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 fox
Trang 1factors 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: s.bird@abdn.ac.uk
(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 (Treg) 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 Treg 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 Tregcell 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; Treg, T-regulatory.
Trang 2responses [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 (Treg) 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 Tregcell 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, Tregcells 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 Treg cells, although the most important appear to be CD4+CD25+Foxp3+Treg [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] Treg
suppressive activity is via IL-10 and TGF-b, although
it remains unclear whether these cytokines are produced by CD4+CD25+Foxp3+Treg 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 Treg 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 Treg 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 Tregcells may exist in fish In this inves-tigation, we have identified, for the first time, t-bet and stat6in zebrafish and, for the first time in any fish spe-cies, foxp3 Lastly, we carried out some preliminary
Trang 3expression 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.
Trang 4Fig 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.
Trang 5t-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.
Trang 6found 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-bindTBK1-bind-ing 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.
Trang 7X 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
Mouse
T-bet
Zebrafish
t-bet
Ginbuna
T-bet
Human
STAT6
Mouse
STAT6
Zebrafish
stat6
Tetraodon
STAT6
Human
Foxp3
Mouse
Foxp3
Zebrafish
foxp3
Above diagonal, identity; below diagonal, similarity.
Trang 8many 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 DOG TBX2 MOUSE TBX2 ZEBRAFISH TBX2 XENOPUSTR TBX2 MOUSE TBX3 HUMANTBX3 CHICKEN TBX3 HUMAN TBX6 MOUSE TBX6 XENOPUSTR TBX6 ZEBRAFISH TBX6 HUMAN T -BET MOUSE T-BET ZEBRAFISH T-BET GINBUNACARP T -BET 58
MOUSE TBX20 HUMAN TBX20 CHICKEN TBX20 XENOPUSTR TBX20 ZEBRAFISH TBX20 MOUSE TBX15 HUMAN TBX15 MOUSE TBX18 HUMAN TBX18 MOUSE TBX1 HUMAN TBX1 XENOPUSTR TBX1 MOUSE TBX10 HUMAN TBX10 60
MOUSE TBX5 RAT TBX5 HUMAN TBX5 CHICKEN TBX5 XENOPUSLA TBX5 ZEBRAFISH TBX5 DOG TBX4 HUMAN TBX4 0.1
TBOX-2/-3
TBOX-6/-21
TBOX-20
TBOX-15/-18
TBOX-1/-10
TBOX-4/-5
HUMAN TBX2 DOG TBX2 MOUSE TBX2 ZEBRAFISH TBX2 XENOPUSTR TBX2 MOUSE TBX3 HUMANTBX3 CHICKEN TBX3 HUMAN TBX6 MOUSE TBX6 XENOPUSTR TBX6 ZEBRAFISH TBX6 HUMAN T -BET MOUSE T-BET ZEBRAFISH T-BET GINBUNACARP T -BET 58
MOUSE TBX20 HUMAN TBX20 CHICKEN TBX20 XENOPUSTR TBX20 ZEBRAFISH TBX20 MOUSE TBX15 HUMAN TBX15 MOUSE TBX18 HUMAN TBX18 MOUSE TBX1 HUMAN TBX1 XENOPUSTR TBX1 MOUSE TBX10 HUMAN TBX10 60
MOUSE TBX5 RAT TBX5 HUMAN TBX5 CHICKEN TBX5 XENOPUSLA TBX5 ZEBRAFISH TBX5 DOG TBX4 HUMAN TBX4 0.1
TBOX-2/-3
TBOX-6/-21
TBOX-20
TBOX-15/-18
TBOX-1/-10
TBOX-4/-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 accesacces-sion 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.
Trang 9instability 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
XENOPUSLA STAT1 CHICKEN STAT1 MOUSE STAT1 PIG STAT1 HUMAN STAT1 SALMON STAT1 TETRAODON STAT1 HALIBUT STAT1 SNAKEHEAD STAT1 CHICKEN STAT4 MOUSE STAT4 HUMAN STAT4 ZEBRAFISH STAT4 FUGU STAT4 TETRAODON STAT4
MOUSE STAT2 PIG STAT2 HUMAN STAT2
HUMAN STAT6 MOUSE STAT6 ZEBRAFISH STAT6 TETRAODON STAT6 HUMAN STAT5 COW STAT5 PIG STAT5
58
MOUSE STAT5 RAT STAT5 TROUT STAT5 ZEBRAFISH STAT5
63
FUGU STAT5 TETRAODON STAT5 TROUT STAT3
ZEBRAFISH STAT3 TETRAODON STAT3 MEDAKA STAT3
55
CHICKEN STAT3 MOUSE STAT3 RAT STAT3
71
PIG STAT3 HUMAN STAT3
49
0.1
XENOPUSLA STAT1 CHICKEN STAT1 MOUSE STAT1 PIG STAT1 HUMAN STAT1 SALMON STAT1 TETRAODON STAT1 HALIBUT STAT1 SNAKEHEAD STAT1 CHICKEN STAT4 MOUSE STAT4 HUMAN STAT4 ZEBRAFISH STAT4 FUGU STAT4 TETRAODON STAT4
MOUSE STAT2 PIG STAT2 HUMAN STAT2
HUMAN STAT6 MOUSE STAT6 ZEBRAFISH STAT6 TETRAODON STAT6 HUMAN STAT5 COW STAT5 PIG STAT5
58
MOUSE STAT5 RAT STAT5 TROUT STAT5 ZEBRAFISH STAT5
63
FUGU STAT5 TETRAODON STAT5 TROUT STAT3
ZEBRAFISH STAT3 TETRAODON STAT3 MEDAKA STAT3
55
CHICKEN STAT3 MOUSE STAT3 RAT STAT3
71
PIG STAT3 HUMAN STAT3
49
STAT-1
STAT-4
STAT-2 STAT-6
STAT-5
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.
Trang 10were 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
XENOPUS FOXP4
MOUSE FOXP4
HUMAN FOXP4
ZEBRAFISH FOXP3
MOUSE FOXP3
HUMAN FOXP3
MACAQUE FOXP3
XENOPUS FOXP2
HUMAN FOXP2
MACAQUE FOXP2
MOUSE FOXP2
ZEBRAFISH FOXP1
XENOPUS FOXP1
CHICKEN FOXP1
RAT FOXP1
MOUSE FOXP1
COW FOXP1
HUMAN FOXP1 50
0.1
XENOPUS FOXP4
MOUSE FOXP4
HUMAN FOXP4
ZEBRAFISH FOXP3
MOUSE FOXP3
HUMAN FOXP3
MACAQUE FOXP3
XENOPUS FOXP2
HUMAN FOXP2
MACAQUE FOXP2
MOUSE FOXP2
ZEBRAFISH FOXP1
XENOPUS FOXP1
CHICKEN FOXP1
RAT FOXP1
MOUSE FOXP1
COW FOXP1
HUMAN FOXP1 50
FOXP4
FOXP3
FOXP2
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.