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,

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

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

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

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

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-

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-

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

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 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

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

-
-
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

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/
-
-

-
-
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

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

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

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

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

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

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-

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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-

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

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].

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

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

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