The a-Tocopherol Transfer Protein Is Essential for
Galen W. Miller1,2, Lynn Ulatowski6, Edwin M. Labut1, Katie M. Lebold1,3, Danny Manor6,7,
Jeffrey Atkinson8, Carrie L. Barton4, Robert L. Tanguay2,4,5, Maret G. Traber1,2,3,5*
1Linus Pauling Institute, Oregon State University, Corvallis, Oregon, United States of America, 2Molecular and Cellular Biology Program, Oregon State University, Corvallis,
Oregon, United States of America, 3School of Biological and Population Health Sciences, Oregon State University, Corvallis, Oregon, United States of America,
4Department of Environmental and Molecular Toxicology, Oregon State University, Corvallis, Oregon, United States of America, 5Environmental Health Sciences Center;
Oregon State University, Corvallis, Oregon, United States of America, 6Department of Nutrition, Case Western Reserve University, Cleveland, Ohio, United States of
America, 7Department of Pharmacology, School of Medicine, Case Western Reserve University, Cleveland, Ohio, United States of America, 8Department of Chemistry,
Brock University, St. Catharines, Ontario, Canada
The hepatic a-tocopherol transfer protein (TTP) is required for optimal a-tocopherol bioavailability in humans; mutations in
the human TTPA gene result in the heritable disorder ataxia with vitamin E deficiency (AVED, OMIM #277460). TTP is also
expressed in mammalian uterine and placental cells and in the human embryonic yolk-sac, underscoring TTP’s significance
during fetal development. TTP and vitamin E are essential for productive pregnancy in rodents, but their precise
physiological role in embryogenesis is unknown. We hypothesize that TTP is required to regulate delivery of a-tocopherol to
critical target sites in the developing embryo. We tested to find if TTP is essential for proper vertebrate development,
utilizing the zebrafish as a non-placental model. We verify that TTP is expressed in the adult zebrafish and its amino acid
sequence is homologous to the human ortholog. We show that embryonic transcription of TTP mRNA increases .7-fold
during the first 24 hours following fertilization. In situ hybridization demonstrates that Ttpa transcripts are localized in the
developing brain, eyes and tail bud at 1-day post fertilization. Inhibiting TTP expression using oligonucleotide morpholinos
results in severe malformations of the head and eyes in nearly all morpholino-injected embryos (88% compared with 5.6% in
those injected with control morpholinos or 1.7% in non-injected embryos). We conclude that TTP is essential for early
development of the vertebrate central nervous system.
Citation: Miller GW, Ulatowski L, Labut EM, Lebold KM, Manor D, et al. (2012) The a-Tocopherol Transfer Protein Is Essential for Vertebrate Embryogenesis. PLoS
ONE 7(10): e47402. doi:10.1371/journal.pone.0047402
Editor: Harold A. Burgess, National Institutes of Health/NICHD, United States of America
Received March 6, 2012; Accepted September 14, 2012; Published October 15, 2012
Copyright: ? 2012 Miller et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This publication was made possible, in part, by the Aquatic Biomedical Models Facility Core of the Environmental Health Sciences Center, Oregon State
University (National Institute of Environmental Health Sciences, P30 ES000210) and by a grant from The Eunice Kennedy Shriver National Institute of Child Health
and Human Development (HD062109). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
Vitamin E (a-tocopherol) was discovered almost 90 years ago
because rats fed an a-tocopherol deficient diet failed to carry their
offspring to term; the fetuses were resorbed approximately 9 days
into pregnancy . Although the fetal-resorption test is still used to
define the international units for vitamin E , the cause of the
embryonic failure has never been characterized. Likely the
embryonic delivery system for a-tocopherol involves the a-
tocopherol transfer protein (TTP) because in the adult liver TTP
facilitates a-tocopherol transfer into the plasma. Humans with
TTPA gene mutations demonstrate a heritable disorder: ataxia
with vitamin E deficiency (AVED, OMIM #277460), which
manifests in infancy and childhood. TTP, however, is not
exclusively a liver protein; it is expressed in human yolk sac ;
and has been detected in mammalian placental and uterine cells
[4–6]. Previously, we utilized the zebrafish model to separate the
maternal and embryonic requirements, and to characterize the
molecular defect of embryonic vitamin E deficiency. We reported
that a-tocopherol-deficient fish spawn and produce viable eggs,
but within days the embryos and larvae display developmental
impairment and increased risk of mortality , establishing a
critical embryonic need for a-tocopherol. Zebrafish nutrients are
derived solely from the yolk sac for the initial 4–5 days post
fertilization. After demonstrating the embryonic requirement for
vitamin E we next queried how a-tocopherol is transferred into the
embryo during development. We hypothesized that 1) zebrafish
express a protein homologous to the human TTP and 2) TTP is
required for early embryonic development. In the present study,
we test the hypothesis that adult zebrafish express TTP that is
homologous to the human protein. As development is a highly
regulated process with specific spatial and temporal control, we
evaluate the quantity and location of Ttpa during the first day of
zebrafish development. To test for embryonic requirement we
inhibited translation of TTP using antisense morpholinos (MO) to
knockdown protein expression. We conclude that TTP is essential
for early brain and axis development.
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Zebrafish TTP: Identification and mRNA Characterization
The zebrafish (NP_956025.2) and human (NP_000361.1) TTP
amino acid sequences were compared using Align2 (http://
bioinfo.cgrb.oregonstate.edu/fasta2.html. Accessed 2012 Sep 17.)
(Figure 1A). The TTP protein sequences are highly conserved
between the two species, sharing 64% identical and 85% similar
amino acid residues. Even greater conservation (82% identity and
95% similarity) is observed within the ligand binding pockets of the
two orthologs (residues 129–194 of the human proteins and 126–
191 of the fish, highlighted in Figure 1A). Close inspection of the
two sequences revealed that of the 18 residues identified as
relevant to human TTP function (identified from AVED patients
and in vitro studies) [8–13], 15 were identical between the zebrafish
and human sequences, 2 were similar, and only one residue (D64)
was different (Table 1). This latter unmatched residue, an
aspartic acid in the 64thposition of the human sequence, has only
been reported in one AVED patient, who also harbored an
additional point mutation in the TTP coding region . The
aspartic acid residue has not been otherwise implicated in a-
tocopherol binding or TTP function. Thus, it is not likely that this
amino acid substitute should alter the activity of the zebrafish
ortholog. For additional confirmation of homology we tested for
anti-human TTP cross reactivity using a new antibody to human
TTP, CW201P that also recognizes mouse TTP. Adult zebrafish
liver homogenate reacted with the antibody with a single band at
33 kD, the expected size of the zebrafish protein (left lane,
Figure 1B); the antibody reacted with mouse TTP, but not with
homogenate from a TTP2/2 mouse liver (middle and right lanes,
The time course (6–24 hpf) of embryonic zebrafish TTP mRNA
expression shows that initial expression (6 hpf) increases dramat-
ically beginning ,10 hpf (Figure 2A). We chose embryos aged 1-
day post fertilization (dpf) prior to development of the liver to
define the spatial expression pattern of TTP using RNA in situ
hybridization. TTP mRNA is expressed throughout the develop-
ing head, eyes and in the tail bud (Figure 2). Prior to 1 dpf, TTP
mRNA expression is less spatially restricted and appears through-
out the length of the embryo, apparently at greater amounts close
to the yolk sac (Figure 2), these earlier time points are similar to
those noted previously .
Disruption of TTP Expression using Morpholinos
MOs were used to evaluate the requirement for TTP during
zebrafish embryogenesis. Our experiments focused on a transla-
tional blocking MO (TRN), complementary to a region including
the start codon of the mature TTP mRNA (Figure 3A). Embryos
injected with the TRN showed significant developmental defects
along the anterior/posterior axis at 1 dpf, including both cranial
and tail malformations (p,0.0001 by ANOVA; p,0.001 TRN
compared to CTR or NON, Tukey’s multiple comparison test,
Figure 3C). These malformations were noted in .88% of TRN
embryos by 1 dpf, compared with the embryos injected with the
CTR (5.6%) or non-injected (NON) embryos (1.7%, Figure 3B).
It is important to note that these malformations occur in the same
regions as the expression of TTP mRNA at 1 dpf (Figure 2).
To determine the sequence of the observed malformations,
embryos injected with TRN and CTR, or NON-controls were
followed using time-lapse microscopy from ,6 hpf until ,24 hpf
(Videos S1 and S2). Throughout blastula formation, epiboly and
gastrulation (6–11 hpf), all embryos appeared to develop normally.
At ,12 hpf, the nascent eye of embryos injected with TRN begin
to display tissue darkening (Figure 4), indicating the initiation of
improper head growth. At 1 dpf in the TRN embryos, eye or
brain formation was almost completely halted, and a misshapen
tail was evident, whereas the CTR embryos developed normally
(Figure 3). Due to the low level of TTP expression in the
developing embryos and interference by the overabundance of
vitellogenin-derived yolk-proteins  we were not able to verify
TTP knockdown by immunohistochemistry.
To confirm that the TRN specifically knocked down TTP
protein expression, we designed a pair of non-overlapping MOs
that target the second exon in the TTP pre-mRNA. The exon-
exclusion (EXC) MOs are complementary to either end of the
second exon (Figure 3A). These MOs interfere with the splicing
and processing of the pre-mRNA resulting in the deletion of exon
two from the mature product [16,17]. This alteration would result
in a truncated protein product, if the aberrant mRNA were
translated, due to a reading-frame shift caused by the exon
exclusion and resulting in a pre-mature stop codon (Figure S1).
The efficacy of splice inhibition by the EXC MOs was verified by
RT-PCR amplification of a region spanning exon two and size
verification by gel electrophoresis (Figure S2, primer locations
shown as black arrows in Figure S1). The RT-PCR gel shows a
complete loss of proper-size TTP mRNA in the EXC MO-treated
embryos; instead the products are smaller due to the exclusion of
exon two from the final product. Additionally, embryos injected
with the EXC MOs present with a significantly lower amount of
TTP transcript (Figure S3), regardless of mRNA size (primers
complimentary with regions not affected by the EXC MOs,
orange arrows Figure S1). This loss of TTP mRNA is likely due
to nonsense-mediated decay of the aberrant transcripts. Impor-
tantly, employing the EXC MOs compared with the TRN MO
yielded the same phenotype, namely abnormal head and eye
formation, and a truncated tail. These results confirm that TTP
knockdown using either MO targeting strategy disrupts the normal
Non-specific p53 induction has been observed following
injection with some MOs [18,19]. To confirm that the phenotype
observed with TTP knockdown was not a result of off-target p53
induction, co-injections with a p53 knockdown MO were
performed. The p53 MO co-injection did not affect the TTP
phenotype (data not shown), and was not used in subsequent
This study shows that expression of TTP is essential for early
embryonic development in the zebrafish. The high degree of
sequence similarity suggests a functional conservation between the
human and zebrafish TTP orthologs. This conclusion is further
supported by the fact that anti-TTP antibodies recognize a band at
the expected size in zebrafish tissues (Figure 1B). The cross-
reactivity of an anti-human TTP antibody (Figure 1B) coupled
with the sequence comparisons (Table 1) all support that
zebrafish TTP is an ortholog of the human protein.
Having established the existence and putative functional
conservation of TTP in the zebrafish, we examined its role in
development. Expression of TTP mRNA during development is
initially low (6 hpf), but increases dramatically by 9–12 hpf and
remains elevated thru 24 hpf (Figure 2A). Importantly, increased
TTP expression precedes formation of the vascular system, and
days ahead of liver formation , suggesting a critical role for
TTP during development.
The phenotype, especially impaired brain formation in TTP
knockdown zebrafish embryos raises the intriguing possibility that
low vitamin E status has adverse events in early central nervous
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system development in other animals, including humans. The
dramatic phenotype observed in zebrafish embryos, has not been
noted in vitamin E deficient rats likely because their embryos are
resorbed prior to neurogenesis or eye formation . In the case of
TTP knockout mouse models, mothers are infertile unless
supplemented with high doses of vitamin E . However, Jishage
et al.  showed that if the mother was TTP2/2 and not
supplemented embryos (regardless of TTP mutations) developed
neural tube defects and failed to come to term . While the
Jishage study focuses on mouse maternal TTP deficiency, the
embryonic phenotype and link to central nervous system
developmentis similarto ourfindings in thezebrafish
(Figure 3C). In support of this notion, previous studies have
shown a clear association between maternal vitamin E status
during gestation and cognitive function of the offspring [21–23].
The zebrafish model presents an important means to elucidate the
fetal requirements for a-tocopherol, independent of the maternal
needs. Fetal resorption and placental failure have been noted in
TTP knockout mice [4,24], which are similar to outcomes
observed upon diet-induced vitamin E deficiency [1,25,26]. The
TTP protein is expressed in the placental and uterine cells of mice
and humans [3–6,27], and is thought to play an important role in
supplying maternal a-tocopherol to the developing fetus to protect
against oxidative stress . The mammalian studies provide
Figure 1. The zebrafish a-tocopherol transfer protein. A. Alignment of human and zebrafish TTP amino acid sequences is shown. Double dots
indicate identical residues and single dots correspond to similar amino acids. Red text signifies a-tocopherol binding pocket. Align2 software (http://
bioinfo.cgrb.oregonstate.edu/fasta2.html. Accessed 2012 Sep 17.) was used for sequence comparison. Sequences were obtained from NCBI. B. Anti-
human TTP antibody cross-reacts with TTP from adult zebrafish liver homogenate. The 33 kD zebrafish protein (left lane) shown with a Ttp2/2
mouse sample as a negative control (right lane) and a WT mouse sample with the 32 kD mouse homolog (left lane).
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insight into the requirement of TTP for implantation and
placental formation, both of which are linked to maternal transfer
and need, but fail to determine the TTP requirement of the
developing fetus. The mammalian maternal vitamin E require-
ments occur prior to the developmental stage in which TTP is
required in the zebrafish, creating a barrier to the study of TTP in
TTP specifically traffics a-tocopherol, suggesting that its loss
confers an a-tocopherol deficient state in the developing embryo.
Our current methods lack the resolution to determine the
subcellular localization of a-tocopherol, although we theorize that
TTP, which functions as an intracellular transporter of a-
tocopherol , is required to facilitate delivery of a-tocopherol
to critical locations, chiefly within the developing neural tissues.
We attempted to determine the distribution of a-tocopherol in
early zebrafish development by injecting 1–2 cell stage embryos
with the previously characterized fluorescent a-tocopherol analog:
v-nitrobenzoxadiazole-a-tocopherol , but due to technical
difficulties could not demonstrate specific transfer and localization.
MO knockdown has been linked to non-specific p53 activation
in the zebrafish embryo [18,19]. We experienced this first hand
with a MO targeting the Ttpa exon1-intron1-2 junction (data not
shown). The non-specific p53 activation presented with a
phenotype similar to TTP morphant embryos (malformations in
the head and tail). These non-TTP related malformations were be
mitigated (although not rescued entirely) by co-injection with a
MO against p53 . The p53 MO co-injection alleviated the
high occurrence of mortality associated with the Ttpa exon1-
intron1-2 MO, revealing the non-specific p53 activation associat-
ed with this Ttpa MO (data not shown). Co-injection with the p53
MO has recently been called into question, as it may cover specific
p53-dependent processes , and it has been suggested that MO
with phenotypes that are rescued by p53 MO co-injection cannot
be reliably studied . As such, we discontinued use of the
exon1-intron1-2 targeted MO, and used instead the MOs
discussed above. All MO were tested for rescue by co-injection.
Co-injection with matching concentrations of p53 MO , failed
to rescue the phenotype associated with TTP knockdown, allowing
the use of these MO to study TTP function in the developing
We previously demonstrated the requirement of vitamin E
during zebrafish development using diet-induced vitamin E
deficient embryos . The malformations associated with TTP
knockdown are different from those caused by parental diet-
induced vitamin E deficiency. Although the a-tocopherol concen-
tration of the E- embryos was .50-fold decreased from the control
embryos, they still possessed detectable amounts of vitamin E. This
is likely due to the specific allocation of maternal vitamin E, and its
incorporation into the yolk of the developing oocyte. Loss of TTP,
however, precludes the specific trafficking and localization of
vitamin E, mimicking an absolute deficient state regardless of the
ubiquitous yolk sac supply. Furthermore, in our previous studies
vitamin E deficiency was imposed by parental diet, while TTP
knockdown was performed using embryos from fish fed commer-
cial lab diets. This difference in parental diets affects not only the
nutrient composition but the transcriptional profiles as well
(unpublished observation). Notably, as morphologic outcomes
from each study are ultimately due to vitamin E deficiency, they
likely involve common mechanisms.
The loss of TTP function results in malformations along the
anterior/posterior axis (Figure 3C) and early life-stage mortality.
We theorize that TTP mediates a-tocopherol transfer to critical
sites in the embryo during early vertebrate development and thus,
TTP is required for embryogenesis. It is important to note that this
requirement for TTP takes place during a time analogous to the
first 20 days of human gestation. This window is prior to the
detection of most pregnancies, and often before the consumption
of prenatal supplements. This early requirement combined with
Table 1. TTP residues implicated in a-tocopherol binding.
residueComparison AVED associated mutationsRef
R59 R56 Identical R59W- early onsetDecreased binding and transfer 
D64A61 Dissimilar D64G- early onsetna
H101H98 Identical H101Q- late onset Similar to wild type
Y117 Y114Identicalna Binding pocket
A120 G117Similar A120T- late onset Similar to wild type
A129A126 Identical na Binding pocket
F133F130 Identicalna Binding pocket[8,10]
S140S137 Identicalna Binding pocket[8,10]
E141E137 IdenticalE141K- early onset  Decreased transfer
I154L151 Similar naBinding pocket [8,10]
I171I168 Identical naBinding pocket[8,10]
I179I176 Identical na Binding pocket[8,10]
V182 V179 IdenticalnaBinding pocket [8,10]
L183 L180 IdenticalL183P- NR Binding pocket[8,10]
L189 L186Identical na Binding pocket
R192R189 Identical R192H- late onsetSimilar to wild type 
R221R118IdenticalR221W- early onset  Decreased binding and transfer
G246G243 IdenticalG246R- late onset na
na, information not available.
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the inadequate a-tocopherol consumption  could be respon-
sible for early failures in human pregnancy. The role of TTP and
a-tocopherol in post-implantation development needs to be
addressed, as these results highlight the role of TTP and
ramifications of its loss.
In summary, we demonstrate that adult zebrafish express TTP,
which is homologous to the human protein. As development is a
highly regulated process and genes are specifically controlled in
both a spatial and temporal fashion, we assayed both the quantity
and location of Ttpa during the first day of zebrafish development.
The function of TTP was determined through inhibition of TTP
translation using antisense MOs to knockdown protein expression.
We conclude that TTP is essential for early brain and axis
development, likely because it delivers a-tocopherol to the
Materials and Methods
Wild-type zebrafish (Tropical 5D strain) were kept under
standard laboratory conditions at 28.5uC with a 14 h light/10 h
dark cycle . Embryos were obtained through natural group
spawning; embryos were collected and kept in standard fish water.
Figure 2. TTP expression is dynamic in the developing zebrafish. A. Embryonic TTP transcription increases during the first 24 hpf. Expression
normalized to odc1 expression, and values are expressed as fold change compared to 6 hpf. Data shown as mean 6 SEM, 6 hpf n=4, 9–12 n=6, 13–
18 n=9, and 19–24 n=11 replicates (30 embryos per replicate). B-G. Whole mount in situ hybridization of ttpa reveals the patterning of mRNA
expression. B. A lateral view of a whole mount embryo at 12 hpf shows fairly even distribution, however, in C a dorsal view of the rostral region with
the yolk removed shows specific staining along what may be the developing neural tube. D. At 17 hpf expression remains along the length of the
embryo, concentrating in the deeper cells, closer to the yolk sac. E. A dorsal view of the developing head at 17 hpf, the eyes and neural tube is where
the expression appears to be localized (outlined). F. By 24 hpf the staining is seen only in the regions of the developing brain, eyes and tail bud. G.
Dorsal view depicts brain and eye specific patterning. Yolk sacs were manually removed to reduce color interference, and for ease of positioning.
fb=forebrain, mb=midbrain, *=midbrain-hindbrain boundary.
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Adult zebrafish were euthanized by overdose of buffered
tricaine, livers were dissected out, frozen in liquid nitrogen and
homogenized in RIPA buffer (150 mM sodium chloride, 1% NP-
40, 0.5% sodium deoxycholate, 0.1% SDS (sodium dodecyl
sulphate), 50 mM Tris, pH 8.) with 1% Protease inhibitor cocktail
set III, EDTA-free (Calbiochem, Gibbstown, NJ). The protein
concentration was determined using the Bradford assay with the
Coomassie Plus reagent per manufacturer’s instructions (Pierce
Biotechnology, Rockford, IL). Lysates were immunoblotted for
endogenous TTP using a rabbit polyclonal CW201P antibody and
a secondary HRP-conjugated rabbit antibody in combination with
SuperSignal West Dura substrate (Thermo Fisher Scientific, Inc.,
Rockford, Il) for visualization.
Rabbit Anti-human TTP Antibody (CW201P)
Recombinant wildtype human TTP was expressed in bacteria
as described and purified as described [11,33]. Briefly, GST-TTP
fusion protein was isolated from over-expressing bacteria using
glutathione affinity chromatography, cleaved with thrombin, re-
purified by two ammonium sulfate precipitations and stored at
220uC in 20 mM Tris pH 8.0, 150 mM NaCl, 50% (v/v)
glycerol, 1 mM DTT. For antibody preparation, purified TTP
was dialyzed into phosphate-buffered saline; 2 rabbits were
injected with the protein (250 mg at 1 mg/ml) (Covance, Denver,
PA). The initial protein injection was emulsified in Freund’s
Complete Adjuvant (FCA), while the 3 boosts, spaced at 3-week
intervals, were emulsified in Freund’s Incomplete Adjuvant (FIA).
The antibody was purified from crude serum using protein G
sepharose and stored at 220uC until use. For Western blotting,
antibody was diluted 1:1000 with PBS, 2% bovine serum albumin.
TTP reactivity was routinely confirmed as an immunoreactive
band near 32 kDa (the expected size of the mouse TTP), which is
missing from liver extracts prepared from TTP2/2mice .
TTP Knockdown by MO Injection
Morpholinos (MOs) (GeneTools LLC, Philomath, OR) were
designed complementary to the TTP RNA sequence. TRN MO
sequence: 59-TCTCGTCTACTTCTTCGGACTTCAT-39, EXC
and 59-TGTATGTACCTGCCAATCCGATAGA-39. A standard
zebrafish control MO was used as a control for the injection process
(GeneTools LLC). MOs dissolved in UltraPure DNase/RNase-Free
distilled water (Invitrogen, Carlsbad, CA), were injected into 1–2 cell-
stageembryosatconcentrationsof0.96to1.0 mMin2-4 nlinjections
(1.9–2.0 mM total for the EXC MO pair). TRN MO injection
Figure 3. Morpholino knockdown of TTP causes severe malformations. A. MO targeting schematic using a graphic representation of the
ttpa transcript. The translational blocking morpholino (TRN) is complementary to the translation start-site, while the splice blocking morpholinos
(EXC) bind to the intron/exon junctions on each side of the second exon. Arrows mark primers used to verify aberrant mRNA products resulting from
the EXC morpholino (Figure S2). Numbered boxes represent exons, and spanning lines are introns, smaller unnumbered boxes are untranslated
regions. B. TTP knockdown leads to high incidence of malformation within the first day of development. Data shown as mean percent incidence from
seven (TRN, CTR and NON) or three separate experiments (EXC). C. Representative pictures of malformations at 1 dpf due to TTP knockdown.
TRN=translational morpholino injected embryo, CTR=standard control injected embryo, concentration and age-matched to the TRN embryo.
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concentrations were determined experimentally and the concentration
utilized caused nearly 100% penetrance. EXC MOs displayed effects
to maintain ,100% efficacy and match TRN MO concentrations. All
concentrations used were within the range of previously published
studies [34–38]. Phenol red (Sigma Aldrich, St. Louis, MO) was added
to verify injection location. To control for spawn quality and embryo
handling, a group of NON-embryos, which were not injected with
MO, were collected and observed as well. After injections embryos
1 dpf by stereomicroscopy.
Time lapse studies. Embryos (4–7 hpf) into individual wells of a
384-well assay plate, black with 0.9 mm clear bottom (Corning
Inc., Corning, NY) in ,90 ml of standard fish water and sealed
with a MicroAmp Optical Adhesive Film (Life Technologies,
Carlsbad, CA). Images were obtained once every 10 min using an
ImageXpress Micro Imaging System (Molecular Devices, Inc.,
Sunnyvale, CA). Images were analyzed and movies created from
stacked (time-lapse) images using MetaXpress software, version
184.108.40.206 (Molecular Devices, Inc.).
RNA in situ Hybridization
Embryos were allowed to develop until the desired stage ,
euthanized by overdose with buffered tricaine (MS 222, ethyl 3-
aminobenzoate methane sulfonate salt; Sigma-Aldrich, St. Louis, MO,
USA) and fixed overnight with 4% paraformaldehyde in phosphate
buffered saline (PBS) at 4uC, then washed and stored in methanol at
220uC until they were processed. Whole mount in situ hybridization
was performed using digoxygenin-labeled, antisense RNA probes as in
, using the 2010-updated protocol (zfin.org). Embryos were
mounted in glycerol, allowed to clear for .24 h and imaged on glass
slides with a Nikon SMZ (800 or 1500) stereomicroscope, using a
Nikon CoolPix 4500 camera. The zebrafish ttpa transcript was cloned
from embryonic cDNA using a pCR4-Blunt TOPO vector with the
primers: 59-TGGACCGCCCGTCGCAGATA-39 and 59-AGCTG-
CACCATTCAGTCATGTCCA-39. The anti-sense probe was syn-
thesized using a T7 RNA polymerase (Promega, Madison, WI) after
enzymatically digested with Pst1 (Promega).
Quantitative real-time PCR: Embryos (n=30) were collected in
RNAlater (Invitrogen) at noted time points, RNA extraction and
qPCR preformed as described previously . Ornithine decarboxylase
1 (odc1) was used as a reference gene for normalization . Odc1
was previously verified as a stably expressed reference gene by Dr.
Emily Ho’s lab group (unpublished results) and correspondingly
used for their studies .
RT-PCR: Embryos (n=30) were collected at 12 hpf and
processed as described above. PCR was preformed using primers
specifically designed to flank the MO-targeted exons (FOR
[UC580] 59-ATGAAGTCCGAAGAAGTAGAC-39 and REV
[UC1441] 59-GAGCATGAGCAAAACACCAA-39, and arrows
in Figure 3A) and KOD Hot Start DNA polymerase (EMD
Chemicals, San Diego, CA) as per manufacture’s direction.
Product resolution was achieved using the FlashGelTMSystem
(Lonza Group Ltd, Switzerland).
Statistical analyses were performed using GraphPad Prism
software version 5.0d (GraphPad Software, Inc., La Jolla, CA,
USA). Relationships between the MO groups were analyzed using
one-way analysis of variance on the percentage of viable embryos.
Post hoc tests were carried out using paired comparisons (Tukey’s
multiple comparison test). Data are reported as means; differences
were considered significant at P,0.05.
Figure 4. Early morphant malformations. Images of embryo
development from 6–18 hpf demonstrating early effects of TTP
knockdown (right panel) compared to an injected control animal at
the same age (left panel). Embryos from each MO injection type remain
constant through 11 hpf. Beginning at 12 hpf, malformations are
noticeable in the rostral region of the TRN embryo. These initial
malformations occur in the head at the time the developing eye
(marked) becomes distinguishable. The malformations in TRN embryos
are more pronounced at later stages of development (16 and 18 hpf),
while somite formation continues unabated. Images are frames from a
time-lapse video (Videos S1 and S2).
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This study was performed in strict accordance with the
recommendations in the Guide for the Care and Use of
Laboratory Animals of the National Institutes of Health. All
protocols were approved by the Institutional Animal Care and Use
Committee of Oregon State University (ACUP Number: 3903).
All fish were euthanized by tricaine (MS 222, Argent Chemical
Laboratories, Inc., Redmond, WA) overdose prior to sampling,
and every effort was made to minimize suffering.
depicted, with EXC morpholinos (green lines), marked. B. The proper
mature mRNA and associated full-length protein. C. A naturally
occurring splice-variant (inclusion of intron 1–2), recorded as ‘‘non-
coding’’, if translated, results in a truncated protein product due to a
in a premature stop codon, and if translated, a truncated peptide
product. Sequences of interest are marked: splice-block verification
primers (black arrows), qPCR primers (orange arrows) and transcrip-
tion start site (black right-hand arrow).
Putative peptide products. A. TTP transcript is
ucts created using primers flanking exon 2 in the TTP mRNA
sequence are shown. Products from EXC injected embryos (EXC)
display an aberrant transcript when compared to the other TTP
shows the expected three bands (the result of splice variants) all of
which are larger than the EXC induced exon deletion (519–604 bp).
MO splice-blocking confirmation. PCR prod-
mRNA. At 12 hpf, prior to overt malformations, TTP transcripts are
This ,10-fold reduction in TTP mRNA is likely due to nonsense
mediated decay of the aberrant transcript (Gene-tools, personal
communication). The qPCR amplicon does not include the excluded
exon (primers represented as orange arrows in Figure S1), and
therefore does not differentiate between proper and aberrant mRNA.
Shown as mean 6 SD, n=5, EXC and n=3 CTR, biological
replicates from separate experiments. ***, p,0.001 by Student’s t-test.
Splice blocking MO cause decreased TTP
Embryos were injected using the noted concentrations at 1–2 cell
stage with the exon-exclusion (EXC) MOs, which are comple-
mentary to either end of the second exon (Upper rows). MO-
injected embryos were observed at 24 hpf for gross morphologic
effects. Results shown are from three separate injection trials.
Results from a representative set of CTR-injected and NON
embryos are shown for comparison (Bottom rows). Co-injections
with a MO against p53 (+p53 MO) were done at concentrations
matching the EXC MO. Note: 2 mM=8–25 ng/MO per
embryo, 1.4 mM=6–18 ng/MO
0.6 mM=2.5–7.6 ng/MO per embryo (excluding p53 MO where
EXC MO concentration efficacy validation.
tive embryo with TTP knockdown from 4–24 hpf (TRN). Loss of
TTP causes notable malformations beginning at ,12 hpf. The
rostral and caudle parts of the embryo fail to develop, while
somitogenesis continues unabated. Arrow appears next to
beginning eye-spot at ,12 hpf.
TTP knockdown time-lapse video. Representa-
tative control (CTR) MO-injected embryo from 4–17 hpf.
Embryo development proceeds in proper fashion regardless of
the injection process, as compared to non-injected, not shown.
Arrow appears next to beginning eye-spot at ,12 hpf.
Control injected embryo time lapse. Represen-
The authors would like to thank Jane K La Du for assistance with in situ
hybridization techniques, Greg Gonnerman for running the high-content
imaging device, and the staff of the Sinnhuber Aquatic Research
Laboratory (SARL) for fish husbandry and embryo handling/production.
A special thank you to Paul Morcos at GeneTools for help designing MOs.
Conceived and designed the experiments: GWM LU EML KML DM
CLB RLT MGT. Performed the experiments: GWM LU EML KML
CLB. Analyzed the data: GWM LU DM JA RLT MGT. Contributed
reagents/materials/analysis tools: LU DM JA. Wrote the paper: GWM LU
DM JA RLT MGT.
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