© 2015. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
RESOURCE ARTICLE
Apoc2 loss-of-function zebrafish mutant as a genetic model of
hyperlipidemia
ABSTRACT
Apolipoprotein C-II (APOC2) is an obligatory activator of lipoprotein
lipase. Human patients with APOC2 deficiency display severe
hypertriglyceridemia while consuming a normal diet, often manifesting
xanthomas, lipemia retinalis and pancreatitis. Hypertriglyceridemia
is also an important risk factor for development of cardiovascular
disease. Animal models to study hypertriglyceridemia are limited, with
no Apoc2-knockout mouse reported. To develop a genetic model of
hypertriglyceridemia, we generated an apoc2 mutant zebrafish
characterized by the loss of Apoc2 function. apoc2 mutants show
decreased plasma lipase activity and display chylomicronemia and
severe hypertriglyceridemia, which closely resemble the phenotype
observed in human patients with APOC2 deficiency. The
hypertriglyceridemia in apoc2 mutants is rescued by injection of
plasma from wild-type zebrafish or by injection of a human APOC2
mimetic peptide. Consistent with a previous report of a transient apoc2
knockdown, apoc2 mutant larvae have a minor delay in yolk
consumption and angiogenesis. Furthermore, apoc2 mutants fed a
normal diet accumulate lipid and lipid-laden macrophages in the
vasculature, which resemble early events in the development of
human atherosclerotic lesions. In addition, apoc2 mutant embryos
show ectopic overgrowth of pancreas. Taken together, our data suggest
that the apoc2 mutant zebrafish is a robust and versatile animal model to
study hypertriglyceridemia and the mechanisms involved in the
pathogenesis of associated human diseases.
KEY WORDS: Zebrafish, Apolipoprotein C-II, APOC2,
Lipoprotein lipase, Hyperlipidemia
INTRODUCTION
Hypertriglyceridemia is an independent risk factor for cardiovascular
disease (CVD) (Do et al., 2013) and is positively associated with
obesity, insulin resistance, type 2 diabetes and other metabolic
1
Division of Endocrinology and Metabolism, Department of Medicine, University of
2
California, San Diego, La Jolla, CA, USA. Sanford Children’s Health Research
Center, Programs in Genetic Disease and Development and Aging, and Stem Cell
and Regenerative Biology, Sanford-Burnham Medical Research Institute, La Jolla,
3
CA, USA. Lipoprotein Metabolism Section, Cardiopulmonary Branch, NHLBI, NIH,
4
Bethesda, MD, USA. State Key Laboratory of Molecular Developmental Biology,
Institute of Genetics and Developmental Biology, Chinese Academy of Sciences,
Beijing, China.
*Present address: Center for Cardiovascular Regeneration, Department of
Cardiovascular Sciences, Houston Methodist Research Institute, Houston, TX,
USA.
‡
Author for correspondence ([email protected])
This is an Open Access article distributed under the terms of the Creative Commons Attribution
License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use,
distribution and reproduction in any medium provided that the original work is properly attributed.
Received 2 January 2015; Accepted 29 May 2015
syndromes (Watts et al., 2013). Hypertriglyceridemia is the result of
interactions between non-genetic and genetic factors. The most
common non-genetic factors causing hypertriglyceridemia are
obesity, alcohol excess, lack of exercise and unhealthy diets (Miller
et al., 2011; Watts et al., 2013). Genetic factors account for 50% of
individual variation in plasma triglyceride (TG) levels (Goldberg
et al., 2011; Miller et al., 2011; Namboodiri et al., 1985), whereas
severe hypertriglyceridemia (TG>885 mg/dl) is usually caused by
single mutations in the gene encoding lipoprotein lipase (LPL), or
less commonly, in genes affecting proteins involved in LPL activity,
such as apolipoprotein C-II (APOC2), lipase maturation factor 1
(LMF1), apolipoprotein A-V (APOA5) and GPI HDL binding
protein1 (GPIHBP1) (Surendran et al., 2012).
APOC2 is an obligatory co-activating factor for LPL, which is the
key enzyme responsible for hydrolysis of plasma TG (Fukushima and
Yamamoto, 2004; Kei et al., 2012). Patients with APOC2 or LPL
deficiency show severe hypertriglyceridemia and chylomicronemia,
and often manifest eruptive xanthomas, lipemia retinalis and acute
and recurrent pancreatitis, which can be lethal (Breckenridge et al.,
1978; Cox et al., 1978; Ewald et al., 2009; Goldberg and Merkel,
2001; Miller et al., 2011; Scherer et al., 2014; Watts et al., 2013).
Current genetic animal models to study hyperlipidemia include mice
with a conditional deficiency in LPL, deficiency of GPIHBP1, or with
overexpression of human APOC2 and APOC3 (Ebara et al., 1997;
Goulbourne et al., 2014; Shachter et al., 1994; Weinstein et al., 2010).
There are several systemic or tissue specific Lpl-knockout mouse
models that have been developed (Garcia-Arcos et al., 2013; Merkel
et al., 1998; Trent et al., 2014; Weinstock et al., 1995). The systemic
Lpl-knockout mice develop hypertriglyceridemia but die shortly after
birth. The neonatal death, together with the hypertriglyceridemia, are
prevented by overexpression of human LPL in either skeletal or
cardiac muscle (Levak-Frank et al., 1999; Weinstock et al., 1995). In
contrast to extensive work conducted with Lpl-knockout mice,
Apoc2-knockout mouse models have not been reported.
Zebrafish is an emerging animal model to study lipid metabolism
and mechanisms of human disease related to lipid abnormalities
(Anderson et al., 2011; Fang et al., 2014; Holtta-Vuori et al., 2010;
Levic et al., 2015; O’Hare et al., 2014; Stoletov et al., 2009). The
advantages of using zebrafish include large progeny numbers,
optical transparency of zebrafish larvae, easy genetic manipulation
and cost-effective maintenance (Dooley and Zon, 2000).
Importantly, genes involved in lipid and lipoprotein metabolism,
such as APOB, APOE, APOA1, LDLR, APOC2, LPL, LCAT and
CETP, are conserved from zebrafish to humans (Fang et al., 2014;
Holtta-Vuori et al., 2010; Otis et al., 2015). Specifically, the
zebrafish Lpl (NCBI Gene ID: 30354) amino acid sequence is
61.7% identical to its human ortholog, with 79.7% conserved
consensus amino acids. The zebrafish full-length Apoc2 (NCBI
Gene ID: 568972, molecular mass 11.2 kDa) is only 27.8%
989
Disease Models & Mechanisms
Chao Liu1, Keith P. Gates2, Longhou Fang1, *, Marcelo J. Amar3, Dina A. Schneider1, Honglian Geng1, Wei Huang1,
Jungsu Kim1, Jennifer Pattison1, Jian Zhang4, Joseph L. Witztum1, Alan T. Remaley3, P. Duc Dong2 and
Yury I. Miller1,‡
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RESOURCE IMPACT
Background
Blood transports dietary lipids and the lipids produced by the liver around
the body in the form of large lipoprotein particles, such as chylomicrons
and very-low-density lipoproteins (VLDL). These lipoproteins also carry
APOC2, a protein that is needed to activate lipoprotein lipase (LPL),
which hydrolyzes triglycerides to deliver fatty acids to tissues.
Hypertriglyceridemia (high levels of triglycerides in the blood) is an
independent risk factor for cardiovascular disease and is positively
associated with metabolic disorders. Obesity, alcohol excess, lack of
exercise and an unhealthy diets can all cause hypertriglyceridemia but,
in addition, patients with a genetic defect in APOC2 or LPL have severe
hypertriglyceridemia and often manifest skin and eye abnormalities
(eruptive xanthomas and lipemia retinalis, respectively) and acute and
recurrent pancreatitis, which can be lethal. The mechanisms that
underlie hypertriglyceridemia-associated diseases are not well
understood, in part, owing to the limited availability of animal models.
Specifically, although substantial work has been conducted with Lplknockout mice, Apoc2-knockout mouse models have not been reported.
Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
nuclease (TALEN) technique. We chose a target site located at exon
3 of the apoc2 gene, with TALEN-binding sequences of 16 bp and
17 bp nucleotides on the left and right side of the target site,
respectively. The spacer DNA was 21 bp long and contained the
BtsI restriction enzyme site (Fig. 1A). The corresponding protein
coding region of the TALEN target site is located in front of the Lplbinding domain (Fig. 1B). The mRNAs encoding TALENs were
injected into one-cell stage zebrafish embryos. To test whether the
apoc2 TALEN pair disrupted the apoc2 gene at its specific target
site, we extracted genomic DNA from F0 generation zebrafish,
amplified the regions containing the target site with PCR and
conducted BtsI enzyme digestion. Compared with wild-type (WT),
a part of the PCR band amplified from TALEN-injected F0
zebrafish was resistant to enzyme digestion (Fig. 1C), consistent
with modification of the BtsI recognition site in the apoc2 genomic
sequence. Then, F0 zebrafish were outcrossed with WT zebrafish
and the F1 progeny were genotyped and outcrossed again for two
generations, followed by an incross to obtain homozygous mutants
Results
Zebrafish is an emerging animal model for the study of lipid metabolism
and the mechanisms of human disease related to lipid abnormalities.
The advantages of using zebrafish include large progeny numbers,
optical transparency of zebrafish larvae, easy genetic manipulation and
cost-effective maintenance. In this study, the authors generate an apoc2
mutant zebrafish characterized by the loss of Apoc2 function. The
authors report that Apoc2 loss-of-function mutant zebrafish display
chylomicronemia (build-up of chylomicrons in the blood) and severe
hypertriglyceridemia, characteristics that closely resemble those seen in
human patients with APOC2 deficiency. They show that the
hypertriglyceridemia in apoc2 mutants can be rescued by injection of
plasma from wild-type zebrafish with functional Apoc2 or by injection of a
human APOC2 mimetic peptide. Notably, apoc2 mutants fed a normal
diet accumulate lipid and lipid-laden macrophages in the vasculature,
which resembles early events in the development of human
atherosclerotic lesions. Finally, apoc2 mutant embryos show ectopic
overgrowth of pancreas.
identical to human APOC2, with 49.5% conserved consensus
amino acids, but its C-terminal region (residues 67-92, zebrafish
numbering), which mediates Apoc2 binding to Lpl, shows 46.1%
identity to the LPL-binding region of human APOC2. Thus, it is
likely that the APOC2-LPL complex required for TG hydrolysis is
also conserved in zebrafish. To support this hypothesis, we have
developed an Apoc2 loss-of-function zebrafish model. Our results
demonstrate that the apoc2 mutant zebrafish develop severe
hypertriglyceridemia, which is characteristic for human patients
deficient in APOC2, and that the apoc2 mutant is a suitable animal
model to study hyperlipidemia and the mechanisms involved in the
pathogenesis of associated diseases.
RESULTS
Mutation of zebrafish apoc2 gene with TALENs
To create a zebrafish model of hypertriglyceridemia, we mutated the
apoc2 gene in zebrafish using a transcription activator-like effector
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Fig. 1. Generation of apoc2 mutant zebrafish. (A) A diagram of partial apoc2
genomic DNA sequence, showing the TALEN-binding sites (underlined) and
the spacer DNA (lowercase), which contains the BtsI restriction site.
(B) A diagram of the TALEN target site in the Apoc2 protein. (C) Test for
TALEN mutagenesis in F0 zebrafish with BtsI enzyme digestion. The arrow
indicates the modified genomic DNA, which was no longer recognized and cut by
BtsI. (D) Representative genotyping results of the F2 generation. (E) Sequencing
data suggest a frame shift mutation at the site preceding the LPL-binding domain
in the apoc2 mutant. (F,G) apoc2 mRNA expression (as assessed by qPCR) in
adult liver and 5.5 dpf larvae. Results are mean±s.e.m.; n=3 in each group.
**P<0.01 (Student’s t-test).
Disease Models & Mechanisms
Implications and future directions
Taken together, these findings indicate that the new apoc2 mutant
zebrafish generated by the authors display a robust hyperlipidemia
phenotype and could, therefore, be a useful and versatile animal model
in which to study the mechanisms that underlie the human diseases
induced by hypertriglyceridemia. Moreover, small molecule and genetic
screens using the apoc2 mutant zebrafish might suggest new
approaches to treatment of hyperlipidemia and related diseases.
RESOURCE ARTICLE
Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
(Fig. 1D). Sequencing of the apoc2 mutant revealed a onenucleotide replacement and ten-nucleotide deletion, resulting in a
frame shift mutation changing the coding sequence of the Lplbinding domain of the Apoc2 protein (Fig. 1E).
We then isolated total RNA from the apoc2 mutant adult liver or
5.5 days post fertilization (dpf ) larvae and conducted quantitative
real-time PCR (qPCR) with primers specific to both WT and
mutated apoc2 mRNA. The apoc2 mRNA was undetectable in the
adult liver of the mutant (Fig. 1F) and dramatically decreased in
5.5 dpf larvae (Fig. 1G). These data suggest that apoc2 mRNA
transcripts that contain the frame shift are eliminated, as has been
demonstrated for many other premature stop codon and frame shift
transcripts (Baker and Parker, 2004), and suggest that the mutation
might result in loss of Apoc2 function.
Adult apoc2 mutants were viable and fertile, and had a normal
physical appearance (Fig. 2A and supplementary material Fig. S1). To
test whether apoc2 deficiency resulted in hypertriglyceridemia, we
drew blood from adult male zebrafish into micro-capillary tubes at 8 h
after the last feeding and let the capillaries stand overnight at 4°C to
sediment erythrocytes. Plasma from an apoc2 mutant showed a milky
phenotype, with a creamy layer at the top (Fig. 2B), typical of that
found in subjects with APOC2 or LPL deficiency (Baggio et al., 1986;
Fellin et al., 1983). These plasma samples were also subjected to
native agarose gel electrophoresis. Compared with WT, mutant
plasma showed a decrease in HDL and a dramatic increase in the verylow-density lipoprotein (VLDL) and low-density lipoprotein (LDL)
fraction. Chylomicrons, which were never observed in fasted WT
zebrafish, were present in the mutant (Fig. 2C, gel origin). Size
exclusion chromatography of pooled zebrafish plasma from apoc2
mutants showed that there was a dramatic increase of TG and total
cholesterol (TC) in the chylomicron and VLDL fraction, as well as in
LDL fraction, but decreased TC in the high-density lipoprotein (HDL)
fraction, compared with that in WT zebrafish (Fig. 2D,E). For
comparison, plasma from an Ldlr−/− mouse fed a 60% high-fat diet
showed a plasma lipoprotein profile similar to the apoc2 mutant,
although it had lower TG levels. Pooled plasma from overnight fasted
zebrafish was also subjected to ultracentrifugation to float triglyceriderich lipoproteins (TRLs). The uppermost layer, corresponding to
TRLs, collected from WT and apoc2 mutant zebrafish, was then
subjected to electron microscopy to visualize lipoproteins. Although
no TRLs were detected in the WT plasma, TRLs of different sizes,
including large chylomicrons, were abundant in apoc2 mutant plasma
(Fig. 2F). Accordingly, plasma TG levels were dramatically increased
(2488±160 mg/dl versus 67±8 mg/dl, mutant versus WT; mean±
s.e.m., n=6, P<0.001, Fig. 2G) and plasma lipase activity was
significantly decreased in apoc2 mutants compared with WT
(Fig. 2H). Although it has been reported that human patients with
APOC2 deficiency have a mild elevation in plasma cholesterol
(Baggio et al., 1986; Fellin et al., 1983), unexpectedly, plasma TC
levels were increased as much as four-fold in apoc2 mutants compared
with WT zebrafish (1202±63 mg/dl versus 296±26 mg/dl, mutant
versus WT; mean±s.e.m., n=6, P<0.001) (Fig. 2I).
Taken together, these data suggest that the TALEN-induced
mutation in the apoc2 gene results in severe hypertriglyceridemia
and chylomicronemia, confirming that the function of APOC2 to
activate LPL is conserved in zebrafish.
Hyperlipidemia in apoc2 mutant larvae
apoc2 mutant embryos and larvae did not show significant defects
in development, other than minor, but statistically significant,
Fig. 2. Adult apoc2 mutant zebrafish develop hyperlipidemia when fed a
normal diet. (A) No apparent differences between 4-month-old male WT and
apoc2 mutant (mut) zebrafish. Scale bar: 0.5 cm. (B) Clear and milky
appearance of plasma from WT and apoc2 mutant zebrafish, respectively.
(C) Native gel electrophoresis and neutral lipid staining of plasma from WT and
apoc2 mutant zebrafish. CM, chylomicron. (D,E) Fast protein liquid
chromatography (FPLC) lipoprotein profile of pooled WT (45 animals) and
apoc2 mutant (14 animals) zebrafish plasma. Plasma from an Ldlr−/− mouse
fed a 60% high-fat diet (HFD) was used for comparison. (F) Electron
microscopy images of TRLs isolated from pooled WT and apoc2 mutant
zebrafish plasma by ultracentrifugation as described in the Materials and
Methods. (G) Plasma TG levels. Results are mean±s.e.m.; n=6 in each group;
***P<0.001 (Student’s t-test). (H) Plasma lipase activity assayed as described
in the Materials and Methods. Assay buffer was used as negative control.
Results are mean±s.e.m.; n=3 in each group; ***P<0.001. (I) Plasma TC levels.
Results are mean±s.e.m.; n=6 in each group; ***P<0.001 (Student’s t-test).
delays in yolk utilization and growth. At 30 h post fertilization (hpf )
and 3.5 dpf, the yolk in the mutant was larger but the body length
was shorter than that of the WT (Fig. 3A-G). However, the
development stage reached at both 30 hpf and 3.5 dpf, which was
determined by the head position and the pigment patterns, were
similar in mutants and WT (Fig. 3A,D).
It has been reported that transient knockdown of apoc2 inhibits
angiogenesis (Avraham-Davidi et al., 2012). To assess embryonic
angiogenesis, we crossed the apoc2 mutant with fli1-EGFP
transgenic zebrafish, which express EGFP in endothelial cells. At
26 hpf, there were no angiogenesis differences between apoc2
mutant and WT zebrafish (Fig. 3Ha,a′). However, at 52 hpf,
growth of inter-segmental blood vessels (ISV) was delayed in the
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Disease Models & Mechanisms
Hyperlipidemia in adult apoc2 mutants
Fig. 3. Development and angiogenesis in apoc2 mutant larvae.
(A-C) Morphology of WT (n=11) and apoc2 mutant (mut, n=11) embryos
at 30 hpf. Results are mean±s.e.m.; ***P<0.001 (Student’s t-test).
(D-G) Morphology of WT (n=11) and apoc2 mutant (n=10) embryos at 3.5 dpf.
Results are mean±s.e.m.; *P<0.05; **P<0.01; ***P<0.001 (Student’s t-test).
The blue, red and yellow lengths highlighted in D were used to delineate the
size of yolk, yolk extension and the body length, respectively. (H) Angiogenesis
in fli1:EGFP WT and apoc2 mutant embryos at 28 hpf (a,a′), 52 hpf (b,b′) and
80 hpf (c,c′). The numbers in the figure indicate the number of animals
exhibiting the illustrated phenotype and the total number of animals. Scale
bars: 200 µm (A,D); 100 µm (H).
apoc2 mutant (Fig. 3Hb,b′), as was the outgrowth of subintestinal
veins (SIV) at 80 hpf (Fig. 3Hc,c′), consistent with the results
reported by Avraham-Davidi et al. Interestingly, these ISV and
SIV defects in mutants were all corrected at later stages and the
vasculature of 14 dpf mutants was normal (supplementary
material Fig. S2).
Until 5 dpf, zebrafish embryos, unlike adults, receive nutrients
through yolk utilization. To test whether TG and cholesterol levels
were affected at this early stage, which is a suitable time window for
drug screening and genetic manipulation by injection of antisense
morpholino oligonucleotides, we stained embryos at 1-6 dpf with
Oil Red O (ORO), which stains neutral lipids, including TG and
cholesterol esters (CEs). Before 3 dpf, the strongest ORO staining
was in the yolk and no significant differences between WT and
apoc2 mutants were detected. Starting from 3 dpf, we noticed
consistently stronger vascular ORO staining in the mutants, with the
most significant differences between WT and apoc2 mutants
observed in non-fed 6 dpf embryos. At this stage, ORO staining was
weak or absent in the blood vessels of WT zebrafish, but strong
ORO staining was detected in the apoc2 mutants (Fig. 4Aa,a′,b,b′).
The dramatic differences in the levels of circulating neutral lipids
were also monitored with the fluorescent dye BODIPY (Fig. 4Ac,c′).
The advantage of using BODIPY is that live zebrafish can be
immersed in the stain and after a wash, changes in neutral lipid
levels can be monitored and quantified (Fig. 4B). Furthermore, the
BODIPY staining in live zebrafish persisted over a prolonged period
of time (up to 48 h in apoc2 mutants in our experiments). Consistent
with the staining results, TG and TC levels were significantly
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Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
Fig. 4. apoc2 mutant larvae develop hyperlipidemia. (A) Non-fed 6 dpf WT
and apoc2 mutant (mut) larvae were stained with Oil Red O (a-b′) or BODIPY
(c,c′). Scale bar: 200 µm. (B) Quantification of BODIPY staining in A. Results
are mean±s.e.m.; n=3 in each group; ***P<0.001 (Student’s t-test).
(C,D) TG and TC levels in homogenates of 6 dpf WT and apoc2 mutant larvae,
normalized to total protein content. Results are mean±s.e.m.; n=3 in each
group; **P<0.01; ***P<0.001 (Student’s t-test).
increased in the homogenates of 6 dpf apoc2 mutant larvae
compared with WT (Fig. 4C,D).
In addition to the apoc2 mutant characterized in Figs 1-4, we
generated a second zebrafish line with a different apoc2 mutation,
which introduced a stop codon in the target site, causing loss of the
Lpl-binding domain of Apoc2. This apoc2 mutant (line 2) also
displayed increased vascular ORO and BODIPY staining at 6 dpf,
similar to the frame shift mutant, and the loss of apoc2 mRNA
(supplementary material Fig. S3), further supporting the functional
conservation of Apoc2 in zebrafish.
Injection of normal plasma rescues hyperlipidemia in apoc2
mutants
In human patients with APOC2 deficiency, transfusions of normal
plasma result in rapid and dramatic decreases in the plasma TG levels
in patients (Breckenridge et al., 1978; Nordestgaard et al., 1988). To
test whether this effect can be reproduced in our apoc2 mutant
zebrafish, we isolated plasma from adult WT and mutant zebrafish
and transfused them into WT and apoc2 mutant larva recipients
through cardinal vein injection. The plasma was supplemented with
red fluorescent dextran as a tracer to confirm a successful injection. At
24 h after injection, the larvae with positive dextran fluorescence were
selected and stained with BODIPY. The WT larvae injected with WT
or mutant plasma did not show changes in BODIPY staining.
Compared with apoc2 mutant plasma-injected mutant larvae, WT
plasma-injected apoc2 mutant larvae showed a significant decrease
in BODIPY fluorescence (Fig. 5A,B). Following BODIPY imaging,
the same larvae were euthanized and homogenized, and lipids were
measured. Consistent with the BODIPY-staining results, TG levels in
the apoc2 mutants were significantly reduced following the injection
of WT plasma (Fig. 5C,D).
Disease Models & Mechanisms
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Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
Fig. 5. Injections of WT zebrafish plasma rescue the
hyperlipidemia phenotype in apoc2 mutants. (A) apoc2 WT or
mutant larvae at 5 dpf were injected with 5 nl of undiluted plasma
from either WT or mutant (mut) adult male zebrafish, both
supplemented with red fluorescent dextran. At 1 day after
injection, at 6 dpf, larvae were stained with BODIPY (green
fluorescence) to assess the neutral lipid content of plasma. Red
dextran fluorescence indicates successful injection. Scale bar:
100 µm. (B) Quantification of results shown in A. Results are
mean±s.e.m.; n=3 in each group; ***P<0.001; ns, not significant
(Student’s t-test). (C,D) TG and TC levels in homogenates of 6 dpf
WT and apoc2 mutant larvae, normalized to total protein content.
Results are mean±s.e.m.; five embryos pooled per sample, n=3 in
each group; *P<0.05; **P<0.01 (Student’s t-test).
We have recently developed a novel bihelical amphipathic peptide
(C-II-a) that contains an amphipathic helix (18A) for binding to
lipoproteins and stimulating cholesterol efflux, as well as a motif
based on the last helix of human APOC2, which activates lipolysis by
LPL (Amar et al., 2015). An inactive APOC2 peptide (C-II-i), in
which four amino acids are mutated, was used as a negative control.
The C-II-a peptide restored normal lipolysis in plasma from APOC2deficient human patients and dramatically decreased the TG levels in
Apoe−/− mice (Amar et al., 2015). Protein alignment indicated that
nine of 20 amino acids in C-II-a were conserved between zebrafish
and human, including the crucial amino acids that were mutated in
C-II-i (Fig. 6A). To test whether the human APOC2-based mimetic
peptide could rescue hypertriglyceridemia in apoc2 mutant
zebrafish, we injected C-II-a or C-II-i, together with a fluorescent
dextran tracer, into zebrafish larvae and assessed the vascular content
of neutral lipids (using BODIPY staining) at 30 min and 6, 30 and
72 h after injection. Although at 30 min there was no difference, at
6 h post C-II-a injection, there was a modest, but statistically
significant, 20% decrease in BODIPY fluorescence (supplementary
material Fig. S4). Furthermore, at 30 h, the BODIPY signal was
dramatically decreased by 60% in C-II-a-injected, but not in C-II-iinjected, zebrafish (Fig. 6B,C), and the effect of the C-II-a peptide
persisted for at least 72 h (supplementary material Fig. S4).
Biochemical measurements confirmed a significant decrease in TG
levels and a trend towards TC decrease (Fig. 6D,E).
Vascular lipid accumulation in apoc2 mutant larvae
In our previous experiments, we found that supplementing a highcholesterol diet (HCD) with red fluorescent CE enabled monitoring
accumulation of vascular lipid deposits in live transparent zebrafish
larvae (Fang et al., 2011; Stoletov et al., 2009). As apoc2 mutant
zebrafish have higher TG and TC levels even when fed a diet with
normal content of cholesterol (Figs 2, 4; Fig. 7A,B), we tested the
apoc2 mutants for the presence of vascular lipid deposits. WT and
apoc2 mutants were fed a diet with a normal content of fat and
cholesterol from 5 to 14 dpf. To trace lipid accumulation,
fluorescently labeled CE (576/589-CE; the numbers indicate
excitation/emission wavelengths) was added to the diet from 8 to
12 dpf. The number of fluorescent lipid deposits was significantly
higher in the vasculature of apoc2 mutants than in WT zebrafish
(Fig. 7C,D). To test whether these vascular lipid deposits were in
fact intracellular lipid accumulated in macrophages, as observed in
human and mouse early atherosclerotic lesions, we crossed apoc2
mutants to mpeg1-EGFP transgenic zebrafish, which express EGFP
in macrophages. As expected, the majority of the lipid deposits were
localized within macrophages (Fig. 7E,F).
Pancreatic ectopic growth in apoc2 mutants
Patients with extremely high TG levels are at greater risk for the
development of acute pancreatitis (Ewald et al., 2009). However, the
mechanisms by which this occurs are not known with certainty,
although they might be related in part to excess TG accumulation in
the pancreatic vasculature, where they would be subjected to
hydrolysis by pancreatic lipases (Wang et al., 2009). To assess
developmental pancreatic defects, we crossed apoc2 mutants with
ptf1a-EGFP transgenic zebrafish, which express EGFP primarily in
acinar cells (Dong et al., 2008; Godinho et al., 2005). We observed
ectopic outgrowth of the acinar pancreas in apoc2 mutants starting at
4 dpf, which was persistent and was also observed in 6.5 dpf embryos
(Fig. 8A,B). At 6.5 dpf, a WT pancreas usually has a head domain,
which contains the principal islet, and a long tail domain that extends
posteriorly. In contrast, the apoc2 mutant pancreas had additional
short ectopic protrusions along the head of the pancreas (Fig. 8).
These defects found in embryos from apoc2−/− incrosses were
confirmed in apoc2+/− incross experiments in which embryos were
scored for ectopic pancreatic outgrowth prior to genotyping (Fig. 8C).
DISCUSSION
Recent genome-wide association studies provide new strong
evidence and support earlier studies suggesting that abnormal
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Disease Models & Mechanisms
Injection of APOC2 mimetic peptide rescues hyperlipidemia
in apoc2 mutants
RESOURCE ARTICLE
Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
new zebrafish model in which a loss-of-function mutation in
the apoc2 gene results in chylomicronemia and profound
hypertriglyceridemia. The first apoc2 mutant we selected had a
frame shift mutation, which led to the amino acid sequence change in
its Lpl-binding domain (Fig. 1B,E). In a different apoc2 mutant line,
we found a stop codon mutation, which caused deletion of the Lplbinding domain from the Apoc2 protein sequence (supplementary
material Fig. S3). Importantly, both apoc2 mutant zebrafish lines
showed dramatically decreased apoc2 mRNA expression and
developed hypertriglyceridemia, validating the Apoc2 loss-offunction model.
The plasma of adult apoc2 mutant zebrafish shared many
characteristics with the plasma of APOC2- or LPL-deficient human
patients (Cox et al., 1978; Hooper et al., 2014; Okubo et al., 2014).
It has significantly decreased lipase activity, chylomicronemia,
dramatic hypertriglyceridemia and increased VLDL content,
hypercholesterolemia and reduced levels of HDL (Fig. 2). The
advantages of using zebrafish as an animal model include the
optical transparency of embryos and larvae, which makes them
suitable for live high-resolution imaging, and easy genetic
manipulation and drug screening. Our studies demonstrate that as
early as at 3 dpf, apoc2 mutant larvae already display remarkable
hyperlipidemia, and in 6 dpf embryos, dramatic differences in
circulating neutral lipids were detected with ORO staining and
confirmed with biochemical measurements of TG and TC levels in
whole-body homogenates (Fig. 4).
metabolism of TRLs contributes to increased risk of CVD (Do et al.,
2015, 2013; Hokanson and Austin, 1996). In addition, it has been
shown that carriers of APOC3 loss-of-function mutations have 40%
lower plasma TG levels and a 40% lower risk of CVD than the
general population (Jørgensen et al., 2014; The TG and HDL
Working Group of the Exome Sequencing Project, National Heart,
Lung, and Blood Institute et al., 2014). These two Mendelian
randomization studies categorized participants based on their
APOC3 genotype rather than plasma TG levels, theoretically
excluding confounding factors that affect both plasma TG levels and
CVD (Cohen et al., 2014). Although one still cannot exclude the
possibility that APOC3 plays its role in CVD through other
pathways independent from its effects on TG levels, those two
studies clearly link the plasma TG levels with the genetic cause of
CVD risk.
A major mechanism by which APOC3 increases plasma TG is by
its ability to inhibit LPL activity (Larsson et al., 2013), although it
also is known to inhibit the clearance of VLDL and chylomicron
remnants. In contrast, APOC2 is a cofactor of LPL and its loss of
function results in deficient LPL activity and consequent
hypertriglyceridemia and chylomicronemia (Breckenridge et al.,
1978; Cox et al., 1978). In this work, we report the development of a
994
Fig. 7. Enhanced vascular lipid accumulation in apoc2 mutants. (A,B) TG
and TC levels in homogenates of 14 dpf WT and apoc2 mutant (mut) larvae,
normalized to total protein content. Larvae were fed a normal diet. Results are
mean±s.e.m.; n=3 in each group; *P<0.05; ***P<0.001 (Student’s t-test).
(C,D) Vascular lipid deposits in 14 dpf WT (n=8) and apoc2 mutant (n=8)
larvae fed a normal diet; **P<0.01 (Student’s t-test). (E) Colocalization
of vascular lipid deposits with macrophages in 14 dpf apoc2 mutant
mpeg1:EGFP zebrafish. (F) Quantification of lipid deposits associated and
non-associated with macrophages in apoc2 mutant larvae. Results are
mean±s.e.m.; n=7. Scale bars: 50 µm.
Disease Models & Mechanisms
Fig. 6. Injections of human APOC2 mimetic peptide rescue
hyperlipidemia phenotype in apoc2 mutant. (A) Protein alignment of
APOC2 mimetic peptides with the corresponding zebrafish Apoc2 amino acids
sequence. Blue and red asterisks indicate the conserved amino acids in the
C-II-a (active) peptide; red asterisks indicate amino acids that were mutated in
the C-II-i (inactive) peptide. (B) At 30 h after injection of 5 nl of C-II-a or C-II-i, at
6.5 dpf, larvae were stained with BODIPY. Red dextran fluorescence indicates
successful injection. Scale bar: 50 µm. (C) Quantification of BODIPY staining
results. Results are mean±s.e.m.; n=3 in each group; ***P<0.001; ns, not
significant (Student’s t-test). (D,E). TG and TC levels in homogenates of
6.5 dpf WT and apoc2 mutant larvae, normalized to total protein content.
Results are mean±s.e.m.; five embryos pooled per sample, n=3 in each group;
*P<0.05; **P<0.01; ns, not significant (Student’s t-test).
RESOURCE ARTICLE
Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
In human APOC2 patients, chylomicron and VLDL size and
abundance fluctuate widely and depend on the type of food
consumed on the preceding day (Breckenridge et al., 1978). Future
work will determine whether variation of the diet fed to apoc2
mutant zebrafish will help maximize chylomicron or VLDL content
for specific experimental goals. An important difference between
mammalian and fish lipoprotein metabolism is the absence of
APOBEC1 in non-mammalian vertebrates (Conticello et al., 2005;
Teng and Davidson, 1992) and, thus, the absence of intestinal apoB48, which is a major apolipoprotein in human chylomicrons. This
might explain why the majority of TRLs we observed in apoc2
mutant zebrafish were a similar size to human and mouse VLDL.
The lipoprotein profiles shown in Fig. 2D-F suggest that the apoc2
mutant zebrafish are suitable for modeling human dyslipidemia, and
the zebrafish model favorably compares with a large number of
other models, including the Ldlr−/− mouse, as profiled previously
(Yin et al., 2012).
In this work, we developed a simple and convenient protocol for
staining circulating lipoproteins with BODIPY. BODIPY is a
lipophilic fluorescent dye, which preferentially localizes to the
neutral lipid core of lipoproteins and intracellular lipid droplets, and
is widely used in cell biology (Bozaquel-Morais et al., 2010; Gocze
and Freeman, 1994; Grandl and Schmitz, 2010). However, to the
best of our knowledge, BODIPY has not been used to stain and
monitor circulating lipid levels in live zebrafish larvae. In our
experiments, live zebrafish were immersed in a BODIPY solution,
with a subsequent wash in water. This simple procedure resulted in
bright green fluorescent staining of neutral lipids in the circulation
of apoc2 mutant animals. This assay provides a robust readout and
might help conduct genetic and drug screening studies.
We used the BODIPY assay to demonstrate that injections of normal
zebrafish plasma significantly diminished hypertriglyceridemia in
apoc2 mutant zebrafish (Fig. 5). This result is similar to the treatment
of APOC2-deficient patients with severe hypertriglyceridemia in
which transfusions of normal, APOC2-containing plasma leads to
rapid (within 1 day) reductions in plasma TGs (Breckenridge et al.,
1978). Remarkably, the amino acids that are crucial for LPL
activation in the last helix of human APOC2 are conserved in
zebrafish, and the human APOC2 mimetic peptide C-II-a (Amar
et al., 2015), rescued severe hypertriglyceridemia in apoc2 mutant
zebrafish (Fig. 6). These results suggest that our zebrafish model is
suitable for testing potential therapeutic agents for treatment of
human APOC2 deficiency.
Two previous studies in which apoc2 was transiently knocked
down in zebrafish embryos with antisense morpholino
oligonucleotides, have suggested that Apoc2 is involved in yolk
absorption and angiogenesis (Avraham-Davidi et al., 2012; Pickart
et al., 2006). Our results with the apoc2 mutant zebrafish support
these conclusions. Consistent with the results reported by Pickart
et al., apoc2 mutant larvae show a slightly delayed yolk consumption
(Fig. 3A-G) and the mutant larvae are smaller than WT larvae. This
phenotype in the apoc2 mutant can be explained, in part, by the
disruption of the Lpl function, resulting in a defect in TG hydrolysis
and the supply of free fatty acids (FFAs) to non-hepatic tissues, which,
in turn, might cause nutrient deprivation and delayed growth. In
contrast, by monitoring the head angle at 28 hpf and the pattern of
pigmentation at 3 dpf, we did not notice any differences in the stage of
development reached between WT and apoc2 mutants. Because adult
apoc2 mutant zebrafish are the same size as WT and have no apparent
phenotype (Fig. 2A; supplementary material Fig. S1), other than
hyperlipidemia, it is possible that feeding and the activity of other
lipases help restore the energy balance in unchallenged animals.
A previous study has reported that a transient apoc2 knockdown
by antisense morpholino oligonucleotides increases levels of apoBcontaining lipoproteins, which, in turn, inhibits embryonic
angiogenesis (Avraham-Davidi et al., 2012). In our experiments,
the Apoc2 loss of function resulted in dramatically increased levels
of VLDL, an apoB-containing lipoprotein, and indeed delayed ISV
and SIV growth. Nevertheless, the apoc2 mutants were viable and
their vascular structure was normal at 14 dpf despite profound
hyperlipidemia (supplementary material Fig. S2). Thus, the excess
of apoB-containing lipoproteins is likely to delay but not to
completely inhibit angiogenesis in vivo.
In agreement with epidemiologic studies suggesting that
hypertriglyceridemia is a risk factor for CVD (Do et al., 2015;
Jørgensen et al., 2014; The TG and HDLWorking Group of the Exome
Sequencing Project, National Heart, Lung, and Blood Institute
et al., 2014), our results demonstrate that the hypertriglyceridemia
induced by the apoc2 mutation in zebrafish results in vascular lipid
accumulation and macrophage lipid uptake (Fig. 7C-E), which
are important initial events in the development of human
atherosclerosis. The vascular lipid accumulation in zebrafish
induced by hypertriglyceridemia was similar to that induced
by hypercholesterolemia (O’Hare et al., 2014; Stoletov et al.,
2009). Our study is also consistent with mouse studies in which
hypertriglyceridemia induced by Lpl or Gpihbp knockout resulted in
995
Disease Models & Mechanisms
Fig. 8. apoc2 mutants have ectopic pancreas outgrowth. (A) Images of pancreas, labeled with EGFP driven by the ptf1a promoter, in 6.5 dpf WT and apoc2
mutant (mut) larvae. The yellow arrow points to the normal pancreatic growth and white arrows point to ectopic outgrowth. Scale bar: 100 µm. (B,C) At 6.5 dpf, an
independent observer scored a pancreatic outgrowth phenotype as 0, normal, 1, mild and 2, severe. (B) WT (n=13) and apoc2−/− mutant (n=21). Results are
mean±s.e.m.; ***P<0.001 (Student’s t-test). (C) Heterozygous apoc2+/− zebrafish carrying one copy of the ptf1a-EGFP transgene were crossed with
the heterozygous apoc2+/− zebrafish carrying no ptf1a-EGFP. A total of 22 embryos that displayed EGFP fluorescence were selected, scored for the
pancreas outgrowth defect and then genotyped. Random ptf1a-EGFP expression resulted in the following distribution of EGFP-positive embryos: apoc2+/+ (n=3),
apoc2+/− (n=9) and apoc2−/− (n=10). Results are mean±s.e.m.; *P<0.05; ***P<0.001 (Student’s t-test).
spontaneous development of atherosclerosis (Weinstein et al., 2010;
Zhang et al., 2008). In contrast to hypertriglyceridemic mice, in
which atherosclerotic lesions develop late, by the age of 1 year,
vascular lipid deposits in apoc2 mutant zebrafish fed normal diet
developed as early as at 14 dpf. One possible explanation is the
absence of CETP in mice, whereas zebrafish express functional Cetp
(Jin et al., 2011; Kim et al., 2012). Transfer of cholesterol from HDL
to LDL and VLDL (and TG in the opposite direction) might play an
atherogenic role in apoc2 mutant zebrafish, as it does in humans
(Barter and Rye, 2012).
One important clinical complication of hypertriglyceridemia in
patients with LPL or APOC2 deficiency is recurrent pancreatitis, but
the underlying mechanisms are poorly defined (Baggio et al., 1986;
Fellin et al., 1983). In apoc2 mutant zebrafish, we found ectopic acinar
outgrowth in the head of the pancreas (Fig. 8). Acinar cells express
high levels of pancreatic lipase. Chylomicronemia results in increased
FFA uptake and injury in the acinar cells, which might lead to
inflammation and pancreatitis (Scherer et al., 2014; Wang et al.,
2009). Our findings suggest that the Apoc2 deficiency affects early
pancreas development in zebrafish. A recent report shows that
modeling chylomicron retention disease in zebrafish, which manifests
in the reduction of circulating neutral lipids, was associated with
inhibited growth of exocrine pancreas (Levic et al., 2015). We as yet
do not know whether the hyper- or hypo-triglyceridemia effects on
exocrine pancreas growth in apoc2 mutant zebrafish are related to the
pathogenesis of pancreatitis in human APOC2-deficient patients.
In summary, our new apoc2 mutant zebrafish display a robust
hyperlipidemia phenotype and present a useful and versatile animal
model to study mechanisms related to human diseases induced by
hypertriglyceridemia. Small-molecule and genetic screens using the
apoc2 mutant zebrafish might suggest new approaches to treatment
of hyperlipidemia and related disorders.
MATERIALS AND METHODS
Zebrafish maintenance and feeding
Adult zebrafish of the AB strain were maintained at 28°C on a 14-h-light–
10-h-dark cycle and fed brine shrimp twice a day. Zebrafish larvae were fed
Golden Pearls (100–200 µm size from Brine Shrimp Direct, Ogden, UT)
twice a day, starting from 4.5 dpf. For lipid-deposition experiments, Golden
Pearls, supplemented with 1 µg/g of a fluorescent cholesterol ester analog
(cholesteryl BODIPY 576/589-C11 from Invitrogen, Carlsbad, CA; catalog
number C12681), were fed to zebrafish from 8 to 12 dpf as described
previously (Stoletov et al., 2009). fli1:EGFP and mpeg1-EGFP transgenic
zebrafish were from the Weinstein (Lawson and Weinstein, 2002) and
Lieschke (Ellett et al., 2011) laboratories, respectively. ptf1a-EGFP
transgenic zebrafish were developed in the Dong laboratory (Dong et al.,
2008; Godinho et al., 2005). All animal studies were approved by the UCSD
Institutional Animal Care and Use Committee.
TALEN construction, mRNA synthesis, injection and
confirmation of apoc2 mutation
TALEN plasmids targeting apoc2 were constructed from the stock cassette
according to the published protocols (Dong et al., 2011; Huang et al., 2014).
TALEN mRNAs were synthesized with an mMESSAGE mMACHINE Sp6
transcription kit (Ambion, Austin, TX; AM1340). mRNAs encoding the
pair of TALEN proteins were mixed at a 1:1 ratio to a final concentration of
200 ng/µl. A total of 1-2 nl of TALEN mRNAs was injected into the one-cell
stage embryos. Genomic DNA (gDNA) was extracted from whole embryos
or from the tissue clipped off the tail fin of adult zebrafish and was
genotyped by PCR (with the following primers: forward, 5′-GTGGTCGCATAGTTTAAGCT-3′; reverse, 5′-GCAACCATGTAAGAGTTGCA-3′)
and BtsI enzyme digestion, and confirmed by sequencing, according to
published protocols (Dong et al., 2011; Huang et al., 2014). To assess
expression of apoc2 in larvae homogenates and in adult liver, total RNA was
996
Disease Models & Mechanisms (2015) 8, 989-998 doi:10.1242/dmm.019836
isolated, reverse transcribed and subjected to qPCR with the following
primers: forward, 5′-ATGAACAAGATACTGGCTAT-3′; reverse, 5′TTGATGGTCTCTACATATCC-3′, using a KAPA SYBR FAST
Universal qPCR kit (KAPA Biosystems, Wilmington, MA; KK4602) and
a Rotor Gene Q qPCR machine (Qiagen, Valencia, CA). The position of
these qPCR primers relative to the TALEN target site is shown in
supplementary material Fig. S3. Because the mutation does not change
nucleotide sequence beyond the target site, these primers detect expression
of both WT and mutant mRNA, if any is present.
Oil red O staining and BODIPY staining
Oil red O (ORO) staining was conducted according to published protocols
(Schlegel and Stainier, 2006). Briefly, embryos were fixed in 4%
paraformaldehyde (PFA) for 2 h, washed three times in PBS, incubated in
0.3% ORO solution for 2 h and then washed with PBS before imaging. For
BODIPY staining, live larvae were immersed in E3 water (5.0 mM NaCl,
0.17 mM KCl, 0.33 mM CaCl2, 0.33 mM MgSO4) containing 0.1 µg/ml
BODIPY® 505/515 (4,4-difluoro-1,3,5,7-tetramethyl-4-Bora-3a,4a-diazas-indacene, Invitrogen; D-3921) for 1 h in dark and then rinsed with E3
water before imaging.
Triglyceride and cholesterol measurements and lipoprotein
analysis
Blood was collected from adult male zebrafish, after overnight fasting,
through tail amputation and diluted 1:50 (WT) or 1:200 (apoc2 mutant) in
PBS. The supernatants were collected as a plasma fraction after centrifugation
at 2350 g for 10 min. Embryos or larvae were gently homogenized in PBS
with a pestle. After centrifugation at 16,000 g for 10 min, supernatants were
collected and referred to as ‘homogenates’. TG and TC levels were measured
in 25 µl of diluted plasma or embryo or larva homogenates using kits from
BioVision (Milpitas, CA; Triglyceride Quantification Kit, K622-100;
Cholesterol Quantification Kit, K623-100) and according to the
manufacturer’s protocol. To assess undiluted plasma, 5 µl blood was
collected in a tube containing 0.5 µl heparin (5 mg/ml) and then loaded
into a heparin-rinsed capillary, which was kept overnight at 4°C in a vertical
position. Following erythrocyte sedimentation, the capillaries were
photographed. Lipoprotein fractions were assessed using native agarose gel
electrophoresis (Helena Laboratories, Beaumont, TX; 3045) as we
previously described (Stoletov et al., 2009).
Fast protein liquid chromatography lipoprotein profile
A total of 100 µl of pooled plasma from 45 adult WT zebrafish or 40 µl of
pooled plasma from 14 adult apoc2 mutant zebrafish were loaded onto a
Superose 6 PC 3.2/30 column (GE Healthcare Life Science, Pittsburgh, PA;
17-0673-01), and TC and TG levels were determined in each fraction
(250 μl), collected at a flow speed of 0.5 ml/min.
Ultracentrifugation and electron microscopy
Pooled plasma from fasted overnight WT and apoc2 mutant zebrafish was
diluted in PBS, without density adjustment, and the samples were
centrifuged at 200,000 g for 4 h at 4°C. The top layer containing TRLs
was collected, supplemented with 0.1% sucrose, added to a FCF100-Cu 100
mesh (Electron Microscopy Sciences, Hatfield, PA) and negatively stained
with 1% uranyl acetate. Stained samples were imaged with a Tecnai G2
Spirit BioTWIN transmission electron microscope equipped with a 4K
Eagle digital camera (FEI Company, Hillsboro, OR).
Plasma lipase activity assay
Adult zebrafish blood was diluted 1:25 in PBS and after centrifugation, 5 µl
of the diluted plasma was used to measure lipase assay with a Lipase activity
assay kit (Cayman Chemical, Ann Arbor, MI; 700640), following the
manufacturer’s manual. The assay buffer was used as a negative control. The
reaction was conducted at 30°C.
Intravenous injections
Blood was collected by tail amputation from adult male zebrafish at 4 h after
the last feeding, and plasma was separated by centrifugation at 2350 g for
Disease Models & Mechanisms
RESOURCE ARTICLE
10 min. C-II-a and C-II-i peptides (Amar et al., 2015) were dissolved in PBS
( pH 7.4) to a concentration of 2 mg/ml. To ensure visual control of
successful injection, zebrafish plasma or APOC2 mimetic peptides were
supplemented with 2% (v/v) tetramethylrhodamine-conjugated 10 kDa
dextran (0.5 mg/ml miniRuby, Invitrogen, D-3312). For injections, 5 dpf
larvae were aligned in 0.5% low-melting-point agarose. A total of 5 nl of
plasma or APOC2 mimetic peptides were injected through the cardinal vein
above the heart chamber using a FemtoJet microinjector (Eppendorf,
Hamburg, Germany). The red signal from fluorescent dextran confirmed
successful injection.
Imaging of live embryos or larvae
For in vivo live microscopy, anesthetized embryos or larvae were mounted in
low-melting-point agarose (0.5%, Fisher, Pittsburgh, PA; BP1360-100)
containing tricaine (0.02%, Sigma, St Louis, MO; A5040) in 50-mm glassbottom dishes (MatTek, Ashland, MA; P50G-0-14-F). Images were
captured with Leica CTR5000 (Wetzlar, Germany), Olympus FV1000
spectral confocal (Tokyo, Japan) or BZ9000 Keyence (Osaka, Japan)
fluorescent microscopes.
Competing interests
J.L.W. is a consultant for ISIS Pharmaceuticals. Other authors declare no competing
interests.
Author contributions
C.L. and Y.I.M conceived and designed the experiments. C.L., K.P.G., L.F., D.A.S.,
H.G., J.P., W.H. and J.K. performed the experiments. M.J.A., J.Z., J.L.W., A.T.R. and
P.D.S.D. provided crucial materials, ideas and discussion for this work. C.L. and
Y.I.M. wrote the paper.
Funding
The project was supported by the National Institutes of Health (NIH) [grant numbers
HL093767 to Y.I.M., HL055798 to Y.I.M., HL088093 to J.L.W. and DK098092
P.D.S.D.], as well as the UCSD Neuroscience Microscopy Facility [grant number
P30NS0471010].
Supplementary material
Supplementary material available online at
http://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.019836/-/DC1
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Liu et al. Apoc2 loss-of-function zebrafish mutant as a genetic model of hyperlipidemia
Supplementary Figures
Figure S1: Body weight and size of adult WT and apoc2 mutant zebrafish. Physical
appearance (A) and body weight and length (B) of WT (n=14) and mutant (n=14), 10
month old, male and female zebrafish. Scale bar, 0.5 cm.
Figure S2: Angiogenesis in fli1:EGFP WT and apoc2 mutant zebrafish at 14 dpf
Disease Models & Mechanisms | Supplementary Material
Disease Models & Mechanisms 8: doi:10.1242/dmm.019836: Supplementary Material
Figure S3: Additional apoc2 mutant zebrafish line
(A) Sequence results of the apoc2 genomic DNA from the mutant line 1 (shown in Fig. 1)
and the additional line 2. (B) Mutation in line 2 introduced a stop codon in the apoc2 gene.
(C) BODIPY and ORO staining of WT and line 2 apoc2 mutant at 6 dpf. Scale bar, 200
µm. (D) BODIPY staining of WT and line 1 and line apoc2 mutant larvae at 6 dpf. Scale
bar, 200 µm. (E) Quantification of BODIPY staining results shown in panel D. Mean±SEM;
n=3 in each group; ***, p<0.001; **, p<0.01. (F) The diagram shows the position of qPCR
primers relative to the mutation target site. apoc2 mRNA expression (qPCR) in whole body
homogenates of 5.5 dpf WT, mutant line 1 and mutant line 2 larvae. Mean±SEM; n=3 in
each group; **, p<0.01. (Part of this graph is also shown in Fig. 1G.)
Disease Models & Mechanisms | Supplementary Material
Disease Models & Mechanisms 8: doi:10.1242/dmm.019836: Supplementary Material
Figure S4: Injection of human apoC-II mimetic peptide: Time course
The C-II-a and C-II-i peptides were injected as in Fig. 6. (A and B) 30 minutes after
injection; (C and D) 6 hours after injection; (E and F) three days after injection. Larvae
were stained with BODIPY. Red dextran fluorescence indicates successful injection.
Scale bar, 50 µm. (B, D and F) Quantification of BODIPY staining results. Mean±SEM;
n=3; ***, p<0.001; **, p<0.01; *, p<0.05; ns, not significant.
Disease Models & Mechanisms | Supplementary Material