Zebrafish as a model for apolipoprotein biology ... · expression and function of eleven zebrafish...

© 2015. Published by The Company of Biologists Ltd | Disease Models & Mechanisms (2015) 8, 295-309 doi:10.1242/dmm.018754

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ABSTRACTImproved understanding of lipoproteins, particles that transport lipidsthroughout the circulation, is vital to developing new treatments for the dyslipidemias associated with metabolic syndrome.Apolipoproteins are a key component of lipoproteins. Apolipoproteinsare proteins that structure lipoproteins and regulate lipid metabolismthrough control of cellular lipid exchange. Constraints of cell cultureand mouse models mean that there is a need for a complementarymodel that can replicate the complex in vivo milieu that regulatesapolipoprotein and lipoprotein biology. Here, we further establish theutility of the genetically tractable and optically clear larval zebrafishas a model of apolipoprotein biology. Gene ancestry analyses wereimplemented to determine the closest human orthologs of thezebrafish apolipoprotein A-I (apoA-I), apoB, apoE and apoA-IV genesand therefore ensure that they have been correctly named. Theirexpression patterns throughout development were also analyzed, bywhole-mount mRNA in situ hybridization (ISH). The ISH resultsemphasized the importance of apolipoproteins in transporting yolkand dietary lipids: mRNA expression of all apolipoproteins wasobserved in the yolk syncytial layer, and intestinal and liverexpression was observed from 4-6 days post-fertilization (dpf).Furthermore, real-time PCR confirmed that transcription of three ofthe four zebrafish apoA-IV genes was increased 4 hours after theonset of a 1-hour high-fat feed. Therefore, we tested the hypothesisthat zebrafish ApoA-IV performs a conserved role to that in rat in theregulation of food intake by transiently overexpressing ApoA-IVb.1 intransgenic larvae and quantifying ingestion of co-fed fluorescentlylabeled fatty acid during a high-fat meal as an indicator of food intake.Indeed, ApoA-IVb.1 overexpression decreased food intake byapproximately one-third. This study comprehensively describes theexpression and function of eleven zebrafish apolipoproteins andserves as a springboard for future investigations to elucidate theirroles in development and disease in the larval zebrafish model.

KEY WORDS: Zebrafish, Developmental expression patterns,mRNA in situ hybridization, Apolipoprotein A-I, Apolipoprotein B,Apolipoprotein A-IV, Apolipoprotein E, Regulation of food intake

RESOURCE ARTICLE

1Carnegie Institution for Science, Department of Embryology, Baltimore, MD 21218, USA. 2Johns Hopkins University, Department of Biology, Baltimore,MD 21218, USA. 3Weizmann Institute of Science, Department of BiologicalRegulation, Rehovot 7610001, Israel.

*Author for correspondence ([email protected])

This is an Open Access article distributed under the terms of the Creative CommonsAttribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricteduse, distribution and reproduction in any medium provided that the original work is properlyattributed.

Received 10 October 2014; Accepted 22 January 2015

INTRODUCTIONThe current epidemic of metabolic syndrome has resulted inwidespread morbidity and mortality due to population increases inobesity, cardiovascular disease, type II diabetes, hypertension andstroke (Grundy et al., 2005). Developing treatments for thedyslipidemias frequently associated with metabolic syndrome [e.g.high serum triglycerides and low high-density lipoprotein (HDL)cholesterol] is a priority. It is well known that lipoprotein particlestransport lipids throughout the circulation. However, thedevelopment of improved treatments for common dyslipidemiasrequires a deeper understanding of lipoprotein function andregulation. For instance, despite a vast scientific literaturespanning over 50 years, we still lack a basic mechanisticunderstanding of the relationship between HDL and cardiovasculardisease risk (Voight et al., 2012). Furthermore, characterization oflipoprotein biology is impeded without a comprehensive study ofapolipoproteins, the class of over a dozen secreted, lipid-bindingproteins that function as structural backbones of lipoproteinparticles and regulators of cellular lipid flux through theirinteractions with cell surface receptors.

This study focuses on four major serum apolipoproteins:apolipoprotein A-I (APOA-I), apolipoprotein B (APOB),apolipoprotein E (APOE) and apolipoprotein A-IV (APOA-IV)(note that we use capitals to denote the mammalian protein form).APOA-I is the main protein component of HDL particles. Inaddition to its intrinsic, beneficial anti-oxidative, anti-inflammatoryand anti-bacterial properties (Feingold and Grunfeld, 2011; Garneret al., 1998; Navab et al., 2000a; Navab et al., 2000b), APOA-Iregulates cholesterol efflux from cells to HDL particles, the first stepin transporting peripheral cholesterol to the liver for excretion inbile. The liver and intestine produce mammalian APOA-I andsubsequently HDL. Humans have two isoforms of the secondapolipoprotein of interest, APOB, resulting from mRNA editing: theshort form APOB-48 is produced by the intestine, and the long formAPOB-100 by the liver (Fisher and Ginsberg, 2002). APOB-48 isan essential structural component of intestinally secretedchylomicrons, whereas APOB-100 gives structure to very-low-density lipoproteins (VLDL) and low-density lipoproteins (LDL) ofhepatic origin. APOE, the third apolipoprotein of interest, isexchangeable, transferring between most classes of lipoproteins inthe circulation. APOE binds to LDL receptors, thereby regulatinglipid uptake (Mahley, 1988). APOE-deficient mice develop severeatherosclerosis due to increased circulating LDL cholesterol(Nakashima et al., 1994). In addition to being expressed by theintestine, liver and macrophages, APOE is synthesized in themammalian brain and acts as the major apolipoprotein of the lipid-rich central nervous system (CNS). The final apolipoprotein

Zebrafish as a model for apolipoprotein biology: comprehensiveexpression analysis and a role for ApoA-IV in regulating foodintakeJessica P. Otis1, Erin M. Zeituni1, James H. Thierer1,2, Jennifer L. Anderson1, Alexandria C. Brown1, Erica D. Boehm1,2, Derek M. Cerchione1,2, Alexis M. Ceasrine1,2, Inbal Avraham-Davidi3, Hanoch Tempelhof3,Karina Yaniv3 and Steven A. Farber1,2,*

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examined, APOA-IV, is produced by the intestine and secreted as acomponent of chylomicrons following lipid-rich meals. In rats, acuteadministration of APOA-IV suppresses food intake (Fujimoto et al.,1992; Fujimoto et al., 1993b; Wang et al., 2013a).

Our choice to focus on these four apolipoproteins in thisinvestigation was determined by their implication in a diverse rangeof dyslipidemias and neurodegenerative diseases. For example, thereare striking positive correlations in human populations betweenprotection from cardiovascular disease and the levels of APOA-Iand HDL cholesterol, as well as the occurrence of specific APOA-I polymorphisms (Franceschini et al., 1980; Kannel et al., 1964;Toth et al., 2013). By contrast, LDL cholesterol levels, which are inpart dependent on APOB owing to its required structural role inthese particles, are positively associated with cardiovascular diseaserisk (Wilson et al., 1998). Three polymorphisms in human APOEare strongly associated with the efficacy of plasma lipid clearance,and consequently with the risk of cardiovascular disease; thesealleles are also correlated with Alzheimer’s disease risk (Corder etal., 1993; Utermann et al., 1977). Finally, the action of APOA-IV inreducing food intake is reduced upon chronic high-fat feeding and

obesity, potentially furthering the cycle of hyperphagia and weightgain (Tso and Liu, 2004).

Cell culture and mouse studies have provided the bulk of ourcurrent understanding of the mechanistic relationships betweenapolipoproteins and disease. However, cell culture systems do notreplicate the crosstalk between multiple organs, and studiesperformed in whole animals often have to rely on indirect assaysthat measure surrogates for lipid metabolism (e.g. plasma lipidprofile). As a result, the details of the regulatory signals thatcoordinate cellular responses to lipids and production of specificlipoproteins from particular tissues are not well characterized. Forexample, it was long believed that APOA-I and HDL preventedcardiovascular disease through reverse cholesterol transport, thetransport of peripheral cholesterol to the liver; however, micelacking APOA-I have normal biliary cholesterol secretion (Amigoet al., 2011) and some pharmaceutical agents that raise HDL-C failto provide the protection observed with naturally high HDLcholesterol levels (Barter et al., 2007; Boden et al., 2011). Moreover,acute administration of APOA-IV to rats suppresses food intake(Fujimoto et al., 1993b), but chronic overexpression or deletion ofAPOA-IV does not affect food intake in mice (Aalto-Setälä et al.,1994; Weinstock et al., 1997). The seemingly conflicting findingssurrounding how APOA-I and APOA-IV function highlight theneed for experimental model systems that support the study ofglobal apolipoprotein dynamics in living organisms and relate theseobservations back to the response of a particular cell within a livingorgan. Remarkably, studies at this level of resolution can beperformed in the larval zebrafish (Danio rerio).

The larval zebrafish has a highly similar, yet simplified,gastrointestinal tract to that of humans (Carten and Farber, 2009).Zebrafish also have a similar lipoprotein lipid transport system(Babin and Vernier, 1989), although the teleost genome duplicationevent created multiple paralogs of most zebrafish apolipoproteins.Unlike humans, but similar to mice, zebrafish transport greateramounts of plasma cholesterol as HDL than as LDL (Pedroso et al.,2012). Historically an important developmental model, the zebrafishis emerging as an invaluable resource to study metabolic function inhealth and disease (Anderson et al., 2011; Asaoka et al., 2013;Carten and Farber, 2009; Fang and Miller, 2012; Otis and Farber,2013; Schlegel and Stainier, 2007; Seth et al., 2013). This claim issupported by a body of research that has combined the genetictractability and conduciveness to live imaging of larvae with novelzebrafish models of dietary manipulations [high-fat (Carten et al.,2011; Marza et al., 2005), high-cholesterol (Stoletov et al., 2009)and high-carbohydrate diets (Fang et al., 2014; Wang et al., 2013b)],cardiovascular disease (Fang et al., 2011), type II diabetes (Curadoet al., 2007; Pisharath et al., 2007), hepatic steatosis (Matthews etal., 2009; Passeri et al., 2009; Sadler et al., 2005) and obesity (Chuet al., 2012; Oka et al., 2010; Song and Cone, 2007), to providetranslational insights that can be applied to human metabolicdisorders.

Specific discoveries related to apolipoprotein function that havebeen gained from work in larval zebrafish include the findings thatApoB negatively regulates angiogenesis through Vegfr1(Avraham-Davidi et al., 2012), ApoA-I binding protein inhibitsangiogenesis through Vegfr2 (Fang et al., 2013), and ApoA-II isrequired for proper chromosome separation during nuclear divisionof in vivo larval zebrafish cells and cultured human cells (Zhanget al., 2011). Although multiple studies have described expressionpatterns and functions of particular zebrafish apolipoproteins(Babin et al., 1997; Herbomel et al., 2001; Monnot et al., 1999;Poupard et al., 2000; Thisse et al., 2001; Thisse and Thisse, 2005;

RESOURCE ARTICLE Disease Models & Mechanisms (2015) doi:10.1242/dmm.018754

RESOURCE IMPACTBackgroundThe numerous diseases related to metabolic syndrome – such asobesity, cardiovascular disease, type II diabetes, hypertension and stroke– are often associated with severe morbidity and mortality. An improvedunderstanding of lipoproteins, which transport lipids throughout thecirculation, is vital for developing new pharmaceutical treatments for lipidabnormalities associated with metabolic syndrome. Greater knowledgeof apolipoproteins (proteins that bind lipids and that function both asstructural backbones of lipoprotein particles and as regulators of cellularlipid import and export) will prove to be invaluable to our understandingof lipoprotein function. Mouse and cell culture models are often used tostudy dyslipidemias (abnormal levels of lipids in the blood) andapolipoprotein biology. However, constrains of these systems warrantdevelopment of a new model that can replicate the complex in vivo milieuthat regulates lipoproteins biology.

ResultsIn this investigation, the authors took advantage of the highly geneticallytractable and optically accessible larval zebrafish as a model toinvestigate apolipoprotein dynamics and actions. The spatiotemporalmRNA expression of the 11 zebrafish apolipoprotein genes (apoA-I,apoB, apoE and apoA-IV) was characterized from embryogenesisthrough early larval development. All apolipoproteins were expressed inthe yolk syncytial layer, highlighting their importance in transporting lipidsfrom the yolk cell to the developing embryo, and subsequently in thedigestive organs (intestine and liver). Given that apolipoproteins areessential for lipoprotein formation and dietary lipid transport, theirregulation by a high-fat diet was investigated. mRNA expression of threeout of the four apoA-IV paralogs was increased by a high-fat feed. It wasalso shown, by acutely overexpressing ApoA-IVb.1 in transgeniczebrafish and observing a decrease in the consumption of fluorescentlylabeled lipids, that zebrafish ApoA-IV has a role in the regulation of foodintake similar to that of rat APOA-IV.

Implications and future directionsThis model lays the foundation for future studies on apolipoproteins andtheir associated lipoproteins and dyslipidemias in zebrafish. Thediscoveries presented here emphasize the close relationship betweenzebrafish and human apolipoprotein expression, further establishing thezebrafish as a novel model organism for the study of apolipoproteinbiology. These findings encourage researchers to apply the uniqueattributes of the larval zebrafish (rapid development, genetic tractability,live imaging, visualization of fluorescently labeled lipids, and ease oflarge pharmaceutical screens in live animals) to the study of global andsubcellular apolipoprotein dynamics in development, health and disease.

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Tingaud-Sequeira et al., 2006), none have characterized expressionfrom the eight-cell stage comprehensively throughout larvaldevelopment, included all paralogs of a given gene or examinedthe effect of feeding.

For this reason we undertook a study to further establish the useof the zebrafish as a model of apolipoprotein function in lipidmetabolism, embryonic development, dietary nutrient processingand disease. We first validated and clarified the zebrafishapolipoprotein nomenclature by investigating the ancestralrelationships between the four human and 11 zebrafish genesencoding the APOA-I, APOB, APOE and APOA-IV proteins byperforming sequence, syntenic, and phylogenetic analyses. Second,we characterized zebrafish apolipoprotein spatiotemporal expressionpatterns throughout embryogenesis and early larval development,and determined how these metabolic proteins are transcriptionallyregulated by a high-fat feed. Finally, we determined that zebrafishApoA-IV, of which three of four paralogs are upregulated by a high-fat meal, decreases food intake when overexpressed in transgeniclarvae presented with a high-fat meal. These results indicate thatzebrafish ApoA-IV has a conserved role in the regulation of high-fat food intake and further establishes the larval zebrafish as avaluable model of apolipoprotein biology.

RESULTSGene ancestry analysis of zebrafish apolipoproteinsThe application of a common apolipoprotein nomenclature is ofparamount importance to the larger research community in order tocorrectly interpret, replicate and build upon published findings. Byconvention, zebrafish genes are named based on their closest humanorthologs, as determined by predicted amino acid sequencecomparisons. To verify that each zebrafish apolipoprotein gene wascorrectly named, we checked not only that the 11 zebrafish apoA-I,apoB, apoE and apoA-IV genes were most similar in amino acidsequence to their human ortholog, but also investigated sharedsyntenic gene regions and completed phylogenetic analyses. Thesedata were communicated to the Zebrafish Model Organism Database(ZFIN) team to refine the nomenclature and propagate these genenames to zebrafish genome databases.

The gene ancestry analysis began with a comparison of zebrafishapolipoprotein amino acid sequences to the human reference proteinsequence database using the BLASTp algorithm to verify orthology(Fig. 1A). This was followed by pairwise Needleman–Wunschprotein sequence alignments, which showed a range of 17.9-33.3%sequence identity and 29.0-54.2% sequence similarity betweenzebrafish and human orthologs (Fig. 1A; supplementary materialFig. S1).

The phylogenetic relationships between the human (Homosapiens), zebrafish, mouse (Mus musculus), chicken (Gallus gallus)and coelacanth (Latimeria chalumnae) genes encoding the APOA-I, APOB, APOE and APOA-IV proteins were examined bygenerating a multiple sequence alignment with the iterative MAFFTmultiple alignment tool. The resulting alignment was funneled intothe MAFFT tree server and used to build a neighbor-joining treewith bootstrap resampling. The zebrafish genes encoding ApoA-I,ApoE and ApoB fell into well-supported clades with their vertebrateorthologs (Fig. 1B). In contrast, the zebrafish ApoA-IV paralogsgrouped into a distinct subfamily of ApoA-IV genes that is onlytenuously linked to amniote APOA-IV genes. The analysis alsoemphasizes that ApoA-IVb.1, ApoA-IVb.2 and ApoA-IVb.3 areevolutionarily more closely related to each other than to ApoA-IVa,as evidenced by very short branch lengths between the three ApoA-IVb paralogs (Fig. 1B).

The publicly available Genomicus and Synteny DB analysis toolswere used to identify syntenic blocks of gene regions sharedbetween zebrafish and human apolipoprotein orthologs. Thesyntenic analysis revealed that the human and zebrafish APOA-I andAPOE orthologs clearly share syntenic gene regions (Fig. 1C).apoBa and apoBb.1 also share syntenic gene regions with humanAPOB, but apoBb.2 does not (Fig. 1C). The zebrafish apoA-IVgenes share syntenic gene regions with human APOE (Fig. 1C).

Spatiotemporal apolipoprotein expression patternsUtilization of the zebrafish to model dyslipidemias is limited byincomplete knowledge of the developmental expression patterns ofeach apolipoprotein. Although expression patterns for a subset ofzebrafish apolipoproteins have been published, these studiesgenerally do not indicate which apolipoprotein paralog was studied,nor comprehensively describe where and when the genes areexpressed throughout embryonic and larval development. Thus, weperformed a comprehensive mRNA expression analysis of all 11 ofthe apoA-I, apoB, apoE and apoA-IV genes from the eight-cell stageto 6 days post-fertilization (dpf).

In-situ hybridization (ISH) was carried out with antisense andcontrol sense riboprobes specific to each apolipoprotein studied(Figs 2-5; supplementary material Figs S2-S6). All 11apolipoproteins were expressed in the yolk syncytial layer (YSL)during embryogenesis (Figs 2-5), a finding consistent with theunique role of the YSL in processing yolk lipids and secretingVLDL to support embryonic and larval development (Carvalhoand Heisenberg, 2010). The earliest apolipoprotein expression wasobserved for apoEb mRNA which localized to the YSL duringblastulation (30% epiboly) (Fig. 4). Gastrulation (80-100%epiboly) is associated with the earliest expression of apoA-Ia andapoA-Ib mRNA in the YSL (Fig. 2). apoBa, apoBb.1 (Fig. 3A),apoEa (Fig. 4) and the four apoA-IV genes (Fig. 5) are firstexpressed during somitogenesis (15-20 somites). Although manyapolipoproteins are expressed uniformly throughout the YSL,some localize to specific YSL regions or in patterns indicative ofsubcellular niches. For example, at 80-100% epiboly, both apoA-Ia and apoA-Ib mRNA localized to perinuclear YSL regions(Fig. 2); at 15-20 somites, apoBb.2 and apoEa were expressedstrongly in distinct subregions of the YSL (Fig. 3A; Fig. 4); and at1 dpf, apoA-IVa, apoA-IVb.2 and apoA-IVb.3 localize morestrongly to the yolk extension than to the other areas of the YSL(Fig. 5).

As the digestive organs develop (4-6 dpf), the apolipoproteinsexamined begin to be expressed in the intestine and/or liver, oftenwith gene paralogs segregated to different organs. ISH showed thatapoA-Ia mRNA was expressed only in the intestine and that themajority of apoA-Ib mRNA localized to the liver, with only weakexpression in the intestine (Fig. 2). The apoB genes showed similarsegregated mRNA localization: apoBa was expressed in the heart (2dpf), strongly in the liver (4-6 dpf) and weakly in the intestine (5-6dpf), apoBb.1 was expressed predominantly in the intestine andweakly in the liver (5-6 dpf), and apoBb.2 was not expressed in theliver or intestine (Fig. 3A). mRNA of both apoE and all four apoA-IV genes localized to the intestine (apoEa at 6 dpf, all others 4-6dpf) (Figs 4,5). The only apoA-IV paralog transcribed in both theliver and intestine is apoA-IVb.1 (Fig. 5). apoEa and apoEb areexpressed outside the YSL and digestive organs; apoEb localizes tothe tail bud (15-20 somites through 1 dpf), apical ectodermal ridge(AER) (15-20 somites through 6 dpf), olfactory orifices (1-6 dpf),fin buds (2-6 dpf), esophagus and pharynx (2-6 dpf), gill arches (3-6 dpf), macrophages in the head (3-6 dpf) (Herbomel et al., 2001),

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retina (4-6 dpf), periderm (4-6 dpf), and both apoE genes areexpressed in the swim bladder (6 dpf) (Fig. 4).

Through ISH we were able to qualitatively characterizespatiotemporal mRNA expression and localization of the zebrafishapoB paralogs. However, mRNA expression levels do notnecessarily correlate with protein levels. Therefore, we used liquidchromatography tandem mass spectrometry (LC-MS/MS) to

determine whether the apoB mRNA expression observed by ISH ismirrored by ApoB protein levels. Total larval proteins ≥250 kDawere analyzed (all three zebrafish ApoB paralogs run in this sizerange on a SDS-PAGE gel; I.A.D., H.T. and K.Y., unpublishedobservation). LC-MS/MS revealed that ApoBb.1 accounts for themajority of total ApoB protein in both 2- and 15-dpf larvae (96.3%of total ApoB protein at 2 dpf and 94.2% at 15 dpf) (Fig. 3B).


Fig. 1. Apolipoprotein gene ancestry analysis. (A) There is low sequence similarity between zebrafish and human apolipoprotein orthologs. Zebrafish genesare listed next to the top BLASTp hit against the human RefSeq protein database. The percentage similarity between zebrafish and human orthologs wasdetermined by Needleman–Wunsch amino acid pairwise sequence alignment. (B) Phylogentetic tree of human, zebrafish, mouse, chicken and coelacanthAPOA-I, APOB, APOE and APOA-IV assembled from amino acid sequences as described in the Materials and Methods. Bootstrap support for each clade isreported in light blue (≥70 interpreted as significant). (C) Most zebrafish apolipoprotein genes share syntenic gene regions with their human orthologs.Schematic representation of human and zebrafish chromosomes regions with lines connecting human and zebrafish orthologs.

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ApoBa protein accounts for a small percentage of total ApoB at bothdevelopmental time points (3.1% of total ApoB protein at 2 dpf and3.4% at 15 dpf) (Fig. 3B). Finally, at 2 dpf, ApoBb.2 representsextremely little of the total ApoB protein (0.6% of total ApoBprotein), but by 15 dpf, it composes 2.4% of total larval ApoBprotein (Fig. 3B).

A high-fat feed induces apolipoprotein transcriptionThe presence in the liver and/or intestine of every apolipoproteinexamined, except apoBb.2, at the 6-dpf developmental point thatcoincides with exhaustion of yolk nutrients and initiation ofexogenous food intake, suggests that there is a crucial role for theseproteins in dietary lipid transport. Despite this correlation, it iscurrently unknown whether ingestion of dietary lipids transcriptionallyregulates zebrafish apolipoprotein mRNA expression. We predictedthat dietary lipids might induce apolipoprotein expression given thatthe presence of these proteins is essential for lipid processing andtransport. To address the hypothesis that ingestion of dietary lipidswould induce apolipoprotein transcription, we used an establishedhigh-fat feeding paradigm in which chicken egg yolk emulsified inembryo medium (5% chicken egg yolk; 4 hours) is fed to larval

zebrafish (Carten et al., 2011). Initially, we performed ISH on unfedand high-fat fed, 6-dpf larvae. Although ISH is a powerful tool toassess the localization of transcripts, it can only provide a qualitativemeasure of expression levels because the development of thecolorimetric readout is a non-linear process. Despite this limitation,we observed increased intestinal and hepatic signals for someapolipoproteins following a high-fat feed, consistent with increases inmRNA expression (Fig. 6A).

To quantitatively assess the effect of dietary lipids on larvalapolipoprotein mRNA expression, we performed quantitative real-time PCR (qRT-PCR) on the guts (intestine, liver and pancreas) of6-dpf unfed larvae and their fed clutch-mates at 1, 2, 3 and 4 hoursafter the onset of a high-fat feed (10% chicken egg yolk; 1 hour)(Fig. 6B). qRT-PCR revealed a remarkable, ~30-fold increase inapoA-IVb.3 mRNA at 2 hours following the onset of feeding (one-way ANOVA, F(4,10)=9.359, P=0.0021], a ~12-fold increase inapoA-IVa at the same time point [one-way ANOVA, F(4,10)=9.318,P=0.0021], and an ~5-fold increase in apoA-IVb.2 by 4 hours post-feed onset [one-way ANOVA, F(4,10)=5.433, P=0.0137]. Theincreases in gene expression appear to be specific to ingestion ofdietary lipids, as no changes were observed after a high-protein,

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Fig. 2. Developmental mRNA expression patterns of apoA-Ia andapoA-Ib. In situ hybridization of apoA-Ia and apoA-Ib duringgastrulation [80-100% epiboly (E)], somitogenesis [15-20 somite (S)],and daily until 6 dpf. Both genes localize to the YSL fromsomitogenesis through 5 dpf, apoA-Ia localizes to the intestine (I) at 6dpf, and apoA-Ib localizes strongly to the liver (L) and weakly to theintestine at 6 dpf. All zebrafish are wild type except 6-dpf larvae,which are nacre−/−. Larvae studied at 2-5 dpf were treated with PTU toprevent pigment formation. No signal was observed for either gene atthe eight-cell stage or blastulation (supplementary material Fig. S1).Experiments were performed three times for each gene at each stagewith n≥5 embryos or larvae per probe per experiment.

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low-lipid feed (10% chicken egg white; E.M.Z. and S.A.F.,unpublished observation). In contrast, there were no significantchanges in expression of the other apolipoproteins (supplementarymaterial Fig. S7). RT-PCR was not performed for apoBb.2 as theexpression level in the gut at 6 dpf is below the detection limit ofthese assays.

Zebrafish ApoA-IV overexpression reduces high-fat foodintakeUnderstanding the gut to brain signals underlying appetite regulationis crucial in the context of the current obesity epidemic. Rat APOA-IV is secreted from the intestine on chylomicrons following a high-fat feed and acts as a satiety factor, signaling via vagal afferents tocentrally decrease food intake (Fujimoto et al., 1992; Fujimoto et al.,1993a). Therefore we hypothesized that ApoA-IV might have aconserved role in the regulation of food intake in zebrafish,predicting that it would decrease food intake during high-fat feeding.To address this question, we created transgenic zebrafish thatoverexpress ApoA-IVb.1 transiently under an inducible heat shockprotein 70 (hsp70) promoter with a fluorescent reporter[Tg(hsp70:apoA-IVb.1:mCherry)].

We anticipated that if ApoA-IVb.1 induces satiety,Tg(hsp70:apoA-IVb.1:mCherry) larvae overexpressing ApoA-IVb.1

following a heat shock would eat less during a high-fat feed. Tomeasure food intake, larvae were fed a high-fat meal (10% egg yolk;4 hours) containing a fluorescently labeled lipid (BODIPY-C16),total larval lipids were extracted, and the amount of fluorescentlipids ingested was quantified as an indication of total food intake(Fig. 7A). Strikingly, larvae overexpressing ApoA-IVb.1 ingestedapproximately two-thirds of the fluorescent lipids compared withwild-type (WT) larvae (paired Student’s t-test, P=0.004) (Fig. 7B),demonstrating that ApoA-IV might impact food intake in zebrafishas it does in rats.

DISCUSSIONGene ancestry analysis of zebrafish apolipoproteinsThis is a comprehensive study of the zebrafish orthologs of fourmajor human serum apolipoproteins that are associated with diseaseprocesses. Unifying the designations of zebrafish apolipoproteinswill facilitate interpretation of results and translation to humanphysiology. Historically, naming zebrafish genes has beencomplicated by the extensive gene duplications that resulted from awhole genome duplication event in the teleost fish lineage as wellas local gene duplication events (Amores et al., 1998; Gates et al.,1999; Postlethwait et al., 1998; Taylor et al., 2001). Furthermore, thezebrafish community only recently achieved a thorough annotation


Fig. 3. Zebrafish apoB mRNA and proteinexpression. (A) Localization of apoBa, apoBb.1 andapoBb.2 mRNA by ISH from somitogenesis [15-20somite (S)] to 6 dpf. All apoB genes are expressedin the yolk syncytial layer (YSL), but mRNAs localizeto distinct subregions of the YSL at variousdevelopmental stages. apoBa mRNA is present inthe heart (H) at 2 dpf. At 6 dpf apoBa mRNA isexpressed strongly in the liver (L) and weakly in theintestine (I), apoBb.1 is strongly expressed in theintestine and weakly in the liver, and apoBb.2 is notdetectable by ISH. All zebrafish are wild type except6-dpf larvae which are nacre−/−. Larvae collected at2-5 dpf were treated with PTU to prevent pigmentformation. No signal was observed at the eight-cellstage or at 30% or 80-100% epiboly (supplementarymaterial Fig. S2). Experiments were performed threetimes for each gene at each stage with n≥5 embryosor larvae per probe per experiment. (B) Relativeabundance of zebrafish ApoB paralogs as identifiedby peptide spectrum matches (PSM) in 2- and 15-dpf larvae (20-30 pooled larvae per experiment) asdetermined by LC-MS/MS of total larval proteins≥250 kDa. Two experiments were performed foreach developmental stage and both experimentsyielded similar results. Data presented are from oneexperiment.

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of the zebrafish genome (Howe et al., 2013). To ensure consistencybetween future studies and to facilitate translation to mammalianstudies, we verified the ancestral relationships of the 11 zebrafishapoA-I, apoB, apoE and apoA-IV genes with their named humanorthologs through sequence, syntenic and phylogenetic analyses.

This study and others have shown that zebrafish apolipoproteinstend to have low sequence similarity to their mammalian orthologs

(supplementary material Fig. S1) (Babin et al., 1997; Durliat et al.,2000). Despite high sequence divergence, by integrating the resultsof our syntenic and phylogenetic analyses, we are confident that thezebrafish genes investigated in this study are accurately named aftertheir human orthologs. The phylogenetic analysis is consistent withthat of Babin et al., in that it is unable to confidently resolve thebasal relationships between apolipoprotein families, but confidentlygroups zebrafish ApoA-I and ApoE into distinct clades with theirvertebrate orthologs (Babin et al., 1997). The present phylogenybuilds on previous findings by demonstrating that the zebrafishApoB paralogs do share a phylogenetic clade with their humanortholog. The phylogenetic analysis also suggests that there are twosubfamilies of zebrafish ApoA-IV genes that are not strongly linkedby bootstrap support.

We expanded upon our phylogenetic analysis of theapolipoproteins by performing an analysis of syntenic gene regionsin zebrafish and human orthologs. These results clearly show theeffects of the teleost genome duplication, as there are two paralogsof zebrafish apoA-I and apoE that share syntenic gene relationshipswith human APOA-I and APOE. The same is true for zebrafishapoBa and apoBb.1, and human APOB, but the lack of syntenicgene similarity between apoBb.2 and human APOB suggest that thisgene resulted from an independent local duplication event.

The zebrafish apoA-IV paralogs do not share syntenic generegions with human APOA-IV, but do unexpectedly share syntenicgene regions with human APOE. This led us to speculate that APOEand APOA-IV share a common ancestor. Indeed, coelacanth andseveral additional fish species contain a cluster of apolipoproteingenes that contain both apoE and apoA-IV (data not shown).

Retention of duplicate apolipoproteinsIt is noteworthy that zebrafish have retained multiple apolipoproteinparalogs over millions of years of evolution. The duplication-degeneration-complementation (DDC) model (Force et al., 1999)proposes an explanation of why duplicated genes might be retained.The DDC model predicts that, instead of being retained due to theevolution of novel functions (neofunctionalization), degenerativemutations in the regulatory elements of duplicated genes increasethe likelihood of their preservation owing to conservation andpartitioning of their ancestral functions (subfunctionalization). Thezebrafish paralogs of pax6 and the igf1r are clear examples of genesthat have subfunctionalized. The paralogs of these genes performsimilar functions in different tissues due to differential expressionpatterns, likely resulting, as the DDC model predicts, frommutations in regulatory elements (Kleinjan et al., 2008; Schlueter etal., 2006). We speculate that similar subfunctionalization hasoccurred in several of the zebrafish apolipoprotein paralogs. Forexample, although mammalian APOA-I is expressed both in theintestine and liver, zebrafish apoA-Ia is expressed in the intestineand the majority of apoA-Ib expression is observed the liver. Basedon the DDC model, we would predict that there are mutations in theregulatory regions of these genes that restrict their expression tocertain tissues while maintaining their ancestral function. Similarsubfunctionalization might have occurred in the zebrafish apoA-IVparalogs because all four are expressed in the larval intestine, butonly apoA-IVb.3 is expressed in the liver. By comparison, humanAPOA-IV is expressed in the intestine, but rat ApoA-IV is expressedin the liver and intestine, hence, the ancestral gene might have hadboth hepatic and intestinal expression.

Zebrafish and humans might have used unique strategies toachieve tissue-specific APOB gene expression. In zebrafish, it ispossible that the zebrafish apoB paralogs have subfunctionalized,

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Fig. 4. apoEa localization to the YSL and intestine contrasts withwidespread expression of apoEb. apoEa and apoEb mRNA expressionobserved by ISH during blastulation [30% epiboly (E)], gastrulation (80% E),somitogenesis [15-20 somite (S)] and 1-6 dpf. apoEa mRNA is expressed indistinct subregions of the YSL at somitogenesis and 1 dpf, and ubiquitouslythroughout the YSL from 2-5 dpf. At 6 dpf (but not 5 dpf), apoEa is observedin the intestine (I). apoEb is expressed not only throughout the YSL (30% Eto 5 dpf), but also in the tail bud (15-20 S and 1 dpf), the apical ectodermalridge (AER) (1-6 dpf), the head (H) (1-6 dpf), mouth and nose orifices (O) (2-6 dpf), the find buds (FB) (2-6 dpf), pharynx (P) (2-6 dpf), gill arches (GA) (3-6 dpf), periderm (4-6 dpf), retina (R) (4-6 dpf), intestine (6 dpf) and swimbladder (6 dpf). No expression of apoEa was observed during blastulation orgastrulation, or for either gene at the eight-cell stage (supplementary materialFig. S3). Zebrafish are wild type, except for 6-dpf larvae which are nacre−/−;1-6 dpf larvae were treated with hydrogen peroxide to remove pigmentation.ISH was performed in triplicate with n≥5 embryos or larvae per probe perexperiment.

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with apoBa restricted to performing the function of the ancestralgene in the liver and heart [where it is expressed in humans andmice (Nielsen et al., 1998)] and apoBb.1 performing the majority offunction of the ancestral gene in the intestine. By contrast, tissue-specific human APOB expression occurs in the form of differentisoforms of the same gene through mRNA editing; a short isoform,APOB-48, is expressed in the intestine, and a long isoform, APOB-100, is expressed in the liver. Although apoBb.2 is the shortestzebrafish paralog, it is not a C-terminal truncation like humanAPOB-48, in that it contains numerous deletions throughout theprotein (data not shown) and it is not expressed in the larvalintestine. Zebrafish ApoB protein analysis by LC-MS/MS indicatedhigher protein levels of ApoBb.1 as compared to ApoBa orApoBb.2. Therefore ApoBb.1 might have a greater role in lipidtransport, but it is not clear whether higher expression of theApoBb.1 protein provides a functional benefit in lipid transport ascompared to the other paralogs. The mechanisms underlying thelarge differences in zebrafish ApoB protein levels have yet to bedetermined; however, unpublished RNAseq data of 6-dpf larval guts(E.M.Z. and S.A.F., unpublished observations) similarly showsmuch higher mRNA levels for apoBb.1 than for apoBa or apoBb.2,suggesting that this regulation takes place at the mRNA level. It isdifficult to postulate how the differences in protein levels betweenzebrafish ApoB paralogs compare to those of the human APOBisoforms as little data describing whole-body human APOB proteinlevels are available; however, Poapst el al. found that there is asimilar serum protein mass of APOB-48 and APOB-100 in humans(Poapst et al., 1987).

The zebrafish apoE paralogs might also have undergonesubfunctionalization. Here, we show apoEa localizing to the YSLand intestine, and both this and other studies have found widespreadexpression of apoEb (Babin et al., 1997; Herbomel et al., 2001;Monnot et al., 1999). Based on its spatiotemporal expression andlocalization patterns, we predict ApoEb likely retains the non-lipidtransport, extra-digestive organ functions of the ancestral gene.Zebrafish apoEb has similar developmental expression to mouse

APOE [visceral yolk sac endoderm at 8.5 days post-conception(dpc), liver, lung, heart and eye at 9.5 dpc, and cells lining theolfactory vesicle, peripheral neural cells, and the brain at 10.5 dpc(Bächner et al., 1999; Harrison et al., 1995)]. Given that bothzebrafish apoE paralogs are expressed in the YSL, it is unclear fromour data whether they exhibit different lipid transport properties.Monnot et al. (Monnot et al., 1999) observed similar expression ofan unspecified apoE paralog during zebrafish development, as wellas induction during adult fin regeneration.

The segregated expression of several larval apolipoproteinparalogs between organs provides a unique opportunity to studypotential differences in lipoproteins generated from various tissues.Recent studies have emphasized the contribution of HDL particlecomposition, as opposed to solely HDL cholesterol levels, tolipoprotein function (Feig et al., 2014; Khera et al., 2011). It is notknown whether nascent mammalian HDL particles synthesized bythe liver have compositions that are unique to those assembled bythe intestine. It is difficult to address this question with traditionalmouse models because it is technically challenging to identify HDLparticles of different origins. Promisingly, the nearly segregatedexpression patterns of the zebrafish apoA-I paralogs provide asystem to study HDL particles of hepatic versus intestinal origins.The apolipoprotein mRNA localization data presented in this studydoes not show localization to any new organs compared to humanortholog expression, but functional studies will need to be performedin the future to rule out the possibility that any neofunctionalizationhas occurred.

mRNA localization suggests important roles forapolipoproteins in YSL and dietary lipid transportPrevious ISH studies support our finding that nearly all zebrafishapolipoproteins show mRNA expression in the YSL (Babin et al.,1997; Thisse et al., 2001). The YSL is a syncytium containing manyhighly dynamic nuclei that form at the surface of the yolk cell duringblastulation (Carvalho and Heisenberg, 2010). In addition to amultitude of key developmental functions, the YSL serves nutritive


Fig. 5. The zebrafish apoA-IV genes have uniqueexpression patterns in the YSL, intestine andliver. Developmental mRNA expression dynamics ofthe zebrafish apoA-IV genes, as measured by ISH,during somitogenesis [15-20 somite (S)] and at 1-6dpf. All four apoA-IV genes showed earliest mRNAexpression at 15-20 S in the YSL; the mRNAslocalize to distinct subregions of the YSL until 5 dpf.All four genes are expressed in the intestine (I) at 4-6 dpf, with weak intestinal expression observed forapoA-IVa, and apoA-IVb.3 is expressed in the liver(L) at 4-6 dpf. No expression was observed for theapoA-IV genes at the eight-cell stage, 30% epibolyor 80-100% epiboly (supplementary material Fig.S4). Larvae from 15-20 S to 5 dpf are wild type (2-5dpf treated with PTU; 6-dpf larvae are nacre−/−.Experiments were performed in triplicate with n≥5embryos or larvae per probe in every experiment.

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functions in the developing embryo, including the hydrolysis andexport of yolk lipids in lipoproteins. Electron microscopy studieshave visualized the secretion of VLDL particles from the highlydeveloped secretary pathways of the fish YSL (Mani-Ponset et al.,1996; Walzer and Schönenberger, 1979) and studies in zebrafishlacking functional mtp (an apoB chaperone required for VLDLformation) have demonstrated the necessity of lipoprotein for exportof yolk lipids (Miyares et al., 2014). Given these observations, it

was expected that the zebrafish orthologs of mammalianapolipoproteins known to be associated with VLDL andchylomicrons (apoB, apoE and apoA-IV) would be expressed in theYSL. However, both apoA-I paralogs were also expressed in theYSL, similar to a previous report (Babin et al., 1997). The presenceof apoA-I suggests that the YSL might produce HDL particles inaddition to the classically described VLDL particles, or that thepreviously described VLDL particles have an apolipoprotein

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Fig. 6. High-fat feed increases expression of apolipoproteins in larval gut. (A) Representative images of mRNA expression as observed by ISH of thezebrafish apoA-I, apoB, apoE and apoA-IV genes in 6-dpf nacre−/− larvae. Larvae have either never eaten exogenous food (Unfed), or have been fed a high-fat, 5% chicken egg yolk meal for 4 hours (Fed). ISH was performed in triplicate with n≥5 larvae; no staining was observed in sense probed (supplementarymaterial Figs S1-S4). (B) A high-fat meal increases zebrafish apoA-IVa, apoA-IVb.2, and apoA-IVb.3 mRNA expression in the gut (intestine, liver, pancreas).Real-time PCR quantification of apolipoprotein transcriptional response in the 6-dpf zebrafish gut to 1, 2, 3 or 4 hours of a 10% chicken egg yolk feed (n=3; 10pooled larval guts per experiment). Groups with the same letter are not significantly different (one-way ANOVA, P<0.05, n=3).

Fig. 7. Overexpression of zebrafish ApoA-IVb.1decreases food intake. (A) Representative fluorescentimages of spotted lipid extracts; each spot represents thefluorescent lipids of 10 pooled larvae following lipidextraction. Tg represents Tg(hsp70:apoA-IVb.1:mCherry)larvae and WT represents wild-type larvae. Larvae haveeither never eaten exogenous food (Unfed), or have beenfed a high-fat, 10% chicken egg yolk with 6.4 μM BODIPY®FL C16 for 4 hours (Fed). (B) Tg(hsp70:apoA-IVb.1:mCherry) larvae ingest fewer fluorescently labeledlipids as shown by comparison of arbitrary fluorescent units(a.f.u.) to wild type (paired Student’s t-test, P<0.001; n=9,20 larvae per experiment). Total larval lipids were extractedand lipid fluorescence was measured. Natural fluorescentlipid background was corrected using measurements ofunfed larvae. The box represents the 25-75th percentiles,and the median is indicated. The whiskers show the 10-90th percentiles.

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composition unique from classical VLDL, but similar tochylomicrons, which contain APOA-I. To our knowledge,characterization of the apolipoprotein composition of YSLlipoproteins has not been reported; these studies would have thepotential to reveal a wealth of information regarding lipoproteinfunction during embryonic development.

Mammalian embryos also have a yolk sac in early development,prior to the development of a circulatory system. Like the fish YSL,the mammalian yolk sac secretes VLDL (Farese et al., 1995;Terasawa et al., 1999). It is likely that mammalian yolk sacapolipoproteins provide required nutritive contributions duringembryogenesis, as ApoB (Terasawa et al., 1999) and ApoE (Harrisonet al., 1995) mRNA are expressed in the mouse yolk sac and loss ofApoB (Farese et al., 1995) or Mttp (Raabe et al., 1998) is embryoniclethal. Knockdown of YSL lipoproteins is similarly lethal inzebrafish (Avraham-Davidi et al., 2012; Miyares et al., 2014). Takentogether, these findings suggest that future studies of zebrafish YSLlipid metabolism can serve as an informative model of mammalianembryonic metabolism.

A second prominent finding from our spatiotemporalapolipoprotein mRNA expression analysis was that over half of theapolipoproteins were not distributed evenly throughout the YSL, butlocalized to distinct subregions. A cursory analysis of publicallyavailable YSL expression patterns on ZFIN reveals many additionalexamples of regional YSL gene expression, yet the significance ofthese YSL subregions, especially in relationship to embryonic lipidmetabolism, has yet to be explored. Notably, the apolipoproteins donot adhere to the YSL expression patterns of gata5 and gata6,markers of the presumptive YSL regions that will become the liverand intestine (Thisse et al., 2001). This supports the theory thatapolipoprotein mRNA is translated into proteins that perform YSL-specific functions, and that the proteins are not simply beingrecruited to the nascent digestive tract.

Dietary lipids induce apolipoprotein expressionExtensive research has elucidated much of the complex, molecularnetwork that regulates transcription of the major serumapolipoproteins (Zannis et al., 2001), but it is less clear how thepresence of dietary nutrients regulates apolipoprotein transcription.Dietary fats increase transcription of mammalian ApoB and ApoA-IV, two apolipoproteins involved in chylomicron assembly(Hernández Vallejo et al., 2009). ApoA-IV is very sensitive tonutritional status; dietary lipids increase intestinal ApoA-IVexpression through HNF-4 promoter binding (Carrière et al., 2005).While fasting (Hanniman et al., 2006; LeBoeuf et al., 1994), high-fat diet (Hernández Vallejo et al., 2009), diabetes (Hanniman et al.,2006), and hepatic steatosis increase hepatic ApoA-IV expressionthrough CREBH (Xu et al., 2014). Our results indicate that intestinaland hepatic mRNA expression of three of the four zebrafish apoA-IV paralogs is similarly increased in response to high-fat feeding, butwhether the molecular mechanisms of this response mirrors those ofmammals remains open to investigation.

In contrast to ApoA-IV, the effects of dietary lipids on intestinaland hepatic ApoA-I and ApoE transcription are less well studied.Additional questions regarding the effects of dietary lipids onapolipoprotein expression are raised by the paradoxical finding ofYoshioka et al. (Yoshioka et al., 2008) that a high-fat meal decreasesmRNA expression of ApoB and one isoform of ApoA-IV in intestinalmucosa, while increasing mRNA expression of a second ApoA-IVisoform. It is possible that the inconsistent results found in variousmouse models are due in part to the nutritional status of the animals.The mice in previous studies were fasted for various lengths of time

prior to refeeding with high-fat diets of varying compositions anddurations. An advantage of the larval zebrafish model is that thelarvae used in this study were experiencing their first exogenousmeal, previously subsisting on yolk nutrients. Therefore, the larvalzebrafish offers a unique opportunity to study food intake in ananimal that has homogeneity, which is often achieved by fastinganimals, without the effects of starvation and refeeding. Thezebrafish presents an exceptional opportunity to explore thepotential synergistic effects of developmental stage on dietary lipidregulation of apolipoprotein transcription.

Zebrafish apoA-IV decreases high-fat food intakeIt has long been appreciated that APOA-IV is secreted from theintestine as a component of chylomicrons in response to long-chaindietary fatty acids (Hayashi et al., 1990; Kalogeris et al., 1996).Over 20 years ago, Fujimoto et al. (Fujimoto et al., 1992; Fujimotoet al., 1993b) demonstrated that acute, peripheral administration ofAPOA-IV decreases food intake in rats due to reduced meal size.Recent work supports the hypothesis that peripheral APOA-IVsignals through the vagal nerve (Kalogeris et al., 1999; Lo et al.,2012) to the CNS [where ApoA-IV is expressed in the hypothalamus(Liu et al., 2001)] to decrease food intake (Fujimoto et al., 1993a;Shen et al., 2008). Recently, Wang et al. (Wang et al., 2013a)identified a specific portion of the N-terminal region of rat APOA-IV that is capable of decreasing food intake.

In this investigation, we demonstrated that overexpression ofzebrafish ApoA-IV can decrease high-fat food intake. We developeda novel assay that measures total ingested larval fluorescent lipids(by lipid extraction, spotting on a TLC plate and quantification oftotal fluorescence) following a high-fat feed as an indicator ofdietary lipid intake. This assay was used to demonstrate that acuteoverexpression of ApoA-IVb.1 decreases ingestion of fluorescentlylabeled C16 during a 4-hour high-fat feed in 7-dpf larvae. It is likelythat some of the ingested BODIPY-C16 is metabolized into complexlipids, as we have previously demonstrated occurs with BODIPY-C16 fed to 6-dpf larvae (Carten et al., 2011) and BODIPY-C12injected into the yolk of 3-dpf larvae (Miyares et al., 2014).However, metabolism of the BODIPY-C16 should not affectquantification of fluorescent lipid intake because total larval lipidswere extracted and spotted on the TLC plate. Additionally, we donot believe that excretion of ingested lipids might have confoundedour measurements of lipid uptake during the 4-hour feed. Priorstudies of intestinal transit rates suggest that it would take >4 hoursfor excretion to commence: >3 hours is required for 7-dpf larvae totransport fluorescent microspheres to the distal intestine post-gavageand <10% of larvae transported the microspheres to the distalintestine by 6 hours post-gavage (Cocchiaro and Rawls, 2013).

It is possible that the additional zebrafish ApoA-IV paralogs alsoregulate food intake: they are highly transcriptionally responsive todietary lipids, and ApoA-IVb.2 and ApoA-IVb.3 share a high degreeof sequence similarity to ApoA-IVb.1 (supplementary material Fig.S1). Although ApoA-IVa has the most divergent sequence fromApoA-IVb.1, it is also highly induced by a high-fat feed. Hence,ApoA-IVa might share a role in regulating food intake, or, given thatmammalian APOA-IV facilitates the production of smaller, morequickly cleared chylomicrons (Kohan et al., 2012), it might beproduced to structurally support chylomicrons.

Unexpectedly, even though ApoA-IVb.1 overexpressiondecreased food intake, we were not able to detect mRNA expressionin the CNS, where it is thought to regulate mammalian food intake.Therefore, it is possible that the signaling pathway by which ApoA-IVb.1 reduces food intake in larvae differs from that of adults.


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Alternatively, apoA-IVb.1 might be expressed at an extremely lowlevel in the CNS that ISH is not sensitive enough to detect. Analternative explanation of our results is that ApoA-IVb.1 signalingmight vary by developmental stage. In fact, neonatal swineexperience a high degree of ApoA-IV transcriptional induction byintraduodenal lipids before, but not after, weaning (Black et al.,1990). Future studies should define the degree to which endogenouszebrafish ApoA-IV proteins naturally regulate high-fat food intakeand explore the signaling pathways by which this occurs.

Despite the relationship between acute APOA-IV administrationand decreased food intake observed in rats, mice deficient in APOA-IV, or with chronically overexpressed APOA-IV, have no changesin food intake (Aalto-Setälä et al., 1994; Weinstock et al., 1997).The potentially conflicting results found in the mouse models ofAPOA-IV overexpression or loss could result from speciesdifferences between rats and mice. However, evidence showing thatAPOA-IV expression is decreased by a chronic high-fat diet andobesity (Tso and Liu, 2004), suggests that long-term overexpressionor loss of APOA-IV might lead to loss of function. In our study,transient overexpression of zebrafish ApoA-IVb.1 by an induciblepromoter decreased food intake during a lipid-rich feed. This findingsupports a model where transient versus chronic induction ofAPOA-IV could have variable effects on food intake. Future studiescould validate this model by creating transgenic zebrafish thatchronically overexpress ApoA-IVb.1 on a constitutively activepromoter, developing an assay to measure food intake in older larvaethat have experienced long-term ApoA-IVb.1 overexpression, anddetermining whether regulation of lipid-rich food intake is lost.

ConclusionsThis study provides a framework for use in future studies ofapolipoprotein biology, lipoprotein metabolism, dyslipidemias andthe roles of apolipoproteins in development in the larval zebrafish.Presented here are analyses of the gene ancestry and geneticrelationships between four major human serum apolipoproteins(APOA-I, APOB, APOE and APOA-IV) and their 11 zebrafishorthologs. The expression patterns of these zebrafish apolipoproteinswere described throughout development and after a high-fat feed at6 dpf. Finally, we determined that a zebrafish ortholog of ApoA-IV,an apolipoprotein that regulates high-fat food intake in rats,functions similarly in zebrafish. This comprehensivecharacterization of zebrafish apolipoproteins and the quantitativeassay of larval lipid ingestion can serve as a foundation for futurestudies of the contributions of apolipoproteins to intestinaldevelopment and lipid processing in health and disease states, andin screens for pharmaceutical modulators of these proteins.Moreover, the quantitative feeding assay we developed can beapplied to a wide range of studies investigating factors that regulatefood intake. These data contribute to a growing literatureestablishing the larval zebrafish as a model for lipid metabolism inhealth and disease.

MATERIALS AND METHODSApolipoprotein gene ancestry analysisTo determine that zebrafish apolipoproteins share the same ancestral geneas their named human ortholog, we performed sequence, syntenic andphylogenetic analyses. The amino acid sequences of the zebrafishapolipoproteins were obtained from the Ensembl Genome Browser(Ensembl.org). First, the protein sequence of each zebrafish gene was usedto query NCBI using BLASTp (standard settings) restricted to the humanRefSeq protein database (Altschul et al., 1997; Altschul et al., 2005); the topBLAST hit for each query was recorded (Fig. 1A). Optimal pairwise

alignments between zebrafish and human ortholog protein sequences weredetermined with the Needleman–Wunsch pairwise sequence alignment(Needleman and Wunsch, 1970) tool available at EMBL_EBI(http://www.ebi.ac.uk/Tools/psa/emboss_needle/) (McWilliam et al., 2013).Second, to examine the phylogenetic relationship between zebrafishapolipoprotein paralogs and their corresponding vertebrate orthologs, theamino acid sequences of human, zebrafish, mouse, chicken and coelacanthAPOA-I, APOB, APOE and APOA-IV were aligned with using the iterativeMAFFT multiple sequence alignment tool (Kuraku et al., 2013; Thompsonet al., 2011). Specifically, the G-INS-I strategy was selected using standardsettings, as it is recommended for sequences sharing global homology.Following alignment, the MAFFT tree server was used to build a neighbor-joining tree based on all gap-free sites, using the JTT substitution model andallowing estimation of site heterogeneity (alpha). Bootstrap resampling wasset to 100, and bootstrap support ≥70 was interpreted as significant. Third,the syntenic relationships between the zebrafish and human apolipoproteinswere established using the Genomicus (Louis et al., 2013) and SyntenyDB(Catchen et al., 2009) online syntenic analysis tools by searching fororthologous apolipoproteins in the corresponding syntenic region. Accessionnumbers of the zebrafish and human apolipoprotein genes used in this studyare listed in supplementary material Table S1.

ZebrafishAll procedures were approved by the Carnegie Institution Animal Care andUse Committee (protocol number 139) or the Weizmann Institute Animal Careand Use Committee (protocol number 01190212-3). For ISH experiments, WT(AB background) and melanophore-free (nacre−/−) (Lister et al., 1999)embryos were collected from natural spawning and were staged and raised inzebrafish embryo medium (EM) as described previously (Kimmel et al., 1995;Westerfield, 1995). WT larvae older than 1 dpf used for ISH experiments weretreated with 0.003% N-phenylthiourea (PTU) (Sigma, St Louis, MO, USA)(Karlsson et al., 2001) or hydrogen peroxide (Thisse and Thisse, 2008) todecrease pigmentation. Zebrafish were fixed in 4% paraformaldehyde (PFA)in phosphate-buffered saline overnight at 4°C, washed twice in methanol, andstored in methanol at −20°C. Larvae raised to 15 dpf for ApoB proteinmeasurements were fed three times per day from 7-15 dpf [larval AP100 foodis composed of marine, animal and vegetable proteins (50% minimum proteincontent), yeast, vegetable starches, fish and vegetable oils, vitamin and mineralpremixes, pigments, antioxidants and biodegradable binders (larval AP100,number 384709-20, Zeigler, Gardners, PA)].

Preparation of feeding liposomesOur laboratory and others have developed a high-fat feeding paradigm forlarvae using chicken egg yolk (Carten et al., 2011; Marza et al., 2005).Briefly, 5% and 10% chicken egg yolk emulsions were prepared intoliposomes from frozen aliquots of egg yolk resuspended in EM as previouslydescribed (Carten et al., 2011). The egg yolk emulsion was vortexed(2 minutes) and pulse sonicated with a one-quarter inch tapered microtip(5 seconds total processing time, 1 second on, 1 second off; output intensityof 3W) for 40 seconds in a sonicator (Ultrasonic Processor 3000, MisonixInc., Farmingdale, NY).

Feeding assay for ISHLarvae (nacre−/−, 6 dpf) were fed a high-fat meal of 5% egg yolk liposomesfor 4 hours at 29°C on an incubated shaker (Incu-Shaker Mini, Benchmark,Edison, NJ). Fed larvae were washed three times in EM, anesthetized withtricaine (Argent Chemical Laboratories, Redmond, WA), screened for thepresence of food in the intestine, and fixed in 4% PFA as described above.Unfed clutch-mates were treated in tandem in EM and collected as controls.

Whole-mount ISHTo elucidate the spatiotemporal mRNA expression patterns of the zebrafishapolipoproteins, ISH was carried out as previously described (Thisse andThisse, 2008). Briefly, ~400-800 base pairs of unique sequence (consistingof coding sequence and in some cases 5′ or 3′ untranslated regions) of apoA-Ia, apoA-Ib, apoBa, apoBb.1, apoBb.2, apoEa, apoEb, apoA-IVa, apoA-IVb.1, apoA-IVb.2 and apoA-IVb.3 were amplified from cDNA (Primers

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listed in supplementary material Table S2), TOPO® cloned into pCRII(Invitrogen, Grand Island, NY), and used to generate sense and antisensedigoxigenin-labeled riboprobes (digoxigenin was from Roche, Indianapolis,IN). The riboprobes were hybridized against zebrafish of the followingstages: eight-cell (to examine the presence of maternal mRNA transcripts),30% epiboly (blastula), 80-100% epiboly (gastrula), 15-20 somite(somitogenesis), 1 dpf, 2 dpf, 3 dpf, 4 dpf, 5 dpf, and unfed and fed 6 dpf.ISH experiments were carried out three times on n≥5 larvae, for each senseand antisense probe, at each stage.

Feeding assay for qRT-PCR experimentsFor assays measuring the transcriptional responses of zebrafishapolipoproteins to a lipid-rich feed, larvae (AB, 6 dpf, staged) were placedin a 10% egg yolk emulsion on an incubated shaker (29°C). After 1 hour offeeding, larvae were washed three times in EM, anesthetized with tricaine,and examined for full intestines. Larvae representing a 1-hour time pointwere set aside for dissection; remaining larvae were incubated at 29°C inEM for the remainder of the time course and collected for dissection at 2, 3and 4 hours. Unfed clutch-mates were incubated in EM, anesthetized, andcollected in parallel as controls. Larval guts (intestine, liver and pancreas)were dissected, pooled in groups of 10, transferred to 30 μl RNALater(Ambion, Grand Island, NY), and stored at −20°C. Pooled experimentalgroups were collected in triplicate from unique clutches.

qRT-PCR sample preparation and analysisRNA was extracted from larval guts using an RNAqueous Micro Kit(Ambion, Grand Island, NY) and stored at −80°C. cDNA was constructedusing the iScript cDNA Synthesis Kit (Bio-Rad, Hercules, CA). qRT-PCRsamples were prepared using cDNA, SsoAdvanced Univeral SYBR GreenSupermix (Bio-Rad), and primers designed against gene-specific sequence(supplementary material Table S3). qRT-PCR was performed in triplicate foreach sample on the Bio-Rad CFX96 Real-Time System with 45 cycles:95°C for 15 seconds, 59°C for 20 seconds and 72°C for 20 seconds. Resultswere analyzed using the Bio-Rad CFX Manager 3.0 software platform.Following qRT-PCR, representative samples were run on a 1% agarose geland stained with ethidium bromide to verify the absence of multipleamplicons (supplementary material Fig. S8). qRT-PCR gene expressionresults were quantified using the ΔΔCT method (Livak and Schmittgen,2001); rps18 was used as the reference gene.

Total larval protein purification and in-gel digestionRelative protein expression levels of the zebrafish apoB paralogs wasmeasured by purifying total larval protein and performing LC-MS/MS. Topurify total protein, 20-30 embryos (2 and 15 dpf) were anesthetized on ice,washed twice with buffer A (β-glycerophosphate 500 mM, EGTA 15 mM,orthovanadate 1 mM, EDTA 10 mM, benzamidine 1 mM, aprotinin10 mg/ml, leupeptin 10 mg/ml, pepstatin 2 mg/ml) and suspended in 400 μlbuffer H (buffer A supplemented with 1 mM DTT). Embryos werehomogenized with a 27-gauge syringe and sonicated twice on ice for7 seconds at 40% power. Samples were centrifuged at 20,800 g at 4°C for15 minutes, and supernatants were collected.

Total larval proteins were separated by SDS-PAGE using a 6% separatinggel [4.5 ml H2O, 1.7 ml 30% acrylamide, 2.1 ml separating buffer (3M Tris-HCl pH 8.8, APS 1:100, TEMED 1:1000)] and a 4% stacking gel [3 mlH2O, 0.66 ml 30% acrylamide, 1.25 ml separating buffer (0.5M Tris-HClpH 6.9, APS 1:100, TEMED 1:1000)]. GelCode Blue Stain Reagent (24590,Thermo Scientific) was used for protein detections according tomanufacturer’s instructions.

Selected areas of interest (250-550 kDa, supplementary material Fig. S6)were excised from the gel, sliced into small (1-2 mm) pieces and placed ina silicon tube (0.65 ml; PGC Scientific, Frederick, MD). Gel bands were de-stained with 25 mM NH4HCO3 in 50% acetonitrile and dried bycentrifugation under vacuum. Protein disulfide bonds were reduced bysaturating the dry gel bands with 10 mM DTT in 25 mM NH4HCO3 at 56°Cfor 1 hour, and were alkylated with 55 mM iodoacetamide in 25 mMNH4HCO3 in the dark for 45 minutes at room temperature. After washing in25 mM NH4HCO3 in 50% acetonitrile and centrifugation under vacuum,proteins were digested by rehydrating the gel pieces with 12.5 ng/μl trypsin

in 25 mM NH4HCO3 at 4°C for 10 minutes followed by overnightincubation at 37°C. Peptides were extracted by addition of 50% acetonitrileand 5% formic acid, vortexing, sonicating, centrifuging and collection of thesupernatant. Digestions were stopped by addition of trifluroacetic acid (1%).Extracted proteins were stored at −80°C until further analysis.

Liquid chromatographyULC and MS grade solvents were used for all chromatographic steps.Sample were loaded using split-less nano-Ultra Performance LiquidChromatography (10 kpsi nanoAcquity; Waters Corporation, Milford, MA).The mobile phase consisted of (A) H2O and 0.1% formic acid and (B)acetonitrile and 0.1% formic acid. Desalting of the samples was performedonline using a reversed-phase C18 trapping column (180 μm internaldiameter, 20 mm length, 5 μm particle size; Waters Corporation). Next, thepeptides were separated using a T3 HSS nano-column (75 μm internaldiameter, 250 mm length, 1.8 μm particle size; Waters Corporation) at 0.35μl/minute. Peptides were eluted from the column into the mass spectrometerwith the following gradient: 4% to 35% B in 105 minutes, 35% to 90% Bin 5 minutes, maintained at 95% for 5 minutes and return to initialconditions.

Mass spectrometryThe nanoUPLC was coupled online through a nanoESI emitter (10 μm tip;New Objective; Woburn, MA) to a quadruple orbitrap mass spectrometer (QExactive Plus, Thermo Scientific) using a FlexIon nanospray apparatus(Proxeon). Data was acquired in DDA mode, using a Top12 method. MS1resolution was set to 60,000 (at 400 m/z) and maximum injection time wasset to 120 milliseconds. MS2 resolution was set to 17,500 and maximuminjection time of 60 ms.

Data analysis of mass spectrometry experimentsRaw data was processed using Proteome Discoverer v1.41. The MS/MSspectra were searched using Mascot 2.5 (Matrix Sciences, London, UK) andSequest HT against the zebrafish protein database UniprotKB(http://www.uniprot.org/) appended with 125 common laboratorycontaminant proteins. A fixed modification was set to carbamidomethylationof cysteine and a variable modification was set to oxidation of methionine.Search results were then imported back to Expressions to annotate identifiedpeaks. Proteins were grouped based on shared peptides and identificationswere filtered such that the global false discovery rate was a maximum of1%.

Generation of transgenic zebrafishGateway cloning was used to create a construct expressing zebrafish ApoA-IVb.1 and mCherry under the heat shock protein 70 (hsp70) promoter andstable transgenic zebrafish lines were generated as previously reported(Kwan et al., 2007). Zebrafish apoA-IVb.1 was amplified from cDNA(forward, 5ʹ-GGGGACAAGTTTGTACAAAAAAGCAGGCTCCATGA -AACTGTATCTGATA-3ʹ; reverse, 5ʹ-GGGGACCACTTTGTACAAGA -AAGCTGGGTCTTAATATCTCTTGGTGA-3ʹ) and cloned into a Gatewaymiddle entry vector. We then used gateway cloning to create ahsp70:apoA4b.1:mCherry construct flanked by tol2 sites, which wasinjected into the yolk of 1-2-cell embryos with tol2 transposase for genomeincorporation. Transgenic Tg(hsp70:apoA-IVb.1:mCherry) larvae wereidentified at 2 dpf by heat shocking larvae in 15 ml EM at 37°C for 45minutes followed by 42°C for 10 minutes and screening for diffusefluorescence throughout the entire larval body 18 hours later. Larvae wereheat shocked a second time at 6 dpf, 18 hours prior to feeding assays, toinduce ApoA4b.1 and mCherry overexpression. At 7 dpf, dim mCherryfluorescence was visible throughout the larvae at 10× magnification, andmCherry accumulation was visible in the vasculature, notochord andpronephros with confocal microscopy (supplementary material Fig. S9).

Experimental feeding assays for Tg(hsp70:apoA-IVb.1:mCherry)larvaeAs an indicator of food intake, Tg(hsp70:apoA-IVb.1:mCherry) and controlWT larvae were fed liposomes labeled with BODIPY® FL C16 (D-3821,


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Thermo Fisher Scientific, Inc., Rockville, MD), a green fluorescent fattyacid. Labeled liposomes were made by adding BODIPY® FL C16 to 5 ml of10% egg yolk emulsion immediately following sonication, for a finalconcentration of 6.4 μM BODIPY® FL C16, and vortexing for 30 seconds.Tg(hsp70:apoA-IVb.1:mCherry) and WT larvae (7 dpf) were placed in thefluorescent liposome solution in an incubated shaker for 4 hours at 29°C.Larvae were washed three times in EM, anesthetized in tricaine and screenedfor the presence of food in the intestine. Groups of 10 larvae were pooled,flash frozen and stored at −80°C. Feeding assays were performed pairwisewith one pool of 10 transgenic larvae and two pools of 10 WT larvae.Unfed, WT and transgenic larvae were treated in parallel and collected ascontrols. A total of nine paired sets of larvae were collected. Allexperimental and control Tg and WT larvae were treated with the same heatshock protocol at 2 and 6 dpf as described above.

Lipid extraction and analysisTotal lipids were extracted from larvae that were fed fluorescent liposomessimilarly to our previous report (Miyares et al., 2014) following the Blighand Dyer method (Bligh and Dyer, 1959). Briefly, 100 μl of H2O was addedto the frozen pool of 10 larvae and it was sonicated for 4 seconds with a one-quarter-inch inch tapered microtip with an output of 3W. Next, 375 μl ofchloroform:methanol (1:2) was added to the homogenate, vortexed for30 seconds and incubated at 25°C for at least 10 minutes. 125 μl ofchloroform and 125 μl of 200 mM Tris-HCl pH 7, were subsequently added,with 30 seconds of vortexing after each addition. The samples werecentrifuged at 1800 g for 5 minutes. The organic phase was transferred to aclean microfuge tube and stored at −80°C. For analysis, samples were driedin a speed vacuum, resuspended in 12 μl chloroform, and spotted on achanneled TLC plate (Whatman Scientific, Florham Park, NJ). To detectBODIPY® FL C16 (as an indication of food intake), plates were scanned(Typhoon Scanner, GE Healthcare, Pittsburgh, PA) using a blue fluorescencelaser (excitation: 488 nm; emission: 520 nm; band pass, PMT 425). Totalfluorescence of each sample was quantified using ImageQuant software (GEHealthcare, Pittsburgh, PA, USA). Correction for naturally fluorescent lipidbackground was made with paired, unfed larval samples.

StatisticsDifferences in gene expression over the course of a 4-hour feed asmeasured by qRT-PCR were determined by one-way ANOVA followed bya Holm–Sidak test for multiple comparisons. Assays to compare differencein feeding between WT and Tg(hsp70:apoA-IVb.1:mCherry) larvae wereperformed pairwise with one sample of transgenic larvae and two samplesof WT larvae per experiment. For each experiment, the fluorescence of thetransgenic sample, expressed in arbitrary units, was normalized to the twoWT samples averaged together. Difference in the amount of fluorescentlipids ingested between WT and Tg(hsp70:apoA-IVb.1:mCherry) larvaewas analyzed by a paired Student’s t-test. Residuals of all the data were normally distributed before analysis and significance was set atP<0.05.

AcknowledgementsThe authors would like to thank Blake Caldwell, Jui-Ko Chang and Rosa Miyaresfor in situ hybridization advice; Oni Celestin and Amy Kowalski for generatingtransgenic fish; Amy Singer (ZFIN database team) for assistance namingapolipoprotein genes; Chi-Bin Chien for Tol2Kit components; Alon Savidor andYishai Levin for conducting the mass spectrometry experiments and dataanalyses; Eldar Zehorai, Inbal Wortzel, Roni Seger, Gili Ben-Nissan, MariaGabriella Fuzesi-Levi and Michal Sharon for technical advice; and Gabriella Almogand Roy Hofi for superb animal care.

Competing interestsThe authors declare no competing or financial interests.

Author contributionsJ.P.O., E.M.Z., J.H.T., K.Y. and S.A.F. conceived of and designed the experiments;J.P.O., E.M.Z., J.H.T., A.C.B., J.L.A., E.D.B., D.M.C., A.M.C., H.T., I.A.D. andS.A.F. performed the experiments; J.P.O., E.M.Z., J.H.T., A.C.B., J.L.A., E.D.B.,D.M.C., A.M.C., H.T., I.A.D., K.Y. and S.A.F. analyzed the data; J.P.O., E.M.Z.,J.H.T., H.T., I.A.D., K.Y. and S.A.F. wrote the paper.

FundingThis work was supported in part by the National Institute of Diabetes and Digestiveand Kidney (NIDDK) [grant numbers RO1DK093399 to S.A.F., RO1GM63904 toThe Zebrafish Functional Genomics Consortium (Stephen Ekker and S.A.F.),F32DK096786 to J.P.O.].The content is solely the responsibility of the authors anddoes not necessarily represent the official views of the National Institutes of Health(NIH). Additional support for this work was provided by the Carnegie Institution forScience endowment and the G. Harold and Leila Y. Mathers Charitable Foundationto the laboratory of S.A.F.; the European Research Council [grant numberERC/335605 to K.Y.]; the Israel Science Foundation [grant number 861/13 to K.Y.];FP7-PEOPLE-2009-RG 256393 (to K.Y.); and an Israel Cancer ResearchFoundation Postdoctoral Fellowship (to I.A.D.). K.Y. is supported by the WillnerFamily Center for Vascular Biology; the estate of Paul Ourieff; the Carolito Stiftung;Lois Rosen, Los Angeles, CA; and the Adelis Foundation. K.Y. is the incumbent ofthe Louis and Ida Rich Career Development Chair.

Supplementary materialSupplementary material available online athttp://dmm.biologists.org/lookup/suppl/doi:10.1242/dmm.018754/-/DC1

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