MINIREVIEW
Evolutionary changes to transthyretin: developmentally
regulated and tissue-specific gene expression
Kiyoshi Yamauchi and Akinori Ishihara
Department of Biological Science, Faculty of Science, Shizuoka University, Japan
Introduction
Thyroid hormones (THs) are lipophilic hormones that
modulate growth and development. As such, and to
ensure the distribution of adequate amounts of THs to
target tissues, most THs in blood are bound to specific
carrier proteins. In large eutherians, the major TH-car-
rier proteins are thyroxine-binding globulin (TBG),
transthyretin and albumin [1], which are synthesized in
the liver and then secreted into the bloodstream. The
TH-carrier proteins form the TH distribution network
[2] that maintains a pool of THs in the bloodstream
and facilitates the uniform distribution of THs to
target cells. The TH distribution network, in part, is
functionally redundant. This assumption is derived
from the observations that transthyretin-null mice and
patients with albumin and TBG deficiencies are euthy-
roid [2]. If the TH distribution network lacks one
TH-carrier protein, the remaining proteins maintain
the uniform distribution of THs to target cells.
The composition of the TH distribution network dif-
fers among species. TBG has been detected in the
plasma of juvenile and adult large eutherians, rodents
and some marsupials at postnatal or pouch stages [3].
However, the presence of TBG in the plasma of
lower vertebrates has not been reported. By contrast,
Keywords
developmental regulation; thyroid hormone;
tissue specificity; transcriptional control;
transthyretin
Correspondence
K. Yamauchi, Department of Biological
Science, Faculty of Science, Shizuoka
University, Shizuoka 422-8529, Japan
Fax: +81 54 238 0986
Tel: +81 54 238 4777
E-mail: sbkyama@ipc.shizuoka.ac.jp
(Received 2 February 2009, revised 21 June
2009, accepted 21 July 2009)
doi:10.1111/j.1742-4658.2009.07245.x
A survey of the expression of the transthyretin and thyroxine-binding glob-
ulin genes in various species during development provides clues as to how
the present thyroid hormone distribution network in extracellular compart-
ments developed during vertebrate evolution. Albumin may be the ‘oldest’
component of the thyroid hormone distribution network as it is found in
the plasma of all vertebrates investigated. Subsequent to albumin, transthy-
retin appeared as the second component in this network during the evolu-
tion of vertebrates. The strong expression of transthyretin genes in the liver
coincides with the presence of recognition site(s) for liver-enriched tran-
scription factors, such as HNF-3b(Foxa2), in the transthyretin promoter
regions of vertebrates. Finally, the addition of thyroxine-binding globulin
to this network occurred at postnatal stages in some marsupials and
rodents and in perinatal to adult stages in most eutherians. All vertebrates
have defined developmental stages when thyroid hormone-dependent tran-
sition from larval to juvenile forms occurs. The inclusion of transthyretin
and thyroxine-binding globulin in the thyroid hormone distribution
network may be correlated with the increased requirement of thyroid
hormones for thyroid hormone-dependent tissue remodeling during these
stages and ⁄or increased metabolism in thyroid hormone-target tissues with
the acquisition of homeothermy.
Abbreviations
C/EBP, CCAAT/enhancer binding protein; E, embryonic day; HNF, hepatic nuclear factor; P, postnatal day; TBG, thyroxin-binding globulin;
TH, thyroid hormone.
FEBS Journal 276 (2009) 5357–5366 ª2009 The Authors Journal compilation ª2009 FEBS 5357
transthyretin has been detected in the plasma of fetal
and adult eutherians, and of some marsupials and
birds, but has not been detected, or has been detected
only at low levels, in the plasma of adult reptiles,
amphibians and fish. In these species, the expression of
transthyretin is modulated in developmentally specific
and species-specific manners [3]. Albumin is common
to all vertebrates (from cyclostomes to mammals) that
have been investigated, suggesting that from an evolu-
tionary perspective, it is the oldest TH-carrier protein.
The TH-carrier proteins are predominantly synthe-
sized in the liver. Transthyretin is secreted from the
choroid plexus into the cerebrospinal fluid of adult
mammals, birds and reptiles, in which it is a predomi-
nant protein. Transthyretin is also expressed in other
tissues in various species (e.g. in the retina of rats, in
the pancreas of humans and rats, in the heart of devel-
oping chickens and in the visceral yolk sac of rats
during fetal development) [2]. Recent studies have sug-
gested that the extrahepatic expression of transthyretin
is more predominant in lower vertebrates than in
higher vertebrates [4].
In this minireview, from an evolutionary point of
view, we survey (a) the developmental and tissue-spe-
cific regulation of the expression of TH-carrier protein
genes, in particular transthyretin, in various vertebrates
and (b) candidates for transcription factors involved in
transthyretin gene expression in the liver and possible
cis-elements in the transthyretin promoter regions, and
demonstrate the close link between the high expression
of TH-carrier protein genes in the liver and develop-
mental stages when THs are absolutely required for
TH-dependent tissue remodelling in each vertebrate
group.
Regulation of transthyretin gene
expression during development
In this section of our article, we will discuss the regula-
tion of transthyretin gene expression during develop-
ment in rat, marsupials, chicken, reptiles, amphibians
and fish (Fig. 1).
Rat
Transthyretin is expressed in extra-embryonic and
embryonic tissues during early embryogenesis, and its
expression is gradually confined to the liver and to the
choroid plexus during late embryogenesis. In rat
embryos, transthyretin transcripts were detected (by
in situ hybridization) in the visceral endoderm as early
as embryonic day (E) 7 [5]. At E9, the expression of
transthyretin was confined to the visceral extraembry-
onic endoderm and the foregut endoderm [5]. Trans-
thyretin transcripts were detected at increasingly higher
levels in the visceral yolk sac endoderm and in the fetal
liver from E10 to E13 [5], and throughout gestation
until birth (E14–E20) [6]. The expression of transthyre-
tin (detected using northern blot analysis) was rela-
tively higher in the visceral yolk sac than in the fetal
liver from E14 to E20 [6]. As the visceral yolk sac is
the site of true placentation in rodents, the expression
of transthyretin in the visceral yolk sac may assist the
transport and delivery of THs from the maternal blood
to the developing fetuses.
In other tissues of the developing rat, transthyretin
gene expression occurs in the brain when neuroepithe-
lial cells derived from the neural tube differentiate into
the epithelial cells of the choroid plexus in both the
hindbrain and forebrain. Transthyretin transcripts first
appear in the fourth ventricle at E12.5, in the lateral
ventricles at E13.5 and subsequently in the third ven-
tricle [7]. Transthyretin expression has also been
reported in the eye, heart, pancreas, skeletal muscle,
spleen and stomach in developing or adult rats [4,8].
Marsupials
The expression and synthesis of transthyretin in marsu-
pials is evident in the evolutionarily older polyproto-
dont marsupials, which inhabit America and Australia,
and in the recently diverged diprotodont marsupials,
which inhabit Australia [2]. In adult marsupials, trans-
thyretin was detected in the plasma from American
polyprotodont marsupials (e.g. Virginia opossum) and
Australian diprotodont marsupials (e.g. tammar wal-
laby, scaly-tailed possum and common wombat) but
not in the plasma from Australian polyprotodont mar-
supials (e.g. fat-tailed dunnart and Tasmanian devil),
even though albumin was detected in all marsupials
investigated [2,3,9,10]. Transthyretin gene expression
was observed in the choroid plexus of all adult mar-
supials studied, in the American and Australian
polyprotodonts (e.g. short-tailed grey opossum and
stripe-faced dunnart, respectively) and in an Australian
diprotodont (e.g. sugar glider) [11].
As a typical example, transthyretin gene expression
during development of the tammar wallaby (Macro-
pus eugenii, an Australian diprotodont marsupial) is
shown in Fig. 1. Transthyretin transcripts were
detected in the liver during pouch life [postnatal day
(P) 3 to P200], when the young animal does not have a
mature thyroid gland and obtains THs from its
mother’s milk. Transthyretin expression in the liver
continues through pouch life to adulthood [12]. This
was confirmed by binding studies of [
125
I]thyroxine
Transthyretin gene expression during development K. Yamauchi and A. Ishihara
5358 FEBS Journal 276 (2009) 5357–5366 ª2009 The Authors Journal compilation ª2009 FEBS
with blood plasma [12]. However, in the fat-tailed
dunnart (an Australian polyprotodont marsupial),
transthyretin was detected in plasma during a defined
developmental stage of pouch life, from P27 to P62 [3].
This defined developmental stage corresponds to that
in the young tammar wallaby during which thyroxine-
binding protein was detected in plasma [3].
Chicken
Transthyretin is known to accumulate in oocytes from
the circulation [13]. THs also accumulate in the yolk
during oogenesis. As THs are necessary for normal
embryonic development, these observations suggest
that before the vascular system and the hypothalamic–
pituitary–thyroid axis are established, transthyretin
plays a role in the transport and distribution of THs
from the yolk to specific embryonic sites.
Transthyretin may have a role in heart development
in the chicken as it was identified as one of the main
proteins secreted from cultured anterior lateral endo-
derm (which may play a part in cardiogenesis), in the
chick embryo at E1.5–E3.5 (corresponding to stages
3–21) before the onset of vascular system development
Fig. 1. Developmentally regulated transthy-
retin expression profile in various verte-
brates. Developmental windows and tissues
of various vertebrates in which transthyretin
expression has been reported, are summa-
rized with those for TBG. Data are from in
the rat (Rattus norvegicus) [5–8,55], marsu-
pial [tammar wallaby (Macropus eugenii)]
[3,12], chicken (Gallus gallus) [14–16], reptile
[saltwater crocodile (Crocodylus porosus)]
[3,20], frog [African clawed toad (Xeno-
pus laevis)] [25] and fish [sea bream (Spa-
rus aurata)] [4,26,27]. Black and gray bars
represent the developmental stages or peri-
ods during which transthyretin or its tran-
scripts, and TBG or its transcripts, are
detected, respectively. In this figure, the
ages at weaning in eutherians, initial pouch
exit in marsupials, at hatch in birds and rep-
tiles, completion of metamorphosis in
amphibians and transition of larva to juvenile
in fish are adjusted as a period of postem-
bryonic transformation in vertebrates. The
day of development: E, embryonic day; P,
postnatal day; dph, day post-hatch. AP,
anterolateral portion of embryo; CP, choroid
plexus; EE, extraembryonic endoderm; FE,
foregut endoderm; MA, midline in the ante-
rior half of embryo; PC, pyloric cacea; PE,
posterior endoderm; VE, visceral endoderm;
VY, visceral yolk sac; B, brain; E, eye; Gi,
gill; Gia, gill arch; Gif, gill filaments; Go,
gonad; H, heart muscle; I, intestine; K,
kidney; Li, liver; Lu, lung; Pa, pancreas; Pi,
pituitary; Sp, spleen; Sk, skin; Sm, skeletal
muscle; St, stomach; T, testis.
K. Yamauchi and A. Ishihara Transthyretin gene expression during development
FEBS Journal 276 (2009) 5357–5366 ª2009 The Authors Journal compilation ª2009 FEBS 5359
[14]. This transthyretin is not of maternal origin
and is synthesized de novo in the embryo. In situ
hybridization located transthyretin transcripts to the
extraembryonic endoderm at E1.0 (stage 6), to the
anterolateral portion of the embryo proper at E1.0–
E1.1 (stage 7) and near the midline in the anterior half
of the embryo at E1.1–E1.4 (stages 8–9). At E1.8–E2.1
(stages 11–14), transthyretin transcripts were also
detected in myocardial cells of the definitive heart.
During later developmental stages in chicks, the
main sites of transthyretin expression are the choroid
plexus and the liver. The amount of transthyretin tran-
scripts in the choroid plexus increased gradually from
E9 (stage 35) to 3 weeks after hatching [15]. By con-
trast, the amount of transthyretin transcripts in the
liver increased to adult levels by E7 (stage 30). At E6
and E8 (stages 28–29 and 33–34), transthyretin expres-
sion was also detected (by northern blot analysis) in
the eye, but not in the intestine and lung [15]. In adult
chickens, transthyretin gene expression was detected
(by RT-PCR) in the kidney, lung, spleen and intestine
[16]. Transthyretin expression was also observed in the
choroid plexus and liver of adult ducks, quails and
pigeons [15].
Reptiles
Transthyretin was first detected in reptiles by culturing
choroid plexus dissected from the Australian stumpy-
tailed lizards (Tiliqua rugosa) with [
14
C]leucine,
followed by analyses using SDS ⁄PAGE and fluorogra-
phy [17]. Interestingly, transthyretin transcripts were
detected (by northern blot analysis) in the choroid
plexus, but not in the liver, of adult lizards [18], and
were detected (by RT-PCR) in the eye and kidney, but
not in the liver [18]. Transthyretin was found to be
highly expressed in the choroid plexus of the turtle
(Trachemys scripta) [19] and the juvenile saltwater croc-
odile (Crocodylus porosus) [20]. By contrast, only small
amounts of transthyretin transcripts were detected in
the liver, and none in the kidney, of the turtles. In the
crocodiles, the second major expression site of the
transthyretin gene was the eye. Transthyretin tran-
scripts were not detected in the heart and liver of the
crocodiles. Recently, transthyretin was detected in the
plasma from embryonic saltwater crocodiles before and
at hatching (E60 to 1 day after hatching), but not in
plasma from juvenile or adult saltwater crocodiles [3].
Amphibians
During early development of amphibians, transthyretin
is expressed in embryonic endoderm. Transthyretin
gene expression was investigated as a tissue marker of
the differentiating liver in the African clawed toad,
Xenopus laevis, during embryonic development by
whole-mount in situ hybridization and RT-PCR
[21,22]. Transthyretin transcripts were expressed in a
more posterior stripe of endoderm (intestine) but not
the liver diverticulum of the tail bud embryo at E1.5
(stages 30–35) [21,22]. Initially, the expression of the
transthyretin transcripts was widespread and included
the mesoderm of the kidney, at E3 (stages 41–42), but
was gradually restricted to the liver by E7 (stages 46–
47) [21]. According to the expression characteristics,
genes for embryonic endoderm development in X.la-
evis were categorized into four groups: (a) the liver-
specific group, (b) the liver and intestine group A, (c)
the liver and intestine group B and (d) the intestine-
specific group [22]. Expression of genes in the liver and
intestine group A are required for the continuous and
specific cell–cell interactions of the endoderm and
includes the transthyretin gene.
During metamorphosis, the predominant site of
transthyretin expression is the liver. Transthyretin gene
expression was detected (by in situ hybridization) in
the liver but not in the choroid plexus of the American
bullfrog, Rana catesbeiana, from premetamorphic to
prometamorphic stages [23]. These stages correspond
to the point in time when the concentrations of TH in
plasma increase and reach maximum levels [24]. How-
ever, transthyretin transcript levels decreased gradually
during the metamorphic climax stages in X.laevis [25]
and R.catesbeiana [23]. Transthyretin transcripts have
not been detected (by northern blot analysis) in adult
tissues of either species.
Fish
During the development of fish, transthyretin is
expressed in the liver. Transthyretin was first detected
in juvenile fish: the sea bream Sparus aurata [26, 27],
and in smolting masu salmon Oncorhynchus masou [28].
In the sea bream, transthyretin transcripts were
expressed in hatching larvae as early as 1 day post-
hatch (48 h after fertilization). The expression of
transthyretin transcripts in the whole body of larva
increased gradually during development (from 1 day
post-hatch to 22 days post-hatch) [27]. RT-PCR
demonstrated that the transthyretin transcripts were
expressed predominantly in the liver of the sea bream
larvae, with low levels of expression found in the brain,
pituitary, gills, kidney, intestine and testis [26].The
amounts of transthyretin transcripts in tissues from the
sea bream fingerling were greatest in the liver, followed
by the brain, then the eye, gill filaments, heart and
Transthyretin gene expression during development K. Yamauchi and A. Ishihara
5360 FEBS Journal 276 (2009) 5357–5366 ª2009 The Authors Journal compilation ª2009 FEBS
kidney [27]. In adult sea breams, the major site of
expression of the transthyretin transcripts was the liver.
Transthyretin was also expressed at moderate levels in
the skin, heart, muscle and gill filaments, with minor
expression found in the spleen, kidney, gonad, pyloric
cacea, intestine, pituitary, eye, brain and gill arch [27].
Expression of the transthyretin gene in the liver dur-
ing smoltification in salmonid fish may facilitate the
transition of the freshwater dwelling parr into a smolt
adapted to the life in salt water. In the masu salmon,
transthyretin was purified from plasma obtained dur-
ing smoltification [28] when the plasma concentrations
of endogenous THs are highest. Transthyretin was also
detected in the plasma from smolting Atlantic salmon
Salmo salar and the Chinook salmon Onchorhyn-
chus tshawytscha [3].
Recently, transthyretin cDNAs were cloned from the
livers of the Pacific bluefin tuna Thunnus orientalis [29]
and from the livers of two genera of the lampreys
Petromyzon marinus and Lampetra appendix [30].
Transthyretin transcripts were detected in the liver of
1-year-old bluefin tuna, concomitant with the ovary
but not the testis. The amount of transthyretin tran-
scripts in the liver of 3-year-old bluefin tuna was less
than that in the liver of 1-year-old bluefin tuna. Trans-
thyretin transcripts were not detected in the ovaries of
sexually mature 3-year-old female bluefin tuna [29]. In
the lamprey larvae, transthyretin transcripts were
detected predominantly in the liver, with low levels of
expression in the gill, brain, heart, gut, kidney and
blood [30]. The expression of transthyretin reached a
maximum level in the liver during metamorphosis
when serum TH levels are at their lowest. In lamprey,
in contrast to the bony fish (such as flatfish) and
amphibians, the onset of metamorphosis is triggered
by a decrease in circulating TH levels. The role of TH
receptors in controlling metamorphosis must be differ-
ent in lamprey than in other vertebrates.
Control of transthyretin gene
expression
Analysis of the transthyretin promoter in mice
The upstream regulatory region of the mouse transthy-
retin gene has been investigated extensively as a model
for understanding gene expression in the liver. The
proximal region of the transthyretin promoter and a
distal enhancer are sufficient for transthyretin gene
expression in the liver, but not in the choroid plexus
[31]. These regions, as well as the regulatory regions of
other genes in the liver such as albumin, a1-antitrypsin
and a-fetoprotein, contain several recognition sites for
liver-enriched transcription factors including hepatic
nuclear factor-4 (HNF-4), three distinct HNF-3 pro-
teins (a,band c; recently renamed Foxa1, Foxa2 and
Foxa3), HNF-1 and CCAAT/enhancer binding protein
(C ⁄EBP) [32,33]. A combination of transcription
factors acting on multiple recognition sites in the
upstream regulatory region of the transthyretin gene is
required for the activation and maintenance of its tran-
scription in the liver. Of these transcription factors,
HNF-3 proteins are essential for transthyretin expres-
sion in human hepatoma HepG2 cells. HNF-3 proteins
belong to a family of transcription factors that are
homologous to the winged helix ⁄forkhead DNA-bind-
ing domain and have important roles in cellular
proliferation and differentiation during embryonic
development. Studies using retinoic acid-induced dif-
ferentiation of mouse F9 embryonal carcinoma cells
revealed that HNF-3a(Foxa1) is a primary target for
retinoic acid action. Activation of HNF-3ainduced
the transcription of HNF-3b(Foxa2), which was fol-
lowed by an increase in the expression of transthyretin,
Sonic hedgehog, HNF-1a, HNF-1band HNF-4agenes
during the differentiation of F9 cells to the visceral
endoderm lineage [34].
Possible recognition sites for liver-enriched
transcription factors in the transthyretin
promoters from various vertebrates
Figure 2A shows the possible recognition sites for
HNF-1, HNF-3band HNF-4 in the 0.2-kbp promoter
regions of transthyretin genes from various species.
The tetrapods [human, rat, chicken and frog (Sil-
urana tropicalis)] have more than one HNF-3bsite –
H3(+) and H3()) – in the promoter regions of the
transthyretin genes. By contrast, the fish (Taki-
fugu rubripes) transthyretin gene has no HNF-3bsite
in the promoter region. Human, rat, frog (Sil-
urana tropicalis) and fish (Takifugu rubripes) have one
HNF-1 site, H1(+), in the promoter regions. Human,
rat, frog (Silurana tropicalis) and fish (Takifugu rub-
ripes), but not chicken, have one HNF-4 site, H4(+).
Of these recognition sites, H3()) (eight of twelve nucle-
otides) and H1(+) (ten of thirteen nucleotides) are the
most conserved throughout vertebrate evolution in
these animals (Fig. 2B). The fish transthyretin gene
may have a distinct promoter for the recognition of
transcription factors, especially HNF-3b, compared
with the transthyretin promoters of other vertebrates.
Prevalent expression of transthyretin in tissues other
than liver of the sea bream [26,27], Pacific bluefin tuna
[29] and lamprey [30] may reflect the unique structure
of the fish transthyretin promoter.
K. Yamauchi and A. Ishihara Transthyretin gene expression during development
FEBS Journal 276 (2009) 5357–5366 ª2009 The Authors Journal compilation ª2009 FEBS 5361
