Tài liệu Báo cáo khoa học: Identification and characterization of the transcription factors involved in T-cell development, t-bet, stat6 and foxp3, within the zebrafish, Danio rerio docx

Identification and characterization of the transcription
factors involved in T-cell development, t-bet, stat6 and
foxp3, within the zebrafish, Danio rerio
Suman Mitra, Ayham Alnabulsi, Chris J. Secombes and Steve Bird
Scottish Fish Immunology Research Centre, School of Biological Sciences, University of Aberdeen, UK
Introduction
Naive CD4
+
T-cells, on antigenic stimulation, become
activated, expand and differentiate into different effec-
tor subsets called T-helper (Th) cells. The differentia-
tion of naive T-cells into Th effector cells depends on
a variety of stimuli, such as antigen nature, antigen
dose and the strength and duration of signals through
the T-cell receptor (TCR)–CD3 complex, as well as the
cytokine microenvironment which activates the cellular
signalling pathways [1]. These Th cell subsets are cru-
cial for the induction of the most appropriate immune
response towards a particular pathogen. In mammals,
three types of CD4
+
Th effector cell populations exist,
Th1, Th2 and Th17, characterized by their cytokine
repertoire and how they regulate B-cell and T-cell
Keywords
adaptive immunity; fish immunology; T-cells;
transcription factors; zebrafish
Correspondence
S. Bird, Scottish Fish Immunology Research
Centre, School of Biological Sciences,
Zoology Building, University of Aberdeen,

Aberdeen AB24 2TZ, UK
Fax: +44 1224 272396
Tel: +44 1224 272881
E-mail:
(Received 25 August 2009, revised
16 October 2009, accepted 27 October
2009)
doi:10.1111/j.1742-4658.2009.07460.x
The discovery of cytokines expressed by T-helper 1 (Th1), Th2, Th17 and
T-regulatory (T
reg
) cells has prompted speculation that these types of
responses may exist in fish, arising early in vertebrate evolution. In this
investigation, we cloned three zebrafish transcription factors, T-box
expressed in T cells (t-bet), signal transducer and activator of transcription
6(stat6) and fork-head box p3 (foxp3), in which two transcripts are pres-
ent, that are important in the development of a number of these cell types.
They were found within the zebrafish genome, using a synteny approach in
the case of t-bet and foxp3. Multiple alignments of zebrafish t-bet, stat6
and foxp3 amino acids with known vertebrate homologues revealed regions
of high conservation, subsequently identified to be protein domains impor-
tant in the functioning of these transcription factors. The gene organiza-
tions of zebrafish t-bet and foxp3 were identical to those of the human
genes, with the second foxp3 transcript lacking exons 5, 6, 7 and 8. Zebra-
fish stat6 (21 exons and 20 introns) was slightly different from the human
gene, which contained 22 exons and 21 introns. Immunostimulation of
zebrafish head kidney and spleen cells with phytohaemagglutinin, lipo-
polysaccharide or Poly I:C, showed a correlation between the expression of
t-bet, stat6 and foxp3 with other genes involved in Th and T
reg

responses
using quantitative PCR. These transcription factors, together with many of
the cytokines that are expressed by different T-cell subtypes, will aid future
investigations into the Th and T
reg
cell types that exist in teleosts.
Abbreviations
foxp3 ⁄ Foxp3, fork-head box p3; IFN-c, interferon-c; IL, interleukin; LPS, lipopolysaccharide; OSBPL7, oxysterol-binding protein-like 7; PHA,
phytohaemagglutinin; PPP1R3F, protein phosphatase 1, regulatory (inhibitor) subunit 3F; RACE, rapid amplification of cDNA ends;
stat6 ⁄ STAT6, signal transducer and activator of transcription 6; t-bet ⁄ T-bet, T-box expressed in T cells; TCR, T-cell receptor; TGF-b,
transforming growth factor-b; Th, T-helper; T
reg
, T-regulatory.
128 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
responses [2]. Th1 cells produce interferon-c (IFN-c)
and lymphotoxin, activating cell-mediated immunity
and providing protection against intracellular patho-
gens and viruses. Th2 cells secrete interleukin-4 (IL-4),
IL-13 and IL-25 (also known as IL-17E), which are
important in the generation of the correct class of
antibodies by B-cells, and for the elimination of
extracellular pathogens, such as helminths and other
extracellular parasites [2]. Th17 is the most recently
identified Th cell subset and secretes pro-inflammatory
cytokines, such as IL-17A, IL-17F, IL-21 and IL-22
[3,4]. Th17 cells play an important role in host defence
against extracellular pathogens, in particular extra-
cellular bacteria, which are not efficiently cleared by
Th1- and Th2-type immunity [5]. Finally, in addition
to Th cells, there is a population of CD4

+
T-cells that
is involved in the regulation of Th responses via the
secretion of cytokines, called T-regulatory (T
reg
) cells,
which help to inhibit harmful immunopathological
responses directed against self- or foreign antigens
[6,7]. The majority of these cells constitutively express
the CD25 cell surface marker and secrete two powerful
anti-inflammatory cytokines: IL-10 and transforming
growth factor-b (TGF-b).
Whether a naive T-cell becomes a Th1, Th2, Th17
or T
reg
cell is influenced by the cytokines that are pro-
duced within the microenvironment, which, in turn,
influence transcription factors and key signalling path-
ways [8]. Th1 differentiation is initiated by coordinate
signalling through the TCR and cytokine receptors,
for cytokines such as type I and II IFNs or IL-27,
which are associated with STAT1 [9,10]. Activation of
STAT1 induces the transcription factor, T-box
expressed in T cells (T-bet), which is a master regula-
tor of Th1 differentiation [11]. T-bet potentiates the
expression of IFN-c, which, in turn, upregulates the
inducible chain of the IL-12 receptor (IL-12Rb2).
Binding of IL-12 to its receptor induces signalling
through STAT4, which further enhances IFN-c pro-
duction and induces the expression of IL-18Ra, allow-

ing the responsiveness of these now mature Th1 cells
to IL-18 [12]. Th2 differentiation is initiated by TCR
signalling, together with IL-4 receptor signalling via
signal transducer and activator of transcription 6
(STAT6). This, in turn, up-regulates the low-level
expression of GATA3, the master regulator of Th2 dif-
ferentiation [13]. GATA3 autoactivates its own expres-
sion, eventually enabling mature Th2 cells to express
the Th2 cytokine cluster, IL-4, IL-5 and IL-13, as a
result of epigenetic changes [14]. Th1 and Th2 cells
negatively regulate each other’s development. GATA3
suppresses STAT4 and the IL-12Rb2 chain expression,
factors which are critical to the Th1 pathway [15],
whereas IL-27 suppresses Th2 development [16].
Th17 differentiation is slightly more complex
because of differences between mice and humans [17].
In mice, Th17 differentiation is initiated by TCR
signalling, together with TGF-b1 and IL-6 receptor
signalling, which activates STAT3 and induces the
expression of the transcription factor retinoic acid-
related orphan receptor ct. IL-23 also activates STAT3
but, in addition, serves to maintain Th17 cells in vivo.
In contrast, human cells do not require TGF-b1, and
it is IL-1, IL-6 and IL-23 that promote human Th17
differentiation [17]. Lastly, T
reg
cells are crucial players
in the regulation⁄ suppression of each of the Th
responses and self-reactive T-cells. It is now known
that there is more than one subtype of T

reg
cells,
although the most important appear to be
CD4
+
CD25
+
Foxp3
+
T
reg
[18]. These cells are affected
by the transcription factor fork-head box p3 (Foxp3),
whose induction is initiated by TCR signalling,
together with TGF-b1 receptor signalling [19]. T
reg
suppressive activity is via IL-10 and TGF-b, although
it remains unclear whether these cytokines are
produced by CD4
+
CD25
+
Foxp3
+
T
reg
or whether
they induce the production of these cytokines from
another population of cells [20].
To date, our knowledge about the different types of

Th and T
reg
responses relates to studies performed in
mammals, especially mice and humans [12]. In fish,
there has been a considerable amount of work under-
taken on immunity over the last few decades, and a
large number of genes involved in immune responses
have been discovered. However, although we know a
lot about the innate and inflammatory immune
responses of fish [21], relatively little is known about
the lymphocyte subpopulations involved in the adap-
tive immune responses in fish, and whether Th subsets
exist. Speculation that Th1, Th2, Th17 and T
reg
responses may exist in fish, and arose early in verte-
brate evolution, has been prompted by the discovery
of many of the cytokines that are expressed by these
cell types [22,23]. However, it is important to note that
not all the cytokines known in mammals have been
found in fish, and it remains to be determined whether
the regulation of adaptive immunity in fish is similar
to that found in mammals, and if it is equally complex.
In addition, the key transcription factors involved in
driving the differentiation of the naive T-cell to Th1,
Th2, Th17 or T
reg
cells may exist in fish. In this inves-
tigation, we have identified, for the first time, t-bet and
stat6 in zebrafish and, for the first time in any fish spe-
cies, foxp3. Lastly, we carried out some preliminary

S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 129
expression analyses to investigate their role in the
immune responses of fish.
Results
Cloning and sequencing
For t-bet, stat6 and foxp3, three overlapping products
were obtained using PCR and specific primers, which
contained the complete cDNA sequence for each gene.
The zebrafish t-bet cDNA (EMBL accession no.
AM942761) consisted of a 36 bp 5¢-UTR, a 419 bp
3¢-UTR and a single open reading frame of 1830 bp,
giving a predicted 609 amino acid t-bet molecule. In
the 3¢-UTR, no obvious polyadenylation signal was
present. The stat6 cDNA transcript (EMBL accession
no. AM941850) consisted of a 135 bp 5¢-UTR, an
809 bp 3¢-UTR and a single open reading frame of
2277 bp, which translated into a predicted 758 amino
acid stat6 molecule. In the 3¢-UTR, two mRNA insta-
bility motifs (attta) were present, and again no obvious
polyadenylation signal was found. The foxp3 cDNA
transcript (EMBL accession no. FM881778) consisted
of a 100 bp 5¢-UTR, a 410 bp 3¢-UTR and a single
open reading frame of 1260 bp, which translated into
a predicted 419 amino acid foxp3 molecule. In the
3¢-UTR, four mRNA instability motifs (attta) were
present upstream of the polyadenylation signal. An
alternative transcript of foxp3 (Fig. 1) was also found
and was shown to be missing the region containing the
zinc-finger and leucine-zipper domain.

Multiple alignment of zebrafish t-bet, stat6 and foxp3
with other known T-bet, STAT6 and Foxp3 amino acid
sequences (Figs 2–4, respectively) revealed areas of
amino acid conservation throughout the vertebrates.
Significant homology was seen in the putative T-box
DNA-binding domain of t-bet, the STAT protein inter-
action domain, STAT protein all-alpha domain, STAT
protein DNA-binding domain and SH2 domain of
stat6, and the zinc-finger domain, leucine-zipper
domain and fork-head domain of foxp3. In addition,
for stat6 and foxp3, there were a few other conserved
features. Within the zebrafish stat6 sequence is an
important tyrosine residue (Tyr664), which was con-
served in all sequences. Within the foxp3 molecule,
some homology was found within the predicted
transcriptional repressor domains, with domain 2
containing a large number of proline residues. As with
other t-bet, stat6 and foxp3 molecules sequenced to
date, the zebrafish t-bet, stat6 and foxp3 peptides did
not possess a signal peptide, as predicted by SignalP
v1.1 (data not shown). Zebrafish t-bet had the highest
amino acid identity and similarity (Table 1) to Ginbuna
crucian carp t-bet (91.0% and 95.4%, respectively),
zebrafish stat6 to Tetraodon stat6 (52.9% and 71.5%,
respectively) and zebrafish foxp3 to mouse foxp3
(31.6% and 49.0%, respectively). Phylogenetic analysis
of t-bet, stat6 and foxp3 (Figs 5–7, respectively)
grouped t-bet, stat6 and foxp3 with their mammalian
homologues, all of which were strongly supported
statistically, when all known vertebrate T-box, STAT

family and Foxp family members were compared.
Fig. 1. Pairwise alignment of the full-length
Danio rerio foxp3 (ZFfoxp3) and an obtained
alternative transcript (ZFfoxp3b). The puta-
tive transcriptional repressor domains 1 and
2, fork-head (FKH), leucine-zipper and zinc-
finger domains are highlighted. The EMBL
accession number of the foxp3b alternative
transcript gene is FM881779.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
130 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
Fig. 2. Multiple alignment of the predicted Danio rerio t-bet (T-box21) with selected known vertebrate T-bet molecules. Identical (*) and sim-
ilar (: or.) residues identified by the
CLUSTALX program are indicated. The putative T-bet DNA-binding domain is highlighted. The EMBL acces-
sion numbers of the T-box21 genes are as follows: human, Q9UL17; mouse, Q9JKD8; Ginbuna crucian carp, AB290187; zebrafish,
AM942761.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 131
t-bet, stat6 and foxp3 gene organization and
chromosome synteny
Using the zebrafish t-bet, stat6 and foxp3 cDNA
sequences elucidated by PCR and the regions of the
zebrafish genome that contained these sequences,
chromosomes 8, 12 and 23, the gene organizations
were obtained (Fig. 8; t-bet GenBank accession no.
FN435332, stat6 GenBank accession no. FN435334,
foxp3 GenBank accession no. FN435333). t-bet was
Fig. 3. Multiple alignment of the predicted
Danio rerio stat6 with selected known
vertebrate STAT6 molecules. Identical (*)

and similar (: or.) residues identified by the
CLUSTALX program are indicated. The putative
STAT interaction, STAT all-alpha, STAT DNA-
binding and SH2 domains are highlighted.
Boxed is an important tyrosine residue
(Tyr664 in zebrafish). The EMBL accession
numbers of the STAT6 genes are as
follows: human, P42226; mouse, P52633;
zebrafish, AM941850.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
132 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
found to have six exons and five introns, stat6 was
found to have 21 exons and 20 introns, and foxp3
was found to have 13 exons and 12 introns. In the
genomic sequence, the intron splicing consensus
(GT ⁄ AG) is conserved at the 5¢ and 3¢ ends of the in-
trons. The gene organization was found to be similar
to that of human t-bet and foxp3 genes (Fig. 8), with
human stat6 having a slightly different gene organiza-
tion of 22 exons and 21 introns. Generally, the sizes
of the zebrafish t-bet, stat6 and foxp3 coding exons
matched well with the corresponding mammalian
exons (Fig. 8). Using the Genscan [24], fasta [25]
and blast [26] suite of programs, other genes were
discovered on zebrafish chromosomes 8, 12 and 23
around the discovered zebrafish t-bet, stat6 and foxp3
genes (Fig. 9). On comparison with the human gen-
ome, some degree of synteny was found between the
two organisms for the regions containing the t-bet
and foxp3 genes. Around t-bet, the genes TBK1-bind-

ing protein 1, oxysterol-binding protein-like 7 (OS-
BPL7) and mitochondrial ribosomal protein L10 were
found in the same order on zebrafish chromosome 12
and human chromosome 17 and, around foxp3, the
gene protein phosphatase 1, regulatory (inhibitor)
subunit 3F (PPP1R3F) was found in the same order
on zebrafish chromosome 8 and human chromosome
Fig. 4. Multiple alignment of the predicted
Danio rerio foxp3 with known Foxp3 mole-
cules. Identical (*) and similar (: or.) residues
identified by the
CLUSTALX program are indi-
cated. The putative transcriptional repressor
domains 1 and 2, fork-head (FKH),
leucine-zipper and zinc-finger domains are
highlighted. Proline residues within the
transcriptional repressor domains are
underlined. The EMBL accession numbers
of the Foxp3 genes are as follows: human,
Q9BZS1; mouse, Q99JB6; crab-eating
macaque, Q6U8D7; zebrafish, FM881778.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 133
X. For stat6, no synteny was found between this
locus on zebrafish chromosome 23 with the stat6
locus on human chromosome 12.
Quantification of expressed stat6, t-bet and
foxp3 genes in spleen or head kidney tissues
stimulated with immunostimulants (quantitative
real-time PCR)

Using RT-PCR, the constitutive expression of t-bet,
stat6 and foxp3 was observed in the spleen, kidney,
gill, gut, liver and skin tissue of healthy fish (data not
shown). After stimulation of kidney cells with a variety
of immunostimulants, the expression of t-bet, stat6
and foxp3, together with other selected zebrafish tran-
scription factors and cytokines, was compared using
quantitative PCR (Fig. 10). Stimulation of kidney cells
with phytohaemagglutinin (PHA) led to a significant
increase in il-4 and gata3 expression, stimulation with
lipopolysaccharide (LPS) led to a significant increase
in il-10, and stimulation with Poly I:C led to a signifi-
cant increase in ifn-c, mx and t-bet. Stimulation of
spleen cells with PHA led to a significant increase in
ifn-c, whereas stimulation with LPS led to a significant
increase in il-10 and foxp3, and stimulation with Poly
I:C led to a significant increase in mx and t-bet.
Up-regulation was observed for a number of other
genes investigated, but expression was not statistically
significant.
Discussion
This paper reports the isolation and sequencing of
three zebrafish transcription factors, which are known
to be important in T-cell subtype differentiation in
mammals. T-bet has already been sequenced within
bony fish, in the Ginbuna crucian carp [27], and
STAT6 in mandarin fish [28], whereas Foxp3 has been
characterized for the first time in fish. The availability
of sequenced fish genomes has allowed the discovery
of a number of immune relevant genes using the synte-

ny (conservation of gene order) found between the
human and fish genomes [29–32] and, in some cases,
has helped determine whether the gene is a true homo-
logue of a mammalian gene. To begin with, we used a
synteny approach to identify the chromosomal location
containing the zebrafish t-bet, stat6 and foxp3 tran-
scription factors. We used this approach for t-bet as,
at the time of discovery, the Ginbuna crucian carp
sequence was unknown. This approach enabled t-bet
and foxp3 to be obtained quickly, as a major difficulty
in the identification of transcription factors is that
Table 1. Amino acid identity ⁄ similarity of zebrafish t-bet, stat6 and foxp3 with other vertebrate T-bet, STAT6 and Foxp3 molecules.
Human
T-bet
Mouse
T-bet
Zebrafish
t-bet
Ginbuna
T-bet
Human
STAT6
Mouse
STAT6
Zebrafish
stat6
Tetraodon
STAT6
Human
Foxp3

Mouse
Foxp3
Zebrafish
foxp3
Human
T-bet
86.90 43.40 42.50 17.50 16.20 15.60 16.40 19.50 18.10 17.60
Mouse
T-bet
91.80 43.90 43.80 17.20 17.50 16.60 15.60 19.20 18.80 16.80
Zebrafish
t-bet
57.00 58.00 91.00 17.30 15.80 16.70 17.00 16.90 17.20 16.30
Ginbuna
T-bet
57.20 58.70 95.40 17.50 16.40 16.20 26.30 16.80 18.10 16.90
Human
STAT6
29.20 28.20 28.70 28.90 84.20 34.20 35.40 14.40 14.00 14.10
Mouse
STAT6
28.90 29.20 31.20 30.60 90.00 34.40 35.30 15.30 15.80 12.10
Zebrafish
stat6
28.60 30.10 32.70 32.80 53.40 54.50 52.90 15.10 14.50 15.20
Tetraodon
STAT6
28.50 28.40 30.60 29.90 55.50 55.20 71.50 14.40 14.80 13.50
Human
Foxp3

33.50 32.50 28.90 30.60 24.30 23.80 26.40 25.20 86.10 31.50
Mouse
Foxp3
31.00 33.20 28.70 29.60 24.10 24.50 24.40 25.40 91.40 31.60
Zebrafish
foxp3
31.40 29.10 27.60 29.10 24.10 23.80 25.20 26.80 48.00 49.00
Above diagonal, identity; below diagonal, similarity.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
134 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
many of them belong to gene families, with members
having high sequence identity, making it hard to find
the correct sequence in the zebrafish genome. This
approach was not used for stat6 as the region in which
this gene was found in the zebrafish genome shared no
synteny with the human genome. The zebrafish gen-
ome was searched using the human stat6 amino acid
sequence directly for identification.
The zebrafish t-bet homologue is predicted to contain
609 amino acids, the stat6 homologue 758 amino acids
and the foxp3 homologue 419 amino acids. None of
these molecules was found to contain a signal peptide
(data not shown), indicating that the molecules are not
secreted through the classical pathway and will remain
cytosolic. Also found in the 3¢-UTR of zebrafish stat6
and foxp3 were numerous copies of an mRNA instabil-
ity motif (attta) which plays a role in mRNA degrada-
tion [33], typical of genes coding for inflammatory
mediators [34], and suggesting that these genes are tran-
siently transcribed. It is unknown whether these

HUMAN TBX2
HUMAN TBX2
DOG TBX2
DOG TBX2
MOUSE TBX2
MOUSE TBX2
ZEBRAFISH TBX2
ZEBRAFISH TBX2
XENOPUSTR TBX2
XENOPUSTR TBX2
MOUSE TBX3
MOUSE TBX3
HUMANTBX3
HUMANTBX3
CHICKEN TBX3
CHICKEN TBX3
HUMAN TBX6
HUMAN TBX6
MOUSE TBX6
MOUSE TBX6
XENOPUSTR TBX6
XENOPUSTR TBX6
ZEBRAFISH TBX6
ZEBRAFISH TBX6
HUMAN T
HUMAN T
-
-
BET
BET

MOUSE T
MOUSE T
-
-
BET
BET
ZEBRAFISH T
ZEBRAFISH T
-
-
BET
BET
GINBUNACARP T
GINBUNACARP T
-
-
BET
BET
58
58
MOUSE TBX20
MOUSE TBX20
HUMAN TBX20
HUMAN TBX20
CHICKEN TBX20
CHICKEN TBX20
XENOPUSTR TBX20
XENOPUSTR TBX20
ZEBRAFISH TBX20
ZEBRAFISH TBX20

MOUSE TBX15
MOUSE TBX15
HUMAN TBX15
HUMAN TBX15
MOUSE TBX18
MOUSE TBX18
HUMAN TBX18
HUMAN TBX18
MOUSE TBX1
MOUSE TBX1
HUMAN TBX1
HUMAN TBX1
XENOPUSTR TBX1
XENOPUSTR TBX1
MOUSE TBX10
MOUSE TBX10
HUMAN TBX10
HUMAN TBX10
60
60
MOUSE TBX5
MOUSE TBX5
RAT TBX5
RAT TBX5
HUMAN TBX5
HUMAN TBX5
CHICKEN TBX5
CHICKEN TBX5
XENOPUSLA TBX5
XENOPUSLA TBX5

ZEBRAFISH TBX5
ZEBRAFISH TBX5
DOG TBX4
DOG TBX4
HUMAN TBX4
HUMAN TBX4
0.1
0.1
TBOX
TBOX
-
-
2/
2/
-
-
3
3
TBOX
TBOX
-
-
6/
6/
-
-
21
21
TBOX
TBOX

-
-
20
20
TBOX
TBOX
-
-
15/
15/
-
-
18
18
TBOX
TBOX
-
-
1/
1/
-
-
10
10
TBOX
TBOX
-
-
4/
4/

-
-
5
5
HUMAN TBX2
HUMAN TBX2
DOG TBX2
DOG TBX2
MOUSE TBX2
MOUSE TBX2
ZEBRAFISH TBX2
ZEBRAFISH TBX2
XENOPUSTR TBX2
XENOPUSTR TBX2
MOUSE TBX3
MOUSE TBX3
HUMANTBX3
HUMANTBX3
CHICKEN TBX3
CHICKEN TBX3
HUMAN TBX6
HUMAN TBX6
MOUSE TBX6
MOUSE TBX6
XENOPUSTR TBX6
XENOPUSTR TBX6
ZEBRAFISH TBX6
ZEBRAFISH TBX6
HUMAN T
HUMAN T

-
-
BET
BET
MOUSE T
MOUSE T
-
-
BET
BET
ZEBRAFISH T
ZEBRAFISH T
-
-
BET
BET
GINBUNACARP T
GINBUNACARP T
-
-
BET
BET
58
58
MOUSE TBX20
MOUSE TBX20
HUMAN TBX20
HUMAN TBX20
CHICKEN TBX20
CHICKEN TBX20

XENOPUSTR TBX20
XENOPUSTR TBX20
ZEBRAFISH TBX20
ZEBRAFISH TBX20
MOUSE TBX15
MOUSE TBX15
HUMAN TBX15
HUMAN TBX15
MOUSE TBX18
MOUSE TBX18
HUMAN TBX18
HUMAN TBX18
MOUSE TBX1
MOUSE TBX1
HUMAN TBX1
HUMAN TBX1
XENOPUSTR TBX1
XENOPUSTR TBX1
MOUSE TBX10
MOUSE TBX10
HUMAN TBX10
HUMAN TBX10
60
60
MOUSE TBX5
MOUSE TBX5
RAT TBX5
RAT TBX5
HUMAN TBX5
HUMAN TBX5

CHICKEN TBX5
CHICKEN TBX5
XENOPUSLA TBX5
XENOPUSLA TBX5
ZEBRAFISH TBX5
ZEBRAFISH TBX5
DOG TBX4
DOG TBX4
HUMAN TBX4
HUMAN TBX4
0.1
0.1
TBOX
TBOX
-
-
2/
2/
-
-
3
3
TBOX
TBOX
-
-
6/
6/
-
-

21
21
TBOX
TBOX
-
-
20
20
TBOX
TBOX
-
-
15/
15/
-
-
18
18
TBOX
TBOX
-
-
1/
1/
-
-
10
10
TBOX
TBOX

-
-
4/
4/
-
-
5
5
Fig. 5. Unrooted phylogenetic tree showing the relationship between the Danio rerio t-bet amino acid sequence for the full-length molecule
with other known vertebrate T-box (TBX) family member sequences. This tree was constructed by the neighbour-joining method using the
CLUSTALX and TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accession
numbers of the TBX-1 amino acid sequences are as follows: human, O43435; mouse, P70323; Xenopus tropicalis, Q3SA49. The accession
numbers of the TBX-2 amino acid sequences are as follows: human, Q13207; mouse, Q60707; dog, Q863A2; X. tropicalis, Q3SA48; zebra-
fish, Q7ZTU9. The accession numbers of the TBX-3 amino acid sequences are as follows: human, O15119; mouse, P70324; chicken,
O73718. The accession numbers of the TBX-4 amino acid sequences are as follows: human, P57082; dog, Q861Q9. The accession numbers
of the TBX-5 amino acid sequences are as follows: human, Q99593; mouse, P70326; rat, Q5I2P1; chicken, Q9PWE8; Xenopus laevis,
Q9W7C2; zebrafish, Q9IAK8. The accession numbers of the TBX-6 amino acid sequences are as follows: human, O95947; mouse, P70327,
X. tropicalis, Q66JL1; zebrafish, P79742. The accession numbers of the TBX-10 amino acid sequences are as follows: human, O75333;
mouse, Q810F8. The accession numbers of the TBX-15 amino acid sequences are as follows: human, Q96SF7; mouse, O70306. The acces-
sion numbers of the TBX-18 amino acid sequences are as follows: human, O95935; mouse, Q9EPZ6. The accession numbers of the TBX-20
amino acid sequences are as follows: human, Q9UMR3; mouse, Q9ES03; chicken, Q8UW76; X. tropicalis, Q3SA46; zebrafish, Q9I9K7. The
accession numbers of the TBX-21 (T-BET) amino acid sequences are as follows: human, Q9UL17; mouse, Q9JKD8; Ginbuna crucian carp,
AB290187; zebrafish, AM942761.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 135
instability motifs will be found within the t-bet 3¢-UTR
as it remains to be fully sequenced. Phylogenetic analy-
sis was carried out using the amino acid sequences of
zebrafish t-bet, stat6 and foxp3 plus those of all known
vertebrate T-box, STAT family and Foxp family

members. The zebrafish genes grouped well with their
vertebrate T-bet, STAT6 and Foxp3 homologues,
which was supported by bootstrap values greater than
75%, providing further evidence of their identity.
Multiple alignments of the zebrafish t-bet, stat6 and
foxp3 amino acids with their vertebrate homologues
revealed regions of high conservation. These regions
0.1
0.1
XENOPUSLA STAT1
XENOPUSLA STAT1
CHICKEN STAT1
CHICKEN STAT1
MOUSE STAT1
MOUSE STAT1
PIG STAT1
PIG STAT1
HUMAN STAT1
HUMAN STAT1
SALMON STAT1
SALMON STAT1
TETRAODON STAT1
TETRAODON STAT1
HALIBUT STAT1
HALIBUT STAT1
SNAKEHEAD STAT1
SNAKEHEAD STAT1
CHICKEN STAT4
CHICKEN STAT4
MOUSE STAT4

MOUSE STAT4
HUMAN STAT4
HUMAN STAT4
ZEBRAFISH STAT4
ZEBRAFISH STAT4
FUGU STAT4
FUGU STAT4
TETRAODON STAT4
TETRAODON STAT4
MOUSE STAT2
MOUSE STAT2
PIG STAT2
PIG STAT2
HUMAN STAT2
HUMAN STAT2
HUMAN STAT6
HUMAN STAT6
MOUSE STAT6
MOUSE STAT6
ZEBRAFISH STAT6
ZEBRAFISH STAT6
TETRAODON STAT6
TETRAODON STAT6
HUMAN STAT5
HUMAN STAT5
COW STAT5
COW STAT5
PIG STAT5
PIG STAT5
58

58
MOUSE STAT5
MOUSE STAT5
RAT STAT5
RAT STAT5
TROUT STAT5
TROUT STAT5
ZEBRAFISH STAT5
ZEBRAFISH STAT5
63
63
FUGU STAT5
FUGU STAT5
TETRAODON STAT5
TETRAODON STAT5
TROUT STAT3
TROUT STAT3
ZEBRAFISH STAT3
ZEBRAFISH STAT3
TETRAODON STAT3
TETRAODON STAT3
MEDAKA STAT3
MEDAKA STAT3
55
55
CHICKEN STAT3
CHICKEN STAT3
MOUSE STAT3
MOUSE STAT3
RAT STAT3

RAT STAT3
71
71
PIG STAT3
PIG STAT3
HUMAN STAT3
HUMAN STAT3
49
49
0.1
0.1
0.1
0.1
XENOPUSLA STAT1
XENOPUSLA STAT1
CHICKEN STAT1
CHICKEN STAT1
MOUSE STAT1
MOUSE STAT1
PIG STAT1
PIG STAT1
HUMAN STAT1
HUMAN STAT1
SALMON STAT1
SALMON STAT1
TETRAODON STAT1
TETRAODON STAT1
HALIBUT STAT1
HALIBUT STAT1
SNAKEHEAD STAT1

SNAKEHEAD STAT1
CHICKEN STAT4
CHICKEN STAT4
MOUSE STAT4
MOUSE STAT4
HUMAN STAT4
HUMAN STAT4
ZEBRAFISH STAT4
ZEBRAFISH STAT4
FUGU STAT4
FUGU STAT4
TETRAODON STAT4
TETRAODON STAT4
MOUSE STAT2
MOUSE STAT2
PIG STAT2
PIG STAT2
HUMAN STAT2
HUMAN STAT2
HUMAN STAT6
HUMAN STAT6
MOUSE STAT6
MOUSE STAT6
ZEBRAFISH STAT6
ZEBRAFISH STAT6
TETRAODON STAT6
TETRAODON STAT6
HUMAN STAT5
HUMAN STAT5
COW STAT5

COW STAT5
PIG STAT5
PIG STAT5
58
58
MOUSE STAT5
MOUSE STAT5
RAT STAT5
RAT STAT5
TROUT STAT5
TROUT STAT5
ZEBRAFISH STAT5
ZEBRAFISH STAT5
63
63
FUGU STAT5
FUGU STAT5
TETRAODON STAT5
TETRAODON STAT5
TROUT STAT3
TROUT STAT3
ZEBRAFISH STAT3
ZEBRAFISH STAT3
TETRAODON STAT3
TETRAODON STAT3
MEDAKA STAT3
MEDAKA STAT3
55
55
CHICKEN STAT3

CHICKEN STAT3
MOUSE STAT3
MOUSE STAT3
RAT STAT3
RAT STAT3
71
71
PIG STAT3
PIG STAT3
HUMAN STAT3
HUMAN STAT3
49
49
STAT-1
STAT-1
STAT-4
STAT-4
STAT-2
STAT-2
STAT-6
STAT-6
STAT-5
STAT-5
STAT-3
STAT-3
Fig. 6. Unrooted phylogenetic tree showing the relationship between the Danio rerio stat6 amino acid sequence for the full-length molecule
with other known vertebrate STAT family member sequences. This tree was constructed by the neighbour-joining method using the
CLUSTALX and TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accession
numbers of the STAT-1 amino acid sequences are as follows: human, P42224; mouse, P42225; pig, Q764M5; chicken, CAG32090; Xeno-
pus tropicalis, AAM51552; salmon, ACI33829; Tetraodon, AAL09414; halibut, ABS19629; snakehead, ABK60089. The accession numbers of

the STAT-2 amino acid sequences are as follows: human, P52630; mouse, Q9WVL2; pig, O02799. The accession numbers of the STAT-3
amino acid sequences are as follows: human, P40763; mouse, P42227; rat, P52631; pig, Q19S50; chicken, Q6DV79; trout, AAB60926;
zebrafish, AAH68320; Tetraodon, AAL09415; medaka, AAT64912. The accession numbers of the STAT-4 amino acid sequences are as
follows: human, Q14765; mouse, P42228; chicken, BAF34318; zebrafish, CAD52132; Fugu, AAS10464; Tetraodon, AAL09416. The
accession numbers of the STAT-5 amino acid sequences are as follows: human, P51692; mouse, P42232; rat, P52632; pig, Q9TUZ0; cow,
Q9TUM3; trout, AAG14946; Tetraodon, AAL09417; Fugu, AAS80167; zebrafish, AAT95391. The accession numbers of the STAT-6 amino
acid sequences are as follows: human, P42226; mouse, P52633; Tetraodon, AAO22057; zebrafish, AM941850.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
136 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
were subsequently identified to be protein domains
important in the functioning of these transcription
factors. T-bet (also known as Tbox-21) belongs to the
T-box family of genes, consisting of over 20 members
characterized in mammals [35]. They contain a con-
served sequence, around 200 amino acids in length,
called the ‘T-box’, which, in T-bet, is centrally located,
whereas, in other members, it is located at the amino-
terminus [36]. This region is known to be a
DNA-binding domain and is quite clearly conserved in
zebrafish, as the sequence, when compared with human
and mouse T-bet [11,37], shows almost complete
identity in this region.
STAT6 (also known as IL-4-induced transcription
factor) belongs to the STAT family of proteins [38].
STAT proteins share structurally and functionally
0.1
0.1
XENOPUS FOXP4
XENOPUS FOXP4
MOUSE FOXP4

MOUSE FOXP4
HUMAN FOXP4
HUMAN FOXP4
ZEBRAFISH FOXP3
ZEBRAFISH FOXP3
MOUSE FOXP3
MOUSE FOXP3
HUMAN FOXP3
HUMAN FOXP3
MACAQUE FOXP3
MACAQUE FOXP3
XENOPUS FOXP2
XENOPUS FOXP2
HUMAN FOXP2
HUMAN FOXP2
MACAQUE FOXP2
MACAQUE FOXP2
MOUSE FOXP2
MOUSE FOXP2
ZEBRAFISH FOXP1
ZEBRAFISH FOXP1
XENOPUS FOXP1
XENOPUS FOXP1
CHICKEN FOXP1
CHICKEN FOXP1
RAT FOXP1RAT FOXP1
MOUSE FOXP1
MOUSE FOXP1
COW FOXP1
COW FOXP1

HUMAN FOXP1
HUMAN FOXP1
50
50
0.1
0.1
0.1
0.1
XENOPUS FOXP4
XENOPUS FOXP4
MOUSE FOXP4
MOUSE FOXP4
HUMAN FOXP4
HUMAN FOXP4
ZEBRAFISH FOXP3
ZEBRAFISH FOXP3
MOUSE FOXP3
MOUSE FOXP3
HUMAN FOXP3
HUMAN FOXP3
MACAQUE FOXP3
MACAQUE FOXP3
XENOPUS FOXP2
XENOPUS FOXP2
HUMAN FOXP2
HUMAN FOXP2
MACAQUE FOXP2
MACAQUE FOXP2
MOUSE FOXP2
MOUSE FOXP2

ZEBRAFISH FOXP1
ZEBRAFISH FOXP1
XENOPUS FOXP1
XENOPUS FOXP1
CHICKEN FOXP1
CHICKEN FOXP1
RAT FOXP1RAT FOXP1
MOUSE FOXP1
MOUSE FOXP1
COW FOXP1
COW FOXP1
HUMAN FOXP1
HUMAN FOXP1
50
50
FOXP4
FOXP4
FOXP3
FOXP3
FOXP2
FOXP2
FOXP1
FOXP1
Fig. 7. Unrooted phylogenetic tree showing the relationship between the Danio rerio foxp3 amino acid sequence for the full-length molecule
with other known vertebrate Foxp family member sequences. This tree was constructed by the neighbour-joining method using the
CLUSTALX
and TREEVIEW packages, and was bootstrapped 10 000 times. All bootstrap values less than 75% are shown. The EMBL accession numbers
of the Foxp1 amino acid sequences are as follows: human, Q9H334; rat, Q498D1; mouse, P58462; cow, A4IFD2; chicken, Q58NQ4; Xeno-
pus laevis, Q5W1J5; zebrafish, Q2LE08. The accession numbers of the Foxp2 amino acid sequences are as follows: human, O15409;
mouse, P58463; crab-eating macaque, Q8MJ97; Xenopus laevis, Q4VYS1. The accession numbers of the Foxp3 amino acid sequences are

as follows: human, Q9BZS1; mouse, Q99JB6, crab-eating macaque, Q6U8D7; zebrafish, FM881778. The accession numbers of the Foxp4
amino acid sequences are as follows: human, Q8IVH2; mouse, Q9DBY0; X. laevis, Q4VYR7.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 137
conserved domains, including an N-terminal STAT pro-
tein interaction domain, which strengthens interactions
between STAT dimers on adjacent DNA-binding sites,
a coiled-coil STAT protein all-alpha domain, which is
implicated in other protein–protein interactions, a
STAT protein DNA-binding domain and an SH2
domain, which binds phosphorylated tyrosine residues
in the context of a longer peptide motif within a target
protein [38,39]. Within zebrafish stat6, there is good
conservation of the protein sequence in these regions
when compared with mammalian STAT6 [40,41]. Also
conserved in the zebrafish sequence is a tyrosine mole-
cule (Tyr664), which is an important phosphorylation
site, necessary for STAT protein activity [39].
Foxp3 belongs to the fork-head box (FOX) family
of proteins [42], a family of transcription factors that
are both transcriptional repressors and activators. It
contains at least three distinct structural domains [43]:
a fork-head domain at the C-terminus (a sequence of
80–100 amino acids forming a motif that is critical for
DNA binding and nuclear localization), which is
shared by all FOX proteins, a leucine zipper domain
and a C2H2 zinc finger domain, both of which are
thought to help mediate DNA binding and may be
involved in the induction of dimerization [43]. Each of
these three regions appears to be present within zebra-

fish foxp3, as there is very good conservation of
protein sequence in these regions when compared with
mammalian Foxp3. In addition, within human Foxp3,
there are two other domains, not found in other Foxp
subfamily members and possibly specific for T
reg
cell
biology, which are transcriptional repressor domains 1
and 2 [44]. In mammals, these regions contain fairly
high numbers of proline residues [44]. Similar domains
may exist in zebrafish foxp3, as there is some homol-
ogy within this region, with a relatively large number
of proline residues present in the potential second tran-
scriptional repressor domain, but not in the first. Inter-
0 500 1000 1500 2000 2500 3000 3500
Zebrafish
Human
Zebrafish
Human
0 500 1000 1500 2000 2500 3000 3500
Zebrafish
Human
0 1000 2000 3000 4000 5000 6000
**
A
B
C
Fig. 8. Comparison of the gene organization and intron ⁄ exon sizes between known T-bet (A), STAT6 (B) and Foxp3 (C) genes with zebrafish
t-bet, stat6 and foxp3. The exons are drawn to scale but, because of the large size of some of the introns, they are not. For stat6, potential
missing human exons in zebrafish are indicated by ‘*’. The EMBL accession numbers of the T-bet genes are as follows: human, CH471109;

zebrafish, FN435332. The EMBL accession numbers of the STAT6 genes are as follows: human, AF417842; zebrafish, FN435334. The
EMBL accession numbers of the Foxp3 genes are as follows: human, CH471224; zebrafish, FN435333.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
138 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
estingly, two transcripts of foxp3 were sequenced
within zebrafish, with the second transcript lacking
exons 5, 6, 7 and 8 which contain transcriptional
repressor domain 2, the zinc-finger domain and the
leucine-zipper domain. In humans, a similar scenario
exists, with two alternatively spliced isoforms of Foxp3
also being expressed, but here only exon 2 is lacking in
the short form [45]. This splicing variant has not been
reported in mice [44].
The gene organizations of zebrafish t-bet, stat6 and
foxp3 were also determined and found to be very simi-
lar to their human homologues. Both the human and
zebrafish T-bet and Foxp3 genes contained the same
number of exons and introns. t-bet contained six exons
and five introns, whereas foxp3 contained 13 exons
and 12 introns. However, zebrafish stat6 showed some
differences from the human homologue, having 21
exons and 20 introns rather than 22 exons and 21
introns. This difference was found at the 3¢ end of the
gene, where the zebrafish and human sequences appear
to be quite diverse. Together with the well-conserved
gene organization, there is a conservation of synteny
between the human and zebrafish genomes where the
t-bet and foxp3 genes are found. In contrast, there was
no synteny between the human and zebrafish regions
containing the stat6 gene, showing that the use of a

synteny approach to look for genes may not work in
all cases. Studies looking at the Fugu and human
genomes have shown complete conservation of gene
order and content at some loci, whereas other regions
demonstrate variation in the order of linked genes, or
extensive differences in gene order within conserved
regions [36,46,47]. Nevertheless, the fact that some
groups of genes have remained in close proximity dur-
ing evolution and could be found within an ancestral
organization some 450 million years ago, before the
fish–tetrapod divergence, may potentially be of impor-
tance functionally or have evolutionary significance
[47,48].
The localization of zebrafish t-bet, stat6 and foxp3
expression was also investigated, and indicated that
these molecules are biologically relevant to bony fish
immune responses and, in particular, Th and T
reg
cell
responses. PCR analysis of zebrafish t-bet, stat6 and
foxp3 detected constitutive expression in the spleen,
kidney, gill, gut, liver and skin tissue of healthy fish
(data not shown). Previous studies on the expression
of these genes within humans and mice, of T-bet
within Ginbuna crucian carp [27] and STAT6 in
mandarin fish [28] have shown similar widespread
expression. In the case of STAT6, highest expression
in mammals occurs in peripheral blood lymphocytes,
colon, intestine, ovary, prostate, thymus, spleen, kid-
ney, liver, lung and placenta [40]. More specifically, it

NAB2
STAT 6
LRP1
NXPH4
SHMT2
TAC3
MYO1A
TMEM194
NDUFA4L2
STAC3
R3HDM2
ZBTB39
GPR182
MIP
stat6
FIGNL2
SCN8A
TMPRSS12
CACNB3A
DTX3
PTGES3
DIP2B
ATF 7
ASB8
RND1
CACNB3B
TBKBP1
TBET
OSBPL7
MRPL10

LRRC46
C17ORF57
NPEPPS
KPNB1
SCRN2
SP6
SP2
ITGB3
MYL4
TBKBP1
tbet
OSBPL7
MRPL10
PNPO
MMRN2
UMOD9
PDK2
ATAD 4
CDK5RAP3
NFE2L1
MYO1D
CDK5R1
PPP1R3F
FOXP3
CCDC22
CACNA1F
SYP
GAGE12D
GAGE12J
GAGE10

PRICKLE3
PLP2
MAGIX
GAGE8
GAGE8L
GAGE12I
GAGE2A
GPKOW
WDR45
PPP1R3F
foxp3
TSPYL2
SEMA3F
WASL
CACNA1S
TMEM9
SUV39H1
SLC38A3
STK38
TMEM81
TAF10
EFHD2
OPN5
TMEM115
PREX1
KCNQ2
A
B
C
NAB2

STAT 6
LRP1
NXPH4
SHMT2
TAC3
MYO1A
TMEM194
NDUFA4L2
STAC3
R3HDM2
ZBTB39
GPR182
MIP
stat6
FIGNL2
SCN8A
TMPRSS12
CACNB3A
DTX3
PTGES3
DIP2B
ATF 7
ASB8
RND1
CACNB3B
NAB2
STAT 6
LRP1
NXPH4
SHMT2

TAC3
MYO1A
TMEM194
NDUFA4L2
STAC3
R3HDM2
ZBTB39
GPR182
NAB2
STAT 6
LRP1
NXPH4
SHMT2
TAC3
MYO1A
TMEM194
NDUFA4L2
STAC3
R3HDM2
ZBTB39
GPR182
NAB2
STAT 6
LRP1
NXPH4
SHMT2
TAC3
MYO1A
TMEM194
NDUFA4L2

STAC3
R3HDM2
ZBTB39
GPR182
Human
Chr12
55.67 Mb – 55.98 Mb
MIP
stat6
FIGNL2
SCN8A
TMPRSS12
CACNB3A
DTX3
PTGES3
DIP2B
ATF 7
ASB8
RND1
CACNB3B
MIP
stat6
FIGNL2
SCN8A
TMPRSS12
CACNB3A
DTX3
PTGES3
DIP2B
ATF 7

ASB8
RND1
CACNB3B
MIP
stat6
FIGNL2
SCN8A
TMPRSS12
CACNB3A
DTX3
PTGES3
DIP2B
ATF 7
ASB8
RND1
CACNB3B
Zebrafish
Chr23
26.44 Mb – 26.95 Mb
TBKBP1
TBET
OSBPL7
MRPL10
LRRC46
C17ORF57
NPEPPS
KPNB1
SCRN2
SP6
SP2

ITGB3
MYL4
TBKBP1
tbet
OSBPL7
MRPL10
PNPO
MMRN2
UMOD9
PDK2
ATAD 4
CDK5RAP3
NFE2L1
MYO1D
CDK5R1
TBKBP1
TBET
OSBPL7
MRPL10
LRRC46
C17ORF57
NPEPPS
KPNB1
SCRN2
SP6
SP2
ITGB3
MYL4
TBKBP1
TBET

OSBPL7
MRPL10
LRRC46
C17ORF57
NPEPPS
KPNB1
SCRN2
SP6
SP2
ITGB3
MYL4
TBKBP1
TBET
OSBPL7
MRPL10
LRRC46
C17ORF57
NPEPPS
KPNB1
SCRN2
SP6
SP2
ITGB3
MYL4
Human
Chr17
42.64 Mb – 43.37 Mb
TBKBP1
tbet
OSBPL7

MRPL10
PNPO
MMRN2
UMOD9
PDK2
ATAD 4
CDK5RAP3
NFE2L1
MYO1D
CDK5R1
TBKBP1
tbet
OSBPL7
MRPL10
PNPO
MMRN2
UMOD9
PDK2
ATAD 4
CDK5RAP3
NFE2L1
MYO1D
CDK5R1
TBKBP1
tbet
OSBPL7
MRPL10
PNPO
MMRN2
UMOD9

PDK2
ATAD 4
CDK5RAP3
NFE2L1
MYO1D
CDK5R1
Zebrafish
Chr12
23.81 Mb – 24.32 Mb
PPP1R3F
FOXP3
CCDC22
CACNA1F
SYP
GAGE12D
GAGE12J
GAGE10
PRICKLE3
PLP2
MAGIX
GAGE8
GAGE8L
GAGE12I
GAGE2A
GPKOW
WDR45
PPP1R3F
foxp3
TSPYL2
SEMA3F

WASL
CACNA1S
TMEM9
SUV39H1
SLC38A3
STK38
TMEM81
TAF10
EFHD2
OPN5
TMEM115
PREX1
KCNQ2
PPP1R3F
FOXP3
CCDC22
CACNA1F
SYP
GAGE12D
GAGE12J
GAGE10
PRICKLE3
PLP2
MAGIX
GAGE8
GAGE8L
GAGE12I
GAGE2A
GPKOW
WDR45

PPP1R3F
FOXP3
CCDC22
CACNA1F
SYP
GAGE12D
GAGE12J
GAGE10
PRICKLE3
PLP2
MAGIX
GAGE8
GAGE8L
GAGE12I
GAGE2A
GPKOW
WDR45
PPP1R3F
FOXP3
CCDC22
CACNA1F
SYP
GAGE12D
GAGE12J
GAGE10
PRICKLE3
PLP2
MAGIX
GAGE8
GAGE8L

GAGE12I
GAGE2A
GPKOW
WDR45
Human
ChrX
48.82 Mb – 49.13 Mb
PPP1R3F
foxp3
TSPYL2
SEMA3F
WASL
CACNA1S
TMEM9
SUV39H1
SLC38A3
STK38
TMEM81
TAF10
EFHD2
OPN5
TMEM115
PREX1
KCNQ2
Zebrafish
Chr8
26.13 Mb – 26.68 Mb
PPP1R3F
foxp3
TSPYL2

SEMA3F
WASL
CACNA1S
TMEM9
SUV39H1
SLC38A3
STK38
TMEM81
TAF10
EFHD2
OPN5
TMEM115
PREX1
KCNQ2
PPP1R3F
foxp3
TSPYL2
SEMA3F
WASL
CACNA1S
TMEM9
SUV39H1
SLC38A3
STK38
TMEM81
TAF10
EFHD2
OPN5
TMEM115
PREX1

KCNQ2
Fig. 9. Comparative gene location map of the regions in which zebrafish and human Foxp3 (A), T-bet (B) and STAT6 (C) are found. The
zebrafish genome assembly version 8 (Zv8) was used for this analysis.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 139
has been shown that various unstimulated murine and
human T-cells, B-cells, myeloid cells, monocytic cells
and fibroblasts, but not neuronal cells or embryonic
stem cells, express STAT6, indicating that this gene is
expressed in haematopoietic cells and more variably in
other lineages [41]. The expression of T-bet has been
seen within a wide variety of tissues and cells in the
Ginbuna crucian carp [27] and, in mammals, in the
lung tissue of mouse and spleen and thymus of human
and mouse [11,37]. Foxp3 is also highly expressed in
lymphoid organs, such as the thymus and spleen [49],
and its expression within a variety of other tissues has
also been observed, although to a far lesser extent
[50,51]. More recently, several genome-wide human
tissue expression studies [52,53] have shown that the
expression of T-bet, STAT6 and Foxp3 mRNA
levels is constitutive in a wide variety of unstimulated
human tissues taken from immune, nervous, muscle,
secretory, internal and reproductive tissue, although,
relative to T-bet and STAT6, Foxp3 expression is
much lower.
Although the expression of T-bet, STAT6 and Foxp3
is not restricted to any particular cell or tissue, they are
known to play a crucial role in the differentiation of
naive T-cells into Th and T

reg
cell subsets [12]. In this
investigation, we attempted to correlate the expression
of the transcription factors with other genes known to
be expressed by T-cell subsets after the immunostimu-
lation of zebrafish head kidney and spleen cells with
PHA, LPS or Poly I:C. On PHA stimulation, a corre-
lation between il-4 and gata3 expression was seen in
both the spleen and head kidney, although only in the
kidney was expression significantly different. stat6
showed no significant increase in either tissue. STAT6,
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHAPHA LPSLPS Poly I:CPoly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C

PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C

il-4
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
–10
–5
0
5
10
15
20
25
30
35
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
gata3
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C

–12
–10
–8
–6
–4
–2
0
2
4
6
8
PHAPHA LPSLPS Poly I:CPoly I:C
stat6
PHAPHA LPSLPS Poly I:CPoly I:C
–5
–4
–3
–2
–1
0
1
2
3
4
5
PHA
PHA
LPS
LPS
Poly I:C

Poly I:C
il-10
–10
–5
0
5
10
15
20
25
30
35
40
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
foxp3
–5
–4
–3
–2

–1
0
1
2
3
4
5
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
ifn-
g
–5
0
5
10
15
20
Fold change compared to
unstimulated control
Fold change compared to

unstimulated control
Fold change compared to
unstimulated control
Fold change compared to
unstimulated control
Fold change compared to
unstimulated control
Fold change compared to
unstimulated control
Fold change compared to
unstimulated control
Fold change compared to
unstimulated control
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
mx
PHA
PHA
LPS
LPS

Poly I:C
Poly I:C
–4
–2
0
2
4
6
8
10
PHA
PHA
LPS
LPS
Poly I:C
Poly I:C
t-bet
–6
–5
–4
–3
–2
–1
0
1
2
3
4
5
PHA

PHA
LPS
LPS
Poly I:C
Poly I:C
= Kidney
= Spleen
A
B
C
Fig. 10. Expression of Th2 (il-4, gata3 and stat6) (A), T
reg
(il-10 and foxp3) (B) and Th1 (ifn-c, mx and t-bet) (C) relevant molecules in head
kidney or spleen tissues stimulated with PHA, LPS or Poly I:C. Pooled head kidney or spleen tissue from five fish was incubated with
each stimulant at previously optimized concentrations for 4 h, and the total RNA was recovered. Following reverse transcription, the
relative expression of each gene was detected by real-time PCR and normalized to the expression of gapdh. The means of six replicates
are shown ± SEM. Differences between treatments and control groups are significant: *P < 0.05; **P < 0.01.
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
140 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
GATA3 and IL-4 are all involved in Th2 cell develop-
ment [54]. The induction of GATA3 expression has been
shown to be dependent on IL-4-stimulated STAT6 acti-
vation, although it remains unclear whether STAT6
activates GATA3 transcription directly [55]. It has also
been described in a number of papers that activated
STAT6 is required to drive Th2-specific cytokine pro-
duction, which includes IL-4, in mammals [56–58].
However, in the present investigation in zebrafish, no
correlation was seen between stat6 and il-4 on stimula-
tion. This is probably a result of the fact that PHA, a

known T-cell mitogen, selectively induces GATA3
expression [59,60], and this is known to be sufficient to
drive elevated IL-4 cytokine production and to induce
Th2 differentiation [13].
On LPS, but not PHA or Poly I:C, stimulation, a
correlation between il-10 and foxp3 expression was
seen in the spleen, with both being up-regulated signifi-
cantly. Foxp3 is involved in CD4
+
CD25
+
Foxp3
+
T
reg
cell development and IL-10 is an essential cytokine in
the mechanism underlying immune suppression by
these cells [18]. A similar correlation of Foxp3 and
IL-10 mRNA expression has already been observed
in mammals [61], and it has been shown that LPS up-
regulates the expression of Foxp3 in CD4
+
CD25
)
⁄
CD4
+
CD25
+
cells [62,63]. In addition, elevated IL-10

expression has already been found in zebrafish that
have been stimulated with LPS [64]. Whether the
expression of these two genes directly relates to each
other will require further investigation in fish, as it has
been found that IL-10 is produced by other sources on
stimulation. LPS induces an initial burst of inflam-
matory cytokine synthesis in human monocytes and
other cells, which is followed by substantial IL-10
production [65,66]. In addition, IL-10 has been found
to be expressed by B-cells [67], dendritic cells [68] and
by T-cells other than T
reg
[69].
On Poly I:C, but not PHA or LPS, stimulation,
there was a good correlation between t-bet, mx and
ifn-c expression, with all three being up-regulated sig-
nificantly in the kidney. T-bet plays an important role
in Th1 development [70] and IFN-c is produced by
this cell type and is known to play a critical role in
driving Th1 cell responses in mammals [54,70]. In
humans, T-bet gene expression is found to be rapidly
induced by IFN-c in lymphoid and myeloid cells [71],
but not by IFN-a, LPS or IL-1, indicating that the
action of IFN-c is specific. In the present investigation,
high ifn-c, but not t-bet, expression was observed in
response to PHA, but this has also been seen in mam-
mals, where PHA upregulates IFN-c and IL-4 [72,73].
This finding probably relates to a different mechanism
by which IFN-c is released.
In conclusion, this investigation has identified three

important transcription factors in zebrafish, t-bet, stat6
and foxp3, that are expected to be involved in the dif-
ferentiation of T-cell subsets. Our preliminary expres-
sion data suggest that these genes are involved in the
fish immune response, and that there is a relationship
between the expression of these transcription factors
and the cytokine genes known to be produced by dif-
ferent T-cell subtypes in mammals. These transcription
factors, together with many of the important cytokines
that are expressed by different T-cell subtypes [22,23],
will aid future investigations into the types of Th and
T
reg
cells that exist in teleost fish.
Materials and methods
Sequence retrieval
The zebrafish t-bet, stat6 and foxp3 sequences were found
initially using the zebrafish genome (em-
bl.org/Danio_rerio/) assembly version 7 (Zv7) and, in the
case of t-bet and foxp3, by exploiting the conservation of
synteny between the human and zebrafish genomes. Chro-
mosomes within the zebrafish genome database were
searched by basic local alignment search tool (blast) analy-
sis [26] using amino acid sequences for human OSBPL7
and PPP1R3F, known to be located close to the T-bet gene
and the Foxp3 gene, respectively, in the human genome.
The human STAT6 sequence was used to search for the
zebrafish stat6. A zebrafish homologue for OSBPL7 was
found in chromosome 12, for PPP1R3F in chromosome 8
and for stat6 in chromosome 23. Subsequently, the DNA

surrounding these genes was retrieved ( 300 000 bp) for
further analysis using various sequence software programs.
Using Genscan [24], possible coding regions within the
genomic DNA were identified, and the amino acid
sequences were analysed using blast [26] and fasta [25].
This analysis identified regions within the zebrafish genome
that appeared to code for possible t-bet, stat6 and foxp3
homologues, and the predicted cDNA sequences were
exploited by designing primers to obtain the full coding
sequences of these genes.
cDNA production
Zebrafish, Danio rerio ( 10 g), were maintained in 20 L
tanks in a freshwater recirculating system at 28 °C. Fish
were fed frozen bloodworm twice daily. The zebrafish used
for initial cDNA production for the cloning of cytokine
genes were anaesthetized in bezocaine (1%), killed and the
spleen tissue was collected under sterile conditions. The
spleens were cut finely using scalpel blades and cultured in
Nunc six-well plates (Fisher Scientific, Loughborough, UK)
containing 5 mL L-15 medium (Invitrogen Ltd, Renfrew,
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 141
UK) supplemented with 5% fetal bovine serum (Life Tech-
nologies, Paisley, UK) and Gibco 100 UÆmL
)1
penicillin,
100 lgÆmL
)1
streptomycin (Invitrogen) at 28 °C. Within
each well, five spleens were used and the cells were stimu-

lated with 10 lgÆmL
)1
PHA, 5 lgÆmL
)1
LPS or 50 lgÆmL
)1
Poly I:C for 4 h. To prepare cDNA templates for the clon-
ing of t-bet, stat6 and foxp3, total RNA from these stimu-
lated spleens was extracted using RNA-stat60 reagent
(AMS Biotechnology Ltd, Abingdon, UK) according to the
manufacturer’s instructions. Single-strand cDNA was syn-
thesized by reverse transcription with oligodeoxythymidy-
late (oligodT)12–18 (Invitrogen) or adapter-dT primer
(Table 2) for 3¢-rapid amplification of cDNA ends (RACE),
using Bioscript reverse transcriptase (Bioline Ltd, London,
UK). cDNA for 5¢-RACE was prepared by transcribing
from poly(A) mRNA using an oligo-dT primer (Invitro-
gen), which was then treated with Escherichia coli RNase H
(Promega, Madison, WI, USA) following the manufac-
turer’s instructions. The cDNA was then purified using a
PCR purification kit (Qiagen, Crawley, UK), and tailed
with poly(C) at the 5¢ end using terminal deoxynucleotidyl
transferase (Promega, Madison, WI, USA) following the
manufacturer’s instructions. The cDNA was finally diluted
with 10 mm Tris ⁄ EDTA buffer (10 mm Tris, 1 mm EDTA,
pH 8.0) and stored at )20 °C before use.
Cloning and sequencing
Initially, PCR was performed using the cDNA prepared
above, with primers zfstat6-F1, zfstat6-R1, zfstat6-F2 and
zfstat6-R2 for stat6,zftbet-F1 and zftbet-R1 for t-bet, and

zffoxp3-F1, zffoxp3-R1, zffoxp3-F2 and zffoxp3-R2 for
foxp3. These primers amplified part of the initial predicted
sequence which contained the majority of the open reading
frames of zebrafish stat6, t-bet and foxp3 to check they were
correct. Having isolated these partial sequences, the com-
plete zebrafish t-bet, stat6 and foxp3 cDNA sequences were
obtained using 5¢- and 3¢-RACE-PCR, with gene-specific
primers. Initially, 5¢-RACE-PCR was performed to amplify
the 5¢ end of the stat6, foxp3 and t-bet genes using the
cDNA prepared above (see section on cDNA production).
The first round of PCR used zf5¢stat6-R1, zf5¢tbet-R1 or
zf5¢foxp3-R1 primer (Table 2) for the stat6, t-bet and foxp3
genes, respectively, with oligo dG (Table 2). Semi-nested
PCR was performed on the first-round product using
zf5¢stat6-R2, zf5¢tbet-R2 or zf5¢foxp3-R2 primer (Table 2)
for the stat6, t-bet and foxp3 genes, respectively, with oligo
dG. The 3¢ end of the stat6, t-bet and
foxp3 genes was
obtained using 3¢-RACE-PCR performed on the cDNA pre-
pared above (see section on cDNA production). The first
round of PCR used zf3¢stat6-F1, zf3¢tbet-F1 or zf3¢foxp3-F1
primer (Table 2) for stat6, t-bet and foxp3 genes, respec-
tively, with the adapter primer (Table 2). Semi-nested PCR
was performed on the first-round product using zf3¢stat6-F2,
zf3¢tbet-F2 or zf3¢foxp3-F2 primer (Table 2) for the stat6,
t-bet and foxp3 genes, respectively, with the adapter primer
(Table 2). Using the above method, complete transcripts
were obtained for t-bet and foxp3. Additional primers for
3¢-RACE-PCR had to be designed for stat6 (zf3¢stat6-F3
and zf3¢stat6-F4) and a semi-nested PCR performed as

above to obtain the complete 3¢-UTR of zebrafish stat6.
The PCR products obtained were ligated into the
pGEM-T Easy vector (Promega). Following transfection
into competent E. coli cells (ActifMotif, Rixensart, Belgium),
recombinants were identified through red–white colour selec-
tion when grown on MacConkey agar (Sigma-Aldrich,
Poole, UK). Plasmid DNA from at least three independent
clones was recovered using an alkaline lysis-based method
[74] and sequenced using an ABI 377 Automated Sequencer
(Applied Biosystems, Foster City, CA, USA). The sequences
generated were analysed for similarity with other known
sequences using the fasta [25] and blast [26] suite of pro-
grams. Homology analysis of the amino acid sequences was
performed using matgat software v2.02 [75], and multiple
sequence alignments were generated using clustalx v1.81
[76]. Phylogenetic relationships were constructed from
clustalx v1.81-generated alignments of the full-length
amino acid sequences of the known T-box, STAT and Foxp
family molecules using the neighbour-joining method [77].
The tree was drawn using treeview v1.6.1 [78] and confi-
dence limits were added [79].
Table 2. Primers used to amplify zebrafish stat6, t-bet and foxp3
cDNA.
Primer name Sequence (5¢-to3¢) Used for
Zfstat6-F1 AGTGAGATGGATACAGGTGCTAAAC Initial PCR
Zfstat6-R1 TCTGGACCTCAGACATGAACTTACT Initial PCR
Zfstat6-F2 TGTCAGTCCTCTTTAATGCT Initial PCR
Zfstat6-R2 AATGGTATCCTGTTTGGCTCAG Initial PCR
Zf3¢stat6-F1 GGTTGTAATTGTACACGGTAGTC 3¢-RACE
Zf3¢stat6-F2 CGGTAGTCAGGAAATCAATGCC 3¢-RACE

Zf5¢stat6-R1 CCATGTCTGCAGATGGTCGAGG 5¢-RACE
Zf5¢stat6-R2 GGACTGACATTGCTCCAGAGC 5¢-RACE
Zf3¢stat6-F3 GCTTCAGTGACTCAGAAATTGG 3¢-RACE
Zf3¢stat6-F4 GTCCAGAATATTCAGCCTTTCACC 3¢-RACE
Zftbet-F1 CTCCCTCAAACAAACCAGAGTC Initial PCR
Zftbet-R1 CACTGGATGAGACAGGAAGTT Initial PCR
Zf3¢tbet-F1 CTTCTCCAGGACAGTCCAAAGAGTC 3¢-RACE
Zf3¢
tbet-F2 CTGGATTGAAGCGCCCTCGGTTAATC 3¢-RACE
Zf5¢tbet-R1 GCTGCCTTTGTTATTTGTAAGCTTCAG 5¢-RACE
Zf5¢tbet-R2 GGAAACTTCCTGTCTCATCCAGTG 5¢-RACE
Zffoxp3-F1 GGAACACACAGAGGGGATGATA Initial PCR
Zffoxp3-R1 CTTCAACACGCACAAAGCAC Initial PCR
Zffoxp3-F2 TGCCACCTTTTCCATCATACA Initial PCR
Zffoxp3-R2 CTGCTTTTCTGGGGACTTCA Initial PCR
Zf3¢foxp3-F1 TGAAGTCCCCAGAAAAGCAG 3¢-RACE
Zf3¢foxp3-F2 GTGCTTTGTGCGTGTTGAAG 3¢-RACE
Zf5¢foxp3-R1 TGTATGATGGAAAAGGTGGCA 5¢-RACE
Zf5¢foxp3-R2 GGAACACACAGAGGGGATGATA 5¢-RACE
Oligo dG GGGGGGIGGGIIGGGIIG 5¢-RACE
Adapter dT CTCGAGATCGATGCGGCCGCT
17
3¢-RACE
Adapter primer CTCGAGATCGATGCGGCCGC 3¢-RACE
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
142 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
Finally, the predicted amino acid sequences were analy-
sed using SignalP v1.1 [80], and important conserved pro-
tein domains were predicted using the NCBI Conserved
Domain Database v2.16 [81].

stat6, t-bet and foxp3 gene organization and
chromosome synteny
The zebrafish stat6, t-bet and foxp3 gene organization was
elucidated by comparing the zebrafish stat6, t-bet and foxp3
cDNA obtained by PCR with zebrafish chromosomes 23,
12 and 8, respectively, using gap2 [82]. Genscan, blast and
fasta were used to discover a number of other genes,
together with the already found genes (see section on
Sequence retrieval). The order of the genes surrounding
zebrafish t-bet, stat6 and foxp3 was compared with
the human chromosomes in which these genes were
found ( />to determine the level of synteny conserved around the
T-bet, STAT6 and Foxp3 genes.
Expression analysis in normal tissues
PCR with t-bet, stat6 or foxp3 primer combinations
(Table 3), Zftbet-F ⁄ Zftbet-R, Zfstat6-F ⁄ Zfstat6-R or
Zffoxp3-F ⁄ Zffoxp3-R, respectively, was performed using
spleen, head kidney, gill, gut, liver and skin cDNA as pre-
pared above (see section on cDNA production). Primers
for zebrafish b-actin,Zfbactin-F and Zfbactin-R (Table 3),
were used as an internal control for RT-PCR. PCR condi-
tions were as follows: one cycle of 94 °C for 3 min, 32
cycles of 94 °C for 30 s, 54 °C for 30 s and 72 °C for 30 s,
followed by one cycle of 72 °C for 5 min. PCR products
were separated on 1.5% agarose gels and visualized by
staining the gels in TBE buffer (Promega) containing
100 ngÆmL
)1
ethidium bromide (Sigma-Aldrich). RT-PCR
analysis was performed on four individual fish.

Quantification of expressed stat6, t-bet and
foxp3 genes in spleen or head kidney tissues
stimulated with immunostimulants (quantitative
real-time PCR)
Stimulation by immunostimulants and cDNA synthesis
Spleen and head kidney tissues were collected under sterile
conditions from freshly killed zebrafish (90 individuals).
Each spleen and head kidney was cut finely using scalpel
blades and cultured in Nunc six-well plates (Fisher Scienti-
fic) containing 5 mL L-15 medium (Invitrogen) supple-
mented with 5% fetal bovine serum (Life Technologies)
and Gibco 100 UÆmL
)1
penicillin, 100 lgÆmL
)1
streptomy-
cin (Invitrogen) at 28 °C. In each well within a plate, either
five spleens or five head kidneys were used. The three plates
containing the spleens or the head kidneys contained three
wells that were nonstimulated and three that were stimu-
lated with 10 lgÆmL
)1
PHA, 5 lgÆmL
)1
LPS or 50 lgÆmL
)1
Poly I:C for 4 h. After incubation, total RNA from these
stimulated spleens and head kidneys was extracted, and sin-
gle-stranded cDNA was prepared using the methods
detailed above (see section on cDNA production). All

cDNA was finally diluted with 100 lLof10mm Tri-
s ⁄ EDTA buffer (10 mm Tris, 1 mm EDTA, pH 8.0), and
3 lL was used as template for PCR employing the primers
described in Table 3.
Quantitative real-time PCR
Real-time amplification was performed with a single batch
of 2· SYBR green PCR Ready-Mix (Sigma) in glass capil-
laries (20 lL reaction volume) using a Light Cycler real-
time PCR machine (Roche, Burgess Hill, UK). Fluores-
cence outputs were measured and recorded at 80 °C after
each cycle for 40 cycles, and quantified by comparison with
a serial 10-fold dilution of reference samples for each pri-
mer pair used. Three reference samples were amplified dur-
ing each run in order to ensure consistency between PCR
runs. PCR primers were designed so that at least one pri-
mer in each pair straddled the predicted splicing sites, and
the suitability of each primer pair in real-time PCR assays
was tested by conventional PCR using cDNA and genomic
DNA as template. Samples loaded onto an agarose gel
stained with ethidium bromide confirmed primer pairs not
amplifying a product from genomic DNA and confirmed
Table 3. Primers used in quantitative PCR for zebrafish stat6, t-bet
and foxp3.
Primer name Sequence (5¢-to3¢) Used for
Zfbactin-F CGAGCAGGAGATGGGAACC Real-time PCR
Zfbactin-R CAACGGAAACGCTCATTGC Real-time PCR
Zfgapdh-F CGCTGGCATCTCCCTCAA Real-time PCR
Zfgapdh-R TCAGCAACACGATGGCTGTAG Real-time PCR
ZFil4-F CATCCAGAGTGTGAATGGGA Real-time PCR
ZFil4-R TTCCAGTCCCGGTATATGCT Real-time PCR

ZFmx-F TGAGTTACACGTTCAGTCAGCAATATG Real-time PCR
ZFmx-R TCTTGGTCTTTAGTTCTTATCATCTTGAGC Real-time PCR
ZFifnc-F AAGATTCTCAGCTACATAATGCACACC Real-time PCR
ZFifnc-R ATGCTCATCAGTAGATTCTGCTCAC Real-time PCR
ZFil10-F ACGCTTCTTCTTTGCGACTG Real-time PCR
ZFil10-R CACCATATCCCGCTTGAGTT Real-time PCR
Zfgata3-F GCTTCTTCCTCCTCGCTGTC Real-time PCR
Zfgata3-R TGCACTCTTTGTCTTCCTGTCG Real-time PCR
Zfstat6-F CGGTAGTCAGGAAATCAATGC Real-time PCR
Zfstat6-R ATCTGTCCAATAGTCTCGTAGG Real-time PCR
Zftbet-F ACACTGGCACTCACTGGATG Real-time PCR
Zf
tbet-R CTCCTTCACCTCCACGATGT Real-time PCR
Zffoxp3-F GCAACCAGCCTTTTCCACAAGC Real-time PCR
Zffoxp3-R GACTATATGGATGCTTCCCAGTA Real-time PCR
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 143
that a band of the correct size was amplified from cDNA.
A negative control (no template) reaction was also per-
formed for each primer pair tested. A melting curve for
each PCR was performed between 72 and 94 °C to ensure
that only a single product had been amplified. Expression
levels of zebrafish il-4 (EMBL accession no. AM403245),
gata3 [83], stat6, ifn-c [84], mx [85], t-bet, il-10 [64] and
foxp3, using the cDNA prepared above (see section on
Stimulation by immunostimulants and cDNA synthesis),
were normalized to two housekeeping genes, gapdh and b-
actin. Both housekeeping genes showed similar expression
patterns, and so gapdh was employed, and the results were
expressed as the fold change compared with the expression

level in the unstimulated control cells using the Pfaffl
method [86]. IL-4, GATA3, IFN-c, Mx and IL-10 were
included alongside the genes characterized in this investiga-
tion, as they are either involved in mammalian Th1, Th2 or
T
reg
responses or are good markers of an antiviral
response.
Statistical analysis
The assessment of statistical significance was analysed by
independent Student’s t-test. Values were considered to be
significant when P < 0.05 and P < 0.01.
Acknowledgement
This work was supported by a SORAS studentship to
S.M.
References
1 Mosmann TR & Sad S (1996) The expanding universe
of T-cell subsets: Th1, Th2 and more. Immunol Today
17, 138–146.
2 Mosmann TR, Cherwinski H, Bond MW, Giedlin MA
& Coffman RL (2005) Pillars article: two types of
murine helper T cell clone. I. Definition according to
profiles of lymphokine activities and secreted proteins.
J Immunol 175, 5–14.
3 Park H, Li ZX, Yang XO, Chang SH, Nurieva R,
Wang YH, Wang Y, Hood L, Zhu Z, Tian Q et al.
(2005) A distinct lineage of CD4 T cells regulates tissue
inflammation by producing interleukin 17. Nat Immunol
6, 1133–1141.
4 Bettelli E, Korn T & Kuchroo VK (2007) Th17: the

third member of the effector T cell trilogy. Curr Opin
Immunol 19, 652–657.
5 Ouyang WJ, Kolls JK & Zheng Y (2008) The biological
functions of T helper 17 cell effector cytokines in
inflammation. Immunity 28, 454–467.
6 Maloy KJ, Salaun L, Cahill R, Dougan G, Saunders
NJ & Powrie F (2003) CD4(+)CD25(+) T-R cells
suppress innate immune pathology through cytokine-
dependent mechanisms. J Exp Med 197, 111–119.
7 Maloy KJ & Powrie F (2001) Regulatory T cells in the
control of immune pathology. Nat Immunol 2, 816–822.
8 Harrington LE, Hatton RD, Mangan PR, Turner H,
Murphy TL, Murphy KM & Weaver CT (2005) Inter-
leukin 17-producing CD4(+) effector T cells develop
via a lineage distinct from the T helper type 1 and 2
lineages. Nat Immunol 6, 1123–1132.
9 Lucas S, Ghilardi N, Li J & de Sauvage FJ (2003)
IL-27 regulates IL-12 responsiveness of naive CD4(+)
T cells through Stat1-dependent and -independent
mechanisms. Proc Natl Acad Sci USA 100, 15047–
15052.
10 Hibbert L, Pflanz S, Malefyt RD & Kastelein RA
(2003) IL-27 and IFN-alpha signal via Stat1 and Stat3
and induce T-Bet and IL-12R beta 2 in naive T cells.
J Interferon Cytokine Res 23, 513–522.
11 Szabo SJ, Kim ST, Costa GL, Zhang XK, Fathman
CG & Glimcher LH (2000) A novel transcription factor,
T-bet, directs Th1 lineage commitment. Cell 100,
655–669.
12 Weaver CT, Hatton RD, Mangan PR & Harrington LE

(2007) IL-17 family cytokines and the expanding diver-
sity of effector T cell lineages. Annu Rev Immunol 25,
821–852.
13 Ouyang WJ, Lohning M, Gao ZG, Assenmacher M,
Ranganath S, Radbruch A & Murphy KM (2000)
Stat6-independent GATA-3 autoactivation directs IL-4-
independent Th2 development and commitment. Immu-
nity 12, 27–37.
14 Yamashita M, Ukai-Tadenuma M, Miyamoto T,
Sugaya K, Hosokawa H, Hasegawa A, Kimura M,
Taniguchi M, DeGregori J & Nakayama T (2004)
Essential role of GATA3 for the maintenance of type 2
helper T (Th2) cytokine production and chromatin
remodeling at the Th2 cytokine gene loci. J Biol Chem
279, 26983–26990.
15 Zhu JF, Min B, Hu-Li J, Watson CJ, Grinberg A,
Wang Q, Killeen N, Urban JF, Guo LY & Paul WE
(2004) Conditional deletion of Gata3 shows its essential
function in T(H)1–T(H)2 responses. Nat Immunol 5,
1157–1165.
16 Yoshimoto T, Yoshimoto T, Yasuda K, Mizuguchi J &
Nakanishi K (2007) IL-27 suppresses Th2 cell develop-
ment and Th2 cytokines production from polarized Th2
cells: A novel therapeutic way for Th2-mediated allergic
inflammation. J Immunol 179, 4415–4423.
17 Chen Z & O’Shea JJ (2008) Th17 cells: a new fate for
differentiating helper T cells. Immunol Res 41 , 87–102.
18 Romagnani S (2006) Regulation of the T cell response.
Clin Exp Allergy 36, 1357–1366.
19 Fontenot JD, Gavin MA & Rudensky AY (2003)

Foxp3 programs the development and function of
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
144 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
CD4(+)CD25(+) regulatory T cells. Nat Immunol 4,
330–336.
20 Roncarolo MG, Gregori S, Battaglia M, Bacchetta R,
Fleischhauer K & Levings MK (2006) Interleukin-10-
secreting type 1 regulatory T cells in rodents and
humans. Immunol Rev 212, 28–50.
21 Whyte SK (2007) The innate immune response of finfish
– a review of current knowledge. Fish Shellfish Immunol
23, 1127–1151.
22 Bird S, Zou J & Secombes CJ (2006) Advances in fish
cytokine biology give clues to the evolution of a com-
plex network. Curr Pharm Des 12, 3051–3069.
23 Secombes CJ, Zou J & Bird S (2009) Fish cytokines:
discovery, activities and potential applications. In Fish
Defenses, Vol. 1 (Zaccone G, Meseguer J & Garcia-
Ayala AK eds), pp. 1–36. Science Publishers, Enfield,
NH.
24 Burge CB & Karlin S (1998) Finding the genes in geno-
mic DNA. Curr Opin Struct Biol 8, 346–354.
25 Pearson WR & Lipman DJ (1988) Improved tools for
biological sequence comparison. Proc Natl Acad Sci
USA 85, 2444–2448.
26 Altschul SF, Gish W, Miller W, Myers EW & Lipman
DJ (1990) Basic Local Alignment Search Tool. J Mol
Biol 215, 403–410.
27 Takizawa F, Araki K, Kobayashi I, Moritomo T,
Ototake M & Nakanishi T (2008) Molecular cloning

and expression analysis of T-bet in ginbuna crucian
carp (Carassius auratus langsdorfii). Mol Immunol 45,
127–136.
28 Guo CJ, Zhang YF, Yang LS, Yang XB, Wu YY, Liu
D, Chen WJ, Weng SP, Yu XQ & He JG (2009) The
JAK and STAT family members of the mandarin fish
Siniperca chuatsi: molecular cloning, tissues distribution
and immunobiological activity. Fish Shellfish Immunol
27, 349–359.
29 Bird S, Zou J, Kono T, Sakai M, Dijkstra JM &
Secombes C (2005) Characterisation and expression
analysis of interleukin 2 (IL-2) and IL-21 homologues
in the Japanese pufferfish, Fugu rubripes, following their
discovery by synteny. Immunogenetics 56, 909–923.
30 Kono T, Bird S, Sonoda K, Savan R, Secombes CJ &
Sakai M (2008) Characterization and expression
analysis of an interleukin-7 homologue in the Japanese
pufferfish, Takifugu rubripes. Febs J 275, 1213–1226.
31 Bird S, Zou J, Savan R, Kono T, Sakai M, Woo J &
Secombes C (2005) Characterisation and expression
analysis of an interleukin 6 homologue in the Japanese
pufferfish, Fugu rubripes. Dev Comp Immunol 29, 775–
789.
32 Zou J, Bird S, Truckle J, Bols N, Horne M & Secombes
C (2004) Identification and expression analysis of an
IL-18 homologue and its alternatively spliced form in
rainbow trout (Oncorhynchus mykiss). Eur J Biochem
271, 1913–1923.
33 Sachs AB (1993) Messenger-RNA degradation in
eukaryotes. Cell 74, 413–421.

34 Caput D, Beutler B, Hartog K, Thayer R, Brownshimer
S & Cerami A (1986) Identification of a common nucle-
otide-sequence in the 3¢-untranslated region of messen-
ger-RNA molecules specifying inflammatory mediators.
Proc Natl Acad Sci USA 83, 1670–1674.
35 Naiche LA, Harrelson Z, Kelly RG & Papaioannou VE
(2005) T-box genes in vertebrate development. Annu
Rev Genet 39, 219–239.
36 Smith J (1997) Brachyury and the T-box genes. Curr
Opin Genet Dev 7, 474–480.
37 Zhang WX & Yang SY (2000) Cloning and character-
ization of a new member of the T-box gene family.
Genomics 70, 41–48.
38 Lim CP & Cao XM (2006) Structure, function, and
regulation of STAT proteins. Mol Biosyst 2, 536–550.
39 Chen XM, Vinkemeier U, Zhao YX, Jeruzalmi D,
Darnell JE & Kuriyan J (1998) Crystal structure of a
tyrosine phosphorylated STAT-1 dimer bound to DNA.
Cell 93, 827–839.
40 Hou JZ, Schindler U, Henzel WJ, Ho TC, Brasseur M
& McKnight SL (1994) An interleukin-4-induced tran-
scription factor – IL-4 Stat. Science 265, 1701–1706.
41 Quelle FW, Shimoda K, Thierfelder W, Fischer C, Kim
A, Ruben SM, Cleveland JL, Pierce JH, Keegan AD,
Nelms K et al. (1995) Cloning of murine Stat6 and
human Stat6, Stat proteins that are tyrosine-phosphory-
lated in response to IL-4 and IL-3 but are not required
for mitogenesis. Mol Cell Biol 15, 3336–3343.
42 Carlsson P & Mahlapuu M (2002) Forkhead transcrip-
tion factors: key players in development and metabo-

lism. Dev Biol 250, 1–23.
43 Li B, Samanta A, Song XM, Furuuchi K, Iacono KT,
Kennedy S, Katsumata M, Saouaf SJ & Greene MI
(2006) FOXP3 ensembles in T-cell regulation. Immunol
Rev 212, 99–113.
44 Ziegler SF (2006) FOXP3: Of mice and men. Annu Rev
Immunol 24, 209–226.
45 Allan SE, Passerini L, Bacchetta R, Crellin N, Dai
MY, Orban PC, Ziegler SF, Roncarolo MG & Levings
MK (2005) The role of 2 FOXP3 isoforms in the gen-
eration of human CD4(+) Tregs. J Clin Invest 115,
3276–3284.
46 Montpetit A, Wilson MD, Chevrette M, Koop BF &
Sinnett D (2003) Analysis of the conservation of
synteny between Fugu and human chromosome 12.
BMC Genomics 4, 30, doi:10.1186/1471-2164-4-30.
47 McLysaght A, Enright AJ, Skrabanek L & Wolfe KH
(2000) Estimation of synteny conservation and genome
compaction between pufferfish (Fugu) and human.
Yeast 17, 22–36.
48 Postlethwait JH, Woods IG, Ngo-Hazelett P, Yan YL,
Kelly PD, Chu F, Huang H, Hill-Force A & Talbot
WS (2000) Zebrafish comparative genomics and the
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 145
origins of vertebrate chromosomes. Genome Res 10,
1890–1902.
49 Brunkow ME, Jeffery EW, Hjerrild KA, Paeper B,
Clark LB, Yasayko SA, Wilkinson JE, Galas D, Ziegler
SF & Ramsdell F (2001) Disruption of a new fork-

head ⁄ winged-helix protein, scurfin, results in the fatal
lymphoproliferative disorder of the scurfy mouse. Nat
Genet 27, 68–73.
50 Karanikas V, Speletas M, Zamanakou M, Kalala F,
Loules G, Kerenidi T, Barda AK, Gourgoulianis KI &
Germenis AE (2008) Foxp3 expression in human cancer
cells. J Transl Med 6, 19, doi:10.1186/1479-5876-6-19.
51 Chen GY, Chen C, Wang LZ, Chang X, Zheng P &
Liu Y (2008) Cutting edge: broad expression of the
FoxP3 locus in epithelial cells: a caution against early
interpretation of fatal inflammatory diseases following
in vivo depletion of FoxP3-expressing cells. J Immunol
180, 5163–5166.
52 Shmueli O, Horn-Saban S, Chalifa-Caspi V, Shmoish
M, Ophir R, Benjamin-Rodrig H, Safran M, Domany
E & Lancet D (2003) GeneNote: whole genome expres-
sion profiles in normal human tissues. CR Biol 326,
1067–1072.
53 Yanai I, Benjamin H, Shmoish M, Chalifa-Caspi V,
Shklar M, Ophir R, Bar-Even A, Horn-Saban S, Safran
M, Domany E et al. (2005) Genome-wide midrange
transcription profiles reveal expression level relation-
ships in human tissue specification. Bioinformatics 21,
650–659.
54 Murphy KM & Reiner SL (2002) The lineage decisions
of helper T cells. Nat Rev Immunol 2, 933–944.
55 Ranganath S, Ouyang WJ, Bhattarcharya D, Sha WC,
Grupe A, Peltz G & Murphy KM (1998) Cutting edge:
GATA-3-dependent enhancer activity in IL-4 gene
regulation. J Immunol 161, 3822–3826.

56 Mikita T, Campbell D, Wu PG, Williamson K & Schin-
dler U (1996) Requirements for interleukin-4-induced
gene expression and functional characterization of
Stat6. Mol Cell Biol 16, 5811–5820.
57 Kurata H, Lee HJ, O’Garra A & Arai N (1999) Ectopic
expression of activated Stat6 induces the expression of
Th2-specific cytokines and transcription factors in
developing Th1 cells. Immunity 11, 677–688.
58 Kubo M, Ransom J, Webb D, Hashimoto Y, Tada T
& Nakayama T (1997) T-cell subset-specific expression
of the IL-4 gene is regulated by a silencer element and
STAT6. EMBO J 16, 4007–4020.
59 Su RC, Becker AB, Kozyrskyj AL & HayGlass KT
(2008) Epigenetic regulation of established human type
1 versus type 2 cytokine responses. J Allergy Clin Immu-
nol 121, 57–63.
60 Stolte EH, Savelkoul HFJ, Wiegertjes G, Flik G &
Verburg-van Kemenade BML (2008) Differential expres-
sion of two interferon-gamma genes in common carp
(Cyprinus carpio L.). Dev Comp Immunol 32, 1467–1481.
61 Mann MK, Maresz K, Shrives LP, Tan YP & Dittel
BN (2007) B cell regulation of CD4(+)CD25(+) T reg-
ulatory cells and IL-10 via B7 is essential for recovery
from experimental autoimmune encephalomyelitis.
J Immunol 178, 3447–3456.
62 Bryn T, Yaqub S, Mahic M, Henjum K, Aandahl EM
& Tasken K (2008) LPS-activated monocytes suppress
T-cell immune responses and induce FOXP3+T cells
through a COX-2-PGE(2)-dependent mechanism. Int
Immunol 20, 235–245.

63 Crellin NK, Garcia RV, Hadisfar O, Allan SE, Steiner
TS & Levings MK (2005) Human CD4(+) T cells
express TLR5 and its ligand flagellin enhances the
suppressive capacity and expression of FOXP3 in
CD4(+)CD25(+) T regulatory cells. J Immunol 175,
8051–8059.
64 Zhang DC, Shao YQ, Huang YQ & Jiang SG (2005)
Cloning, characterization and expression analysis of
interleukin-10 from the zebrafish (Danio rerio). J Bio-
chem Mol Biol 38, 571–576.
65 Cao SJ, Liu JG, Song LH & Ma XJ (2005) The
protooncogene c-Maf is an essential transcription factor
for IL-10 gene expression in macrophages. J Immunol
174, 3484–3492.
66 Wanidworanun C & Strober W (1993) Predominant
role of tumor-necrosis-factor-alpha in human monocyte
IL-10 synthesis. J Immunol 151, 6853–6861.
67 Moore KW, Malefyt RD, Coffman RL & O’Garra A
(2001) Interleukin-10 and the interleukin-10 receptor.
Annu Rev Immunol 19, 683–765.
68 McGuirk P, McCann C & Mills KHG (2002) Patho-
gen-specific T regulatory 1 cells induced in the respira-
tory tract by a bacterial molecule that stimulates
interleukin 10 production by dendritic cells: A novel
strategy for evasion of protective T helper type 1
responses by Bordetella pertussis. J Exp Med 195,
221–231.
69 O’Garra A & Robinson D (2004) Development and
function of T helper 1 cells. Adv Immunol 83 , 133–162.
70 Rengarajan J, Szabo SJ & Glimcher LH (2000) Tran-

scriptional regulation of Th1 ⁄ Th2 polarization. Immunol
Today 21 , 479–483.
71 Lighvani AA, Frucht DM, Jankovic D, Yamane H,
Aliberti J, Hissong BD, Nguyen BV, Gadina M, Sher
A, Paul WE et al. (2001) T-bet is rapidly induced by
interferon-gamma in lymphoid and myeloid cells. Proc
Natl Acad Sci USA 98, 15137–15142.
72 Asare AL, Kolchinsky SA, Gao Z, Wang R, Raddassi
K, Bourcier K & Seyfert-Margolis V (2008) Differential
gene expression profiles are dependent upon method of
peripheral blood collection and RNA isolation. BMC
Genomics 9, 474, doi:10.1186/1471-2164-9-474.
73 Abdalla AO, Kiaii S, Hansson L, Rossmann ED,
Jeddi-Tehrani M, Shokri F, Osterborg A, Mellstedt H
& Rabbani H (2003) Kinetics of cytokine gene
Zebrafish T-cell transcription factors t-bet, stat6 and foxp3 S. Mitra et al.
146 FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS
expression in human CD4(+) and CD8(+) T-lympho-
cyte subsets using quantitative real-time PCR. Scand
J Immunol 58, 601–606.
74 Birnboim HC (1983) A rapid alkaline extraction
method for the isolation of plasmid DNA. Methods
Enzymol 100, 243–255.
75 Campanella JJ, Bitincka L & Smalley J (2003)
MatGAT: an application that generates similarity ⁄
identity matrices using protein or DNA sequences.
BMC Bioinformatics 4, 29, doi:10.1186/1471-2105-4-29.
76 Thompson JD, Gibson TJ, Plewniak F, Jeanmougin F
& Higgins DG (1997) The CLUSTAL_X windows
interface: flexible strategies for multiple sequence align-

ment aided by quality analysis tools. Nucleic Acids Res
25, 4876–4882.
77 Saitou N & Nei M (1987) The neighbor-joining method
– a new method for reconstructing phylogenetic trees.
Mol Biol Evol 4 , 406–425.
78 Page RDM (1996) TreeView: an application to display
phylogenetic trees on personal computers. Comput Appl
Biosci 12, 357–358.
79 Felsenstein J (1985) Confidence-limits on phylogenies
– an approach using the bootstrap. Evolution 39, 783–
791.
80 Nielsen H, Engelbrecht J, Brunak S & vonHeijne G
(1997) Identification of prokaryotic and eukaryotic
signal peptides and prediction of their cleavage sites.
Protein Eng 10, 1–6.
81 Marchler-Bauer A, Anderson JB, Derbyshire MK,
DeWeese-Scott C, Gonzales NR, Gwadz M, Hao LN,
He SQ, Hurwitz DI, Jackson JD et al. (2007) CDD: a
conserved domain database for interactive domain
family analysis. Nucleic Acids Res 35, D237–D240.
82 Huang XQ (1994) On global sequence alignment.
Comput Appl Biosci 10, 227–235.
83 Neave B, Rodaway A, Wilson SW, Patient R & Holder
N (1995) Expression of zebrafish Gata-3 (Gta3) during
gastrulation and neurulation suggests a role in the
specification of cell fate. Mech Dev 51, 169–182.
84 Zou J, Carrington A, Collet B, Dijkstra JM, Yoshiura
Y, Bols N & Secombes C (2005) Identification and
bioactivities of IFN-gamma in rainbow trout Oncorhyn-
chus mykiss: the first Th1-type cytokine characterized

functionally in fish. J Immunol 175, 2484–2494.
85 Altmann SM, Mellon MT, Johnson MC, Paw BH,
Trede NS, Zon LI & Kim CH (2004) Cloning and
characterization of an Mx gene and its corresponding
promoter from the zebrafish, Danio rerio. Dev Comp
Immunol 28, 295–306.
86 Pfaffl MW (2001) A new mathematical model for
relative quantification in real-time RT-PCR. Nucleic
Acids Res 29, e45.
S. Mitra et al. Zebrafish T-cell transcription factors t-bet, stat6 and foxp3
FEBS Journal 277 (2010) 128–147 ª 2009 The Authors Journal compilation ª 2009 FEBS 147