comp51561_c0_seq9,
comp51561_c0_seq20
comp54952_c0_seq3 serpin h1-like XP_004561658 LG14 10 13 VII n/a 11
comp56596_c1_seq8, serpin h1-like XP_003457702 LG3 n/a 18 VII Un 8
comp56596_c1_seq15
comp26234_c0_seq1 nodal homolog XP_004544784 LG20 n/a 9 XIII 12 21
comp38539_c0_seq1 nodal homolog XP_003965673 LG19 21 12 XIV 4 6
comp47939_c0_seq2 nodal homolog 2-a-like XP_003961216 LG1B n/a 19 V 2_random 1
comp52627_c0_seq6 histone-lysine n- XP_004542769 LG10 24 17 III 15 HE591741
methyltransferase mll3-
like isoform x7
comp54317_c2_seq1, histone-lysine n- XP_004542766 LG10 2 17 III 15 HE591741
comp54317_c2_seq3, methyltransferase mll3-
comp54317_c2_seq7, like isoform x4
comp54317_c2_seq9
comp60664_c0_seq1 histone-lysine n- XP_004570816 UN n/a 20 Un 4 10
methyltransferase
mll3-like
scf, scaffold; Un, not random; n/a, not available.

are either shared between two or more particular sets of
comparisons or unique in just one set of comparison is shown
in the Venn diagram (Fig. 4). In total 271 differentially ex-
pressed transcripts were found to be in common in all six sets
of comparisons. The first comparison (morula and 50% epi-
boly) included 576 unique transcripts, while the second (mo-
rula and late gastrulation-organogenesis), third (morula and
organogenesis-embryo of meridian), fourth (morula and
late gastrulation), fifth (morula and stage of hatching), and
sixth comparison (morula and 24 h after hatching stage) com-

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162 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax

Table 4. Double entry table with numbers of differentially expressed genes (P value 0.05) comparing each pair of
condition
Late Organogenesis-Embryo Late 24 h After
Morula 50 % Epiboly Gastrulation-Organogenesis 3/4 of Meridian Organogenesis Hatching Hatching

Morula 2,008 2,172 1,952 1,960 1,716 1,678

50 % epiboly 3,530 3,571 3,658 3,445 3,075
Late gastrulation-organogenesis 4,328 4,252 4,140 3,593
Organogenesis-embryo 3/4 of 4,350 4,199 3,687
meridian
Late organogeneisis 4,629 4,135
Hatching 4,472
24 h after hatching

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prised 400, 235, 230, 243, and 434 unique transcripts, respec-
tively.
miRNA identification during seabass early developmental
stages. Illumina sequencing of miRNA of European seabass
early developmental stages produced 58,216,215 reads of an
average length of 50 bp. Quality trimming and clustering
decreased the number of unique reads to 745,139. Differential
expression analysis further decreased the amount of miRNA
reads to 1,928. In the present study we aimed to identify new
miRNA (miRNAs described for other species than the Euro-
pean seabass) involved in European seabass development.
Thus, after annotation a total of 42 miRNAs (Supplemental
File 1) were identified, showing differential expression during
the stages studied.1 The expression pattern of these 42
miRNAs is illustrated by the heat map in Fig. 5.
1
The online version of this article contains supplemental material.
Network analysis and miRNA target site prediction. Four-
teen out of 42 known miRNAs were correlated with 173
clusters of a total of 7,740 highly differentially expressed
mRNA transcripts (Supplemental File 4, Fig. 6). LeMoNe
software combined transcripts with common expression pat-
terns in all pair-wise stage comparisons, to create clusters.
Afterward, clusters were associated with the expression pattern
of one or more of the 14 miRNA that were either compatible
or opposite, indicating an indirect or direct regulation of
mRNA expression, respectively. Six of the miRNAs that seem
to have direct regulation of clusters were tested with RNAhy-
brid software (45) and the MiRanda v3.3a software (19)
(Supplemental File 2). The software tests the possible hybrid-
ization of miRNA with a corresponding mRNA, providing a
value for the energy needed. Transcripts assigned to the GO
term developmental process and with a minimum free energy
(mfe) of 25 are shown in Fig. 7.

Fig. 1. k-means clustering of all differentially expressed transcripts. A: the optimal number of clusters were obtained by printing the cluster centroids obtained
with k-means function using R. Number of clusters are given on the x-axes, and the sums of squares within groups on the y-axes. The plot determines 4 clusters
as the best solution to partition the differentially expressed transcripts (red cycle). B: the 4 clusters determined in A are illustrated by heat map imaging (black
arrow). Cluster 1 comprises 2,939 transcripts, cluster 2 842, cluster 3 12,968, and cluster 4 1,391. Stages are indicated under each column of clusters. Each row
represents the expression of 1 transcript where shades of red represent upregulation and shades of green represents downregulation. Heat maps were drawn on
the normalized counts of the transcripts of each developmental stage.

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Fig. 2. Enrichment analysis. A: enrichment analysis with the set of transcripts grouped into the 1st (transcripts downregulated at the morula stage) cluster using
the total of annotated transcriptome as reference set and false discovery rate (FDR) 0.05. Gene Ontology (GO) terms related to biological process are shown
with a red frame. B: enrichment analysis of the transcripts grouped into the 4th cluster (transcripts upregulated at the morula stage) using the annotated
transcriptome as reference set and P 0.005. GO terms found to be enriched with FDR 0.1 and FDR 0.05 are marked * or **, respectively. Red and green
frames include GO terms related to biological process and gene expression. C: enrichment analysis with different term filter modes and values produced different
number of GO terms. Symbol marks the cases that are shown in A and B.

DISCUSSION comprising mRNA as well as miRNA of the European seabass

The onset of next-generation sequencing technologies has
enabled the research community to retrieve genomic and tran-
scriptomic information for any organism of interest (30, 58, 63,
68, 85). The main characteristics of these platforms is their
speed as well as the assessment of high amount of data at low
cost. These advantages clearly have advanced clinical, envi-
ronmental, as well as biotechnological research. Nevertheless,
next-generation sequencing also comprises error rates as well
as pitfalls during analysis and data interpretation. Several RNA
sequencing data have been produced by applying GS454 FLX
pyrosequencing, the first high-throughput next-generation se-
quencing platform. The advantages of GS454 FLX certainly
are the capability to produce long reads (up to 1,000 bp with
GSFLX technology) facilitating the following assembly pro-
cess. However, for robust gene expression results high-
throughput data are necessary, and thus a number of GSFLX
runs have to be executed (25), which in turn results in high
consumable cost. Alternatively, 3=-untranslated region (UTR)
tagged cDNAs for library construction may decrease the
amount of transcripts needed for robust differential gene ex-
pression studies (71). Today mainly the Illumina platform is
applied due to its high throughput, low cost, and low sequenc-
ing error rates (54). In the present study, the transcriptome
was sequenced using paired-end reads and single-end reads,
respectively. Each stage was individually tagged for down-
stream expression analysis. For mRNA sequencing the gener-
ation of paired-end reads resolves assembly problems due to
repetitive regions. A reference transcriptome was constructed,
resulting in 69,794 contigs with an average length of 1,718 bp
and N50 of 2,769 bp (Table 2). The Illumina technology of
sequencing paired-end reads facilitates the assembly, and the
use of a combined assembly and mapping strategy results in a
sound and accurate reference transcriptome that is also strongly
supported by the obtained results of sequence average length
and N50 length. We assessed transcript expression profiles by
mapping paired-end reads against the reconstructed assembly.
To obtain a robust transcript dataset we applied stringent
filters, resulting in 69,794 transcripts. It has to be noted that the
obtained transcripts do not reflect the gene number of an
organism. Next-generation sequencing technologies provide a
number of different transcript variants. Thus, BLAST searches
may result in the same annotation and/or they result in matches
entitled transcript variant X1, transcript variant X2, with-
out any further information whether transcript variants are
paralogous genes or alternative spliced transcript. It has been
shown that paralogous genes arose due to whole genome

Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org

164 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax
Fig. 3. Principal Component Analysis (PCA) of the expression profiles of the
studied developmental stages. The 7 developmental stages are projected in the
2-dimensional plane by the expression profile of all transcripts differentially
expressed. Red ellipses group together the stages that were neighboring both
after PCA and in the developmental process.
duplication events, and some may have altered or acquired
novel functions (sub- or neofunctionalization). However, de-
tection of differential expression of transcript variants alone is
not sufficient to identify putative paralogs as differentially
spliced transcripts may also show stage-specific expression,
such as the ATP-binding cassette subfamily D member 4-like
Fig. 4. Venn diagram. Venn diagram show-
ing the relation between the 4,930 significant
differentially expressed transcripts (P
0.05) after the comparison of morula stage
with the other 6 stages (50% epiboly, late
gastrula-organogenesis, organogenesis-em-
bryo 23 of meridian, late organogenesis,
hatching, and 24 h after hatching). Total
number of transcripts differentially ex-
pressed in each stage comparison is given
next to the Venn diagram.
Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org
protein in the present study, where one transcript is signifi-
cantly upregulated at the morula and the 50% epiboly stage,
whereas the other two are upregulated at the later stage with the
second having a peak at gastrulation and hatching and the third
with an expression peak at the organogenesis-embryo of
the meridian and at the organogenesis stage. Consequently, to
distinguish paralogous genes from simple transcript variants in
the present study we pursued manual evaluation of transcript
alignments as well as a comparative mapping approach. Tran-
scripts mapping on different linkage groups of the European
seabass or on different chromosomes in any of the five model
species were considered as putative paralogous genes (Table 3).
Linkage groups as well as chromosomes found were homologous
Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.6 on August 16, 2017
groups as described in comparative mapping studies in seabass
but also in the gilt-head sea bream compared with the five model
fish species (29, 75). Examples of putative paralogs involved in
developmental processes, found in the present study, are ortho-
denticle 2 (otx2) (59), nodal (13a), the cell surface receptors
junctional adhesion molecule b-like (jamb) (66), diencephalon/
mesencephalon homeobox 1, and orthopedia (otp). The latter
three genes were expressed especially during organogenesis of
seabass embryos. It has been shown that both paralogs of
diencephalon/mesencephalon homeobox play an important
role in brain development of zebrafish embryos (91) and that
both paralogs of otp are essential for the development of a
subset of dopaminergic neurons (20). Jamb on the other hand
has been shown to play a pivotal role along with jamc during
somitogenesis in fusion of myocyte precursors (66). For further
downstream analysis only transcripts significantly differen-
tially expressed in at least one of the studied stages were used.
The in total 18,140 differentially expressed transcript were
clustered into four groups with specific expression patterns.
Among the four clusters, clusters 1 and 4 are the most appeal-
 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax
Fig. 5. Heat map imaging of microRNAs (miRNAs). Heat map of the 42
identified miRNAs throughout the 7 developmental stages of the European
seabass. Stages are indicated under each column. Each row represents the
expression of 1 miRNA, where red represents upregulation and green repre-
sents downregulation. Heat map was drawn on the normalized counts of
miRNAs of each developmental stage.
ing ones, comprising transcripts being either down- (cluster 1)
or up- (cluster 4) regulated at the morula stage (Fig. 1B).
Morulation implements the repeated cleavage resulting in the
morula stage, a bundle of cohering and sticky blastomers
loosely arranged. It has been shown that embryonic stages
Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org
165
before gastrulation are more vulnerable (83a), and as early as
three decades ago in medaka (O. latipes) stage sensitivity and
decrease of mortality after the morula stage was shown (60). It
is also known that the early embryonic processes are carried
out by maternal factors and the zygotic genome is activated
only at the midblastula transition. Enrichment analysis using
transcripts belonging to cluster 1 against the total number of
annotated transcripts showed a significant accumulation of
GO terms involved in the developmental process (Fig. 2A).
Transcripts in cluster 1 were mainly downregulated during
the morula stage, which shows that at this stage transcripts
important for the development of the organism are not yet
activated. Transcripts found to be mainly upregulated during
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the morula stage (cluster 4) were classified in GO terms with
regulative functions (Fig. 2B). In addition, GO terms pole
plasm as well as germ plasm were enriched in cluster 4.
Both terms belong to the ontology of cellular components
and encode for differentiated cytoplasm, which is associated
with a pole of an oocyte, egg, or early embryo (39).
Apparently, the morula stage differentiates itself clearly
form the other six stages. This is shown not only by cluster
analysis as discussed before but also by a PCA plot (Fig. 3),
where transcripts differentially expressed during the morula
stage are isolated from the other six stages. In this line the
morula stage was used as a reference stage to extract
stage-specific differentially expressed transcripts (in total
11,486 transcripts, representing 4,930 unique transcripts)
illustrated by a Venn diagram in Fig. 4. Out of the unique
transcripts, only 5.5% were in common for all six stages,
while transcripts found only at the stages late organogene-
sis, hatching, and l of meridian were between 4.6 and 4.9%.
Transcripts solely detected at the stages late gastrulation and
hatching after 24 h were 8.1 and 8.8%, respectively. The
highest amount of transcripts (11.7%) uniquely found were
at 50% epiboly (Fig. 4). The largest amount of transcripts
significantly regulated only at the 50% epiboly stage was
also found in zebrafish (83). The high amount of unique
transcripts at 50% epiboly detected in zebrafish as well as in
the present study highlights the importance of this stage and
pinpoints an enhanced transcriptional activity.
Besides differential expression of paralogous genes and/or
developmentally specific genes, the importance of miRNAs
during development has been shown in several studies. There-
fore, a first approach to describe new miRNAs for the Euro-
pean seabass was performed in the present study. This is to our
knowledge the first report about miRNAs in the early devel-
opment of the European seabass. miRNAs are small noncoding
RNA regulating gene expression mainly in a suppressive way
at the posttranscriptional level (33). In teleosts, 1,044 of mature
miRNAs have been identified (reviewed in Ref. 6), and it has
been shown that they play an important role in several biolog-
ical process including development. The developmental pro-
files of miRNAs have been reported in zebrafish (64), fugu
Tetraodon (6), medaka (52), Asian seabass (92), Atlantic cod
(46), Japanese flounder (23), and Atlantic halibut (7). The
identification of novel miRNAs, thus not yet described in any
other species, has not been aimed in the present work, instead
miRNAs characterized in other species but not yet described in
the European seabass were detected. In total, 42 miRNAs have
166 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax

Fig. 6. Network analysis. Representation of the net-

work inferred by the LeMoNe algorithm showing the
interaction between differential expressed transcripts

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during developmental stages and the 14 of the 42
identified miRs of European seabass. Clusters of co-
expressed transcripts are shown as black circles, and
miRNAs are shown as red triangles.

been described in the present work, and their stage-specific not critical for the earliest developmental processes (88, 89).
expression is illustrated in Fig. 5. In the present work clearly The low expression of miRNA found in the earlier stages in the
most of the miRNAs are highly expressed at the late organo- present study may also suggest that miRNAs are not critical for
genesis stage. It has been shown that in zebrafish miRNAs are the first developmental processes in European seabass. It has to

mfe
miR Transcript ID Blast match RNAhybrid
(kcal/mol)
miR21

comp54330_c1_seq2 XP_003438118 -29.6*

H+ V0 subunit b
miR30a

comp58836_c0_seq19
XP_003979245 -25.6*
major vault protein
miR221-2

comp53312_c0_seq3 XP_003449875 -30.6*

delta-like protein B-like

comp59023_c0_seq23
XP_003455166 -26.2*
protein strawberry notch homolog 1

comp57660_c1_seq1 XP_003965670 -25.9

fukutin-like
miR455b

comp57361_c0_seq2 XP_003454480 -25.7

transcription factor Sp8-like

comp55922__c0_seq3
thyroid hormone receptor associated CAC44632 -25.5
protein complex component
TRAP230/KIAA0192

Fig. 7. Transcripts assigned to the GO term developmental process were tested for hybridization with corresponding miRNA. *Positive hybridization with both
RNAhybrid software (minimum free energy 25.0) and miRanda software.

Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org

 DYNAMISM IN GENE EXPRESSION OF Dicentrarchus labrax
be noted that of the 42 identified miRNAs, some of the
classical miRNAs like lin-4, let-7, lsy-6, and miR-273 (89)
have not been detected. The two miRNA lin-4 and let-7 are the
best studied ones and regulate developmental timing in Cae-
norhabditis elegans (49, 90), whereas let-7 and lsy-6 are
known to regulate left/right asymmetry in worms (11). A
possible explanation could be that those miRNAs are highly
expressed even earlier in development. On the other hand,
other classical miRNAs involved in important development
processes have been found, such as miR-196. miR-196 is
involved in developmental patterning in mouse, and its target
has been identified to be Hoxb8 (96). Other important miRNAs
identified later on in various teleost fish species are also found
to be differentially expressed in the present work. Ramachan-
dra et al. (67) for instance described the function of miR-21,
23a, 26a, 200b, and 455 during the early embryo, whereas for
example miR-1 has been shown in zebrafish (77), common
carp (Cyprinus carpio) (95), and blunt snout bream (Megalo-
brama amblycephala) to play a distinct role in muscles. Fur-
thermore, Yan et al. (94) showed the involvement of miR-203b
in the muscle development of the Nile tilapia (Astatotilapia
burtoni). The remainder of the discussion will be focused on
the interaction of the identified miRNA with the detected
differentially expressed genes (Supplemental File 4). We
achieved this by performing network analysis based on the
expression data. Network analysis is mostly performed in
human cancer research to reveal regulators, mainly miRNAs,
responsible for cancer outbreak. Molecular networks comprise
nodes and modules, where the nodes are in the present study
miRNAs and the modules coexpressed transcripts. The module
network is a probabilistic model based on probabilistic graph-
ical models and Bayesian networks and aims to identify regu-
latory modules from gene expression data. The procedure
identifies modules of co-regulated genes and their regulators.
Here in total 14 miRNAs were identified as nodes linked to
modules based on expression patterns using the LeMoNe
software package (Fig. 6). One of the nodes presents miR-1
regulating modules, which were revealed to regulate transcripts
involved in the proper function of muscle as well as muscle
development (12, 24, 95). As network analysis does not dis-
tinguish whether regulators repress or enhance possible tran-
scripts it has to be taken into account that not only direct
interaction of miRNAs is illustrated here but also indirect
involvement. Indirect involvement for example is seen in the
case of miR-1, where transcripts clustered in the modules are
upregulated in concordance with the expression of miR-1
(expression data not shown). Direct regulation was found for
22 modules linked to seven miRNAs. The final step to detect
and evaluate interaction between miRNAs and mRNAs of the
present study was to allocate possible targets of the miRNAs.
Therefore, 3=-UTRs of the sequences belonging to the clusters
found to be regulated by one of the miRNAs were searched by
two software packages for putative target sites. Figure 7 shows
the most significant ones comprising miRNA, target site, as
well as structure and mfe values. All miRNAs shown in Fig. 7
have been described to be involved in developmental processes
(89). One of the miRNAs, miR-30a, is linked to several
modules, including one module with putative direct regulation
of miR-196a. Both miRNAs have been previously described to
be involved in development with the former one in early
muscle development (42) and the latter one in developmental
Physiol Genomics doi:10.1152/physiolgenomics.00001.2015 www.physiolgenomics.org
167
patterning (96). In the present study, two putative target sites in
the major vault protein (mvp) and in the tata box binding
protein 1 (tbp1) (mfe 25.6 and mfe 22.7, respectively)
were identified for miR-30a (Fig. 7 and Supplemental File 2).
The former has been shown to be induced during regeneration
(98) and is considered to belong to the highly conserved
ribonucleoprotein particles. It is suggested that mvp might be
involved in the regulation of important cell signaling pathways
including the PI3K/Akt and the MAPK pathways (78). Inter-
estingly, miR-30a is also known to be required for hepatobili-
ary development (31), and MAPK, as well as Akt, is activated
in extrahepatic biliary tract carcinoma (35), a malfunction of
the extrahepatic biliary tract. Nevertheless, further experimen-
Downloaded from http://physiolgenomics.physiology.org/ by 10.220.33.6 on August 16, 2017
tal studies have to be carried out to confirm the predicted target
sites of the present work.
Conclusion
This study reports the characterization of the European
seabass (D. labrax) transcriptome during early development
(from morula to hatching). mRNAs comprising paralogous
genes and differentially spliced transcripts are described, and
their expression patterns during development assessed. In ad-
dition, we identified 42 new miRNA, as well as their expres-
sion during the seven early developmental stages. Putative
direct and indirect regulatory function of the miRNA studied in
the present work were determined by network analysis identi-
fying putative miRNA/mRNA interactions as well as putative
target sites that have not been described before.
ACKNOWLEDGMENTS
We thank the Department of Aquaculture for providing fish eggs and
assistance in sampling and the Informatics group for computational support.
We also acknowledge the Gnopole-Plateforme bio-informatique Centre
INRA de Toulouse-Unit de BIA for providing Venn diagram calculation for
six groups after request.
GRANTS
Financial support for this study has been provided by the Ministry of
Education and Religious Affairs, under the Call ARISTEIA I of the National
Strategic Reference Framework 20072013 (ANnOTATE), cofunded by the
EU and the Hellenic Republic through the European Social Fund.
DISCLOSURES
No conflicts of interest, financial or otherwise, are declared by the author(s).
AUTHOR CONTRIBUTIONS
Author contributions: E.K. and J.X. performed experiments; E.K. and E.S.
analyzed data; E.K. and E.S. prepared figures; E.K. and E.S. drafted manu-
script; E.K., J.X., E.A., C.S.T., and E.S. approved final version of manuscript;
E.A., C.S.T., and E.S. edited and revised manuscript; E.S. conception and
design of research; E.S. interpreted results of experiments.
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