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Dec 20, 2004 - Esther Marza, Christophe Barthe, Miche`le André, Laure Villeneuve, Chantal Hélou, and. Patrick J. ... expressed sequence tag gb GenBank hpf hours postfertilization fabp2 intestinal fatty ... al., 1957; Jersild and Clayton, 1971), .... to 24 hpf, the hybridization signal was ..... man MTP large subunit is the pres-.
DEVELOPMENTAL DYNAMICS 232:506 –518, 2005

PATTERNS & PHENOTYPES

Developmental Expression and Nutritional Regulation of a Zebrafish Gene Homologous to Mammalian Microsomal Triglyceride Transfer Protein Large Subunit Esther Marza, Christophe Barthe, Miche`le Andre´, Laure Villeneuve, Chantal He´lou, and Patrick J. Babin*

The microsomal triglyceride transfer protein (MTP) large subunit is required for the assembly and secretion of apolipoprotein B-containing lipoproteins. We have found a zebrafish mtp homologous gene coding a protein with 54% identity with human MTP large subunit with the most conserved regions distributed in the corresponding predicted ␣-helical and C- and A-sheet domains. In situ hybridizations showed that zebrafish mtp transcripts were distributed in the yolk syncytial layer during early embryogenesis and in anterior intestine and liver from 48 hr postfertilization onward. Real-time quantitative RT-PCR confirmed the developmental regulation and tissue-specificity of mtp expression. A significant pretranslational up-regulation of mtp expression was observed in the anterior intestine after feeding. The nutritional regulation of zebrafish mtp expression observed in the anterior intestine supports the notion that this protein, similar to mammalian MTP large subunit, could be a factor implicated directly or indirectly in large lipid droplets accumulation observed in the fish enterocyte after feeding. Developmental Dynamics 232:506 –518, 2005. © 2004 Wiley-Liss, Inc. Key words: zebrafish; intestine; larval nutrition; lipoprotein; MTP; apolipoprotein B; lipid nutrition; liver; dietary regulation; triacylglycerol; abetalipoproteinemia Received 10 August 2004; Revised 16 September 2004; Accepted 20 September 2004

INTRODUCTION Microsomal triglyceride transfer protein (MTP) large subunit is an intracellular member of the large lipid transfer

protein super family also including apolipoprotein B (apoB), apolipophorin II/I, and vitellogenin (Babin et al., 1999). MTP large subunit is essential for assembly and secretion by liver and intes-

tine of apoB-containing lipoproteins, very low density lipoproteins (VLDLs), and chylomicrons (Gordon et al., 1995). This 97-kDa protein is combined with the multifunctional 58-kDa protein di-

ABBREVIATIONS apoB apolipoprotein B dpf days postfertilization EF1␣ elongation factor 1␣ ef1␣ elongation factor 1␣ gene EST expressed sequence tag gb GenBank hpf hours postfertilization fabp2 intestinal fatty acid-binding protein gene MTP microsomal triglyceride transfer protein mtp zebrafish microsomal triglyceride transfer large subunit gene Mttp mammalian microsomal triglyceride transfer protein large subunit gene PTU 1-phenyl-2-thio-urea q-PCR real-time quantitative RT-PCR RACE rapid amplification of cDNA ends SD standard deviation sp Swiss-Prot TG triglycerides YS yolk sac YSL yolk syncytial layer

Laboratoire Ge´nomique et Physiologie des Poissons, UMR 1067 NUAGE INRA-IFREMER, Universite´ Bordeaux 1, Talence Cedex, France Grant sponsor: Commission of the European Communities, the specific RTD program, “Quality of Life and Management of Living Resources”; Grant number: Q5S-2002-00784, “CRYOCYTE”; Grant sponsor: French Ministry of Research and Education; Grant number: grant AGENAE. *Correspondence to: Patrick J. Babin, Laboratoire Ge´nomique et Physiologie des Poissons, UMR 1067 NUAGE INRAIFREMER, Universite´ Bordeaux 1, Avenue des Faculte´s, Baˆt. B2, 33405 Talence Cedex, France. E-mail: [email protected] DOI 10.1002/dvdy.20251 Published online 20 December 2004 in Wiley InterScience (www.interscience.wiley.com).

© 2004 Wiley-Liss, Inc.

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sulfide isomerase (PDI) in a soluble microsomal heterodimer (Wetterau et al., 1990). MTP transfers neutral lipids and triglycerides (TG) contained in the endoplasmic reticulum membrane to nascent apoB localized in the lumen (Wetterau and Zilversmit, 1984, 1986; Jamil et al., 1995). Furthermore, MTP large subunit physically interacts with apoB acting also as a chaperone by preventing apoB degradation (Patel and Grundy, 1996; Wu et al., 1996; Hussain et al., 2003). Mammalian MTP large subunit gene (Mttp) is expressed during development (Terasawa et al., 1999; Shelton et al., 2000; Levy et al., 2001; Lu et al., 2002) and seems to be essential, as all homozygous Mttp knockout mice have been reported to die during embryonic development (Raabe et al., 1998). Genetic mutations of human Mttp causing the absence or length modifications of the protein are responsible for abetalipoproteinemia (Shoulders et al., 1993; Sharp et al., 1993). This disease is characterized by the absence of apoB-containing lipoproteins in the plasma, intestinal fat, and malabsorption of liposoluble vitamins, leading to spinocerebellar degeneration and retinopathy (Wetterau et al., 1992). Intestinal biopsies of abetalipoproteinemic patients has revealed intestinal steatosis with large lipid droplet accumulation (Herbert et al., 1985; Shoulders et al., 1993). In mice, an accumulation of cytosolic fat in the visceral endoderm of the yolk sac (YS) has been associated with the absence of Mttp (Raabe et al., 1998). Moreover, liver-specific Mttp knockout mice exhibit hepatic steatosis and a striking reduction in VLDL TG (Raabe et al., 1999). The use of a MTP lipid transfer activity inhibitor in murine hepatocytes not only reduces the pool of TG associated with apoB, i.e., VLDL, but also apoB-free TG (Kulinski et al., 2002). MTP could interact with lipid droplets and play a role in their formation and stabilization in the endoplasmic reticulum lumen (Bakillah and Hussain, 2001; Hussain et al., 2003). Fish are useful animal models for studying vertebrate lipid metabolism and transport (Wallaert and Babin, 1994; Farber et al., 2001). Cholesterol and other lipids are carried in fish blood by different lipoprotein classes,

of which the basic molecular organization and role in lipid metabolism are similar to those described in mammals (Babin and Vernier, 1989). In fish (Sire et al., 1981; Honkanen et al., 1985) as in mammals (Borgstrom et al., 1957; Jersild and Clayton, 1971), the proximal third of the intestine is the major site of fat absorption. In Cyprinidae, including zebrafish (Iwai, 1969; Gauthier and Landis, 1972; Noaillac-Depeyre and Gas, 1974, 1976; Rombout et al., 1984; Andre´ et al., 2000), in sea bream (Diaz et al., 1997), in trout (Bergot and Fle´chon, 1970; Sire et al., 1981), and in killifish (Vetter et al., 1985), the columnar epithelial cells of the rostral part of the gut contain large lipid droplets during dietary fat absorption. These enterocytes synthesize and transfer large amounts of VLDL in the plasma (Babin and Vernier, 1989; Wallaert and Babin, 1992). A functional homolog of human MTP large subunit has been identified recently in Drosophila and a Fugu-putative MTP large subunit sequence was deduced from data extracted from the Fugu genomics project (Sellers et al., 2003). However, no data are currently available on the developmental and dietary regulation of the expression of an MTP large subunit homologous gene in a nonmammalian species. In the present study, we cloned and sequenced a zebrafish cDNA encoding a protein homologous to human MTP large subunit and determined the corresponding gene (mtp) structure. By using this useful model for developmental studies, we performed in situ hybridization and real-time quantitative reverse transcriptase-polymerase chain reactions (qPCRs) to investigate the tissue-specific expression and developmental and nutritional regulation of mtp expression in zebrafish. Our data provide the first detailed spatial and temporal expression pattern of a MTP large subunit-related gene in a lower vertebrate.

RESULTS Molecular Cloning of a cDNA Encoding Zebrafish Putative MTP Large Subunit A cDNA containing the entire encoding sequence of a zebrafish protein similar to mammalian MTP large sub-

unit was obtained by PCRs with reverse transcribed total RNAs obtained from adult anterior intestine. An 884-bp fragment (MTP-zeb884) was isolated using forward primer p2 and the degenerated reverse primer p4. MTP-zeb884 was then extended by rapid amplification of cDNA ends (RACE) -PCR. A 431-bp-long 5⬘-end fragment was obtained using reverse primer p1 and the universal adapter UPM. Forward primer p6 and an oligodT adapter were used to amplify the 3⬘-end, generating a 2,048-bp fragment. Total MTP cDNA sequence isolated was 3,054 bp long. GenBank screening by using the 3⬘-end sequence of MTP cDNA identified four overlapping EST clones with accession nos. gb/CN172225/, gb/ CN015783/, gb/AI974191/, and gb/ BF718005/. These draft sequences indicated that total MTP large subunit cDNA sequence was approximately 4,327-bp long, including a polyA tail of 28 bp. A 2,652-bp open reading frame was identified with an ATG codon at position 74 and a stop codon (TAA) at nucleotide 2,726. One transcript of around 4,200 bp corresponding to MTP large subunit cDNA size was detected by Northern blotting performed on total RNAs extracted from liver or anterior intestine of adult zebrafish females (data not shown). This finding confirmed the transcription of the corresponding gene in these tissues. The deduced protein sequence was 884 amino acids long with a predicted signal peptide cleavage site between amino acids 21 and 22. The calculated molecular weight of the unprocessed precursor was 97,041 Da, and its pI was 8.51. The zebrafish MTP large subunit cDNA sequence obtained is available under the accession no. gb/ AJ428850/.

Gene Structure and MTP Large Subunit Sequence Comparison Between Zebrafish and Human Zebrafish mtp structure was deduced from genome sequences extracted from Sanger Institute Ensembl Genome Data Resources after comparison with zebrafish MTP large subunit cDNA sequence. Zebrafish mtp was located on chromosome 12 and consisted

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of 18 translated exons (Fig. 1) with sizes similar to those of human Mttp. In the translated part of the cDNA, both genes contained 17 introns, which were of the same intron types (type 0 introns are located between two codons, type 1 introns are situated between nucleotide positions 1 and 2 of a codon, and type 2 introns lie between nucleotide positions 2 and 3 of a codon) except intron number 13 (type 2 in human and type 1 in zebrafish genes). Introns number 2–5, 7, 9 –11, 14, and 15 were of type 0 in zebrafish mtp. Introns number 1 and 13 were of type 1, and introns number 6, 8, 12, 16, and 17 were of type 2. Between zebrafish and human genes, introns number 13, 16, and 17 were shifted by one codon. The sequences at the intron– exon boundaries were consistent with the usual consensus intron/exon splice junction rule (GT/AG) with the exception of zebrafish intron number 13 (data not shown). Among the five introns contained in the Drosophila MTP-related gene, three of them appeared to be at the same position as introns number 7, 16, and 17 of zebrafish mtp (data not shown). The alignment between zebrafish and human MTP large subunit proteins (Fig. 1) showed 54% amino acid identity and 66% amino acid similarity. The most conserved regions between human and zebrafish MTP large subunit were distributed in the corresponding predicted ␣-helical domain (␣1 to ␣17; 58.6% identity and 69% similarity) and in the C-sheet (C-␤1 to C-␤6; 70.6% identity and 79.8% similarity) and A-sheet (␣A to A-␤4; 68.6% identity and 78.4% similarity) domains of the C-terminus part of human MTP large subunit. A-sheet ␣A (725-736) and ␣B (781-786) helices are involved in human MTP lipid transfer activity (Read et al., 2000). The lipid transfer activity-essential amino acid leucine-734 (Read et al., 2000) is conserved in zebrafish and in all other fish MTP large subunit-deduced partial protein sequences currently available, including trout and Fugu sequences (Fig. 1). On the other hand, valine-782 of helix ␣B, another critical amino acid for MTP transfer activity (Read et al., 2000), is not conserved in lower vertebrate sequences (Fig. 1). A glycine or an alanine is

systematically found at this position in fish and Xenopus sequences.

Expression of mtp During Zebrafish Development The expression pattern of mtp during zebrafish development was followed by whole-mount in situ hybridization (Fig. 2) using sense and antisense digoxigenin-labeled riboprobes synthesized from clone MTP-zeb884. mtp hybridization signal was visible in the blastoderm margin by 4 hours postfertilization (hpf; Fig. 2A), a period associated with the beginning of zygotic transcription, i.e., the mid-blastula transition (Kimmel et al., 1995). Later in embryonic development, from 9 hpf to 24 hpf, the hybridization signal was localized in the yolk syncytial layer (YSL; Fig. 2B–E). By 48 hpf, the intestinal tube began to differentiate as a columnar epithelium ventrally to the first pronephric tubules and dorsally to the YS. At this stage, mtp hybridization signal decreased from the extraembryonic YSL and was detected in the embryonic liver primordium and intestinal tube (Fig. 2F). These embryonic structures were strongly labeled by 4 days postfertilization (dpf; Fig. 2G), before the first feeding occurring by 5 dpf. Between 3 and 5 dpf, the anterior intestine underwent the transition from a straight to a coiled tube leading to gut looping on the left side of the larva. At 6 and 15 dpf, mtp hybridization signal was clearly restricted to the two main liver lobes and the anterior part of the intestine, including the intestinal bulb (Fig. 2H,I), whereas no signal was detected in the pharynx or posterior intestine (Fig. 2I). No signal was detected using sense riboprobe either (Fig. 2J). Cryosections of hybridized 15 dpf larvae (Fig. 2I) confirmed that mtp hybridization signal was localized in the enterocytes of the anterior part of the intestine (Fig. 2K,L) as well as in the hepatic cells (Fig. 2L). On parasagittal sections, the lower staining signal observed in the dorsal versus ventral intestinal epithelium of the intestinal bulb (Fig. 2L) could be attributed to a differential riboprobe tissue penetration. Nonspecific staining signal from the food filling the intestinal lumen was not detected (Fig. 2L). To evaluate the relative quantity of

mtp transcript during zebrafish embryonic development, we conducted qPCRs from total RNAs extracted at different developmental stages. The relative amounts of mtp transcripts were calculated by using ef1␣ transcript amplification as a standard (Fig. 3A). A very small amount, if any, of mtp transcript of maternal origin was detected before 2 hpf. The ratio between mtp and ef1␣ transcript quantities significantly increased between 2 and 5 hpf (0.26 ⫾ 0.08 vs. 13.23 ⫾ 3.99, respectively, P ⬍ 0.001) and remained at a high level at 6 hpf (8.51 ⫾ 1.82) and 9 hpf (10.22 ⫾ 1.28). A significant decrease in the amount of mtp transcript was then observed at 12 hpf (2.05 ⫾ 0.27). This low level was maintained until the end of embryogenesis, which occurred by 72 hpf

Fig. 2. Expression pattern of the microsomal triglyceride transfer protein (MTP) large subunit gene (mtp) during zebrafish embryonic and larval development. A: After the mid-blastula transition, 4 hours postfertilization (hpf). B: During the gastrulation period (9 hpf). C: At the fivesomite stage (12 hpf). D: At the 15-somite stage (16 hpf). E: A 24-hpf embryo. F: A 48-hpf embryo. G: A 4 days postfertilization (dpf) larva. H,J: A 6 dpf larva. I,K,L: A15 dpf larva. Wholemount in situ hybridizations were performed with digoxigenin-labeled sense and antisense riboprobes and are presented in lateral (A–G,I) or dorsal (H,J) views with the anterior part to the left (C–J,L). The 6-dpf and 15-dpf larvae were nourished ad libitum. In some cases (G,H,J), the animals were raised in 0.2 mM 1-phenyl-2-thiourea containing water to prevent pigment formation. The hybridization signal is colored dark brown to blue. J: No staining signal was observed by using the sense probe. A: A specific hybridization signal was first observed in the blastoderm margin (bm). B–F: mtp was then strongly expressed in the yolk syncytial layer (ysl) from the end of the gastrulation period until 48 hpf. C–E: No transcripts were detected in embryonic structures until 24 hpf. F: At 48 hpf, mtp mRNA was detected in the liver (l), intestinal tube (it), and yolk syncytial layer. G: By 4 dpf, the hybridization signal was restricted to the liver and anterior intestine (ai). H: By 6 dpf, the two liver lobes and the anterior intestine were strongly labeled. I: By 15 dpf, mtp mRNA was detected in the liver and the anterior intestine, including the intestinal bulb, while no hybridization signal could be detected in pharynx (ph) and posterior intestine (pi). Transverse (K), indicated by a vertical broken line in (I), and parasagittal (L) cryosections of 15-dpf larvae showed that mtp transcripts were restricted to enterocytes and hepatic cells. Embryos, larvae, and cryosections were mounted in 100% glycerol. yc, yolk cell; op, optic primordium; tb, tail bud; e, eye. Scale bars ⫽ 100 ␮m in A–D,F–L, 500 ␮m in E.

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Fig. 2.

Fig. 1. Fig. 1. Comparison between fish and tetrapods microsomal triglyceride transfer protein (MTP) large subunit predicted amino acid sequences. Amino acid sequences are numbered from the initiator methionine for human (Hum), pig (Pig), mouse (Mou), chicken (Chi), Fugu (Fug), and zebrafish (Zeb) sequences. Xenopus (Xen) and trout (Tro) sequences are numbered according to the amino acid sequence deduced from the partial cDNA sequences currently available. Residues identical or considered conserved with human sequence are colored in cyan and yellow, respectively. The allowed conservative substitutions were defined as follows: A, G; S, T; E, D; R, K, H; Q, N; V, I, L, M; Y, F; W; P; C. A: Zebrafish MTP large subunit predicted amino acid sequence aligned with the corresponding human sequence. The predicted signal peptide sequences are underlined. Human MTPpredicted structure based on the atomic coordinates of lamprey lipovitellin (Mann et al., 1999; Read et al., 2000) is shown with arrows indicating ␤-strands, cylinders depicting ␣-helices, plain lines representing loops, and dashed lines the unassigned coordinates. Human MTP large subunit is structurally divided into four domains: the N-sheet (␤1–␤13) consists of amino acids 22 to 303, the ␣-helical domain (␣1–␣17) is distributed from amino acids 304 to 598, the C-sheet (C-␤1–C-␤6) corresponds to the fragment between amino acids 603 and 712, and the A-sheet (␣A–A-␤4) includes amino acids 725 to 829. Downward and upward black arrowheads indicate intron positions in human and zebrafish MTP large subunit gene, respectively. The number below the zebrafish sequence and attached to the upward black arrow is indicative of the intron number. Gaps inserted to optimize alignments are indicated by dashes. B: Comparison of the C-terminal part of vertebrate MTP large subunit protein sequences encompassing helices ␣A and ␣B of the A-sheet domain of human MTP large subunit. Single dots in the trout sequence indicate an unknown sequence. Numbers in parentheses in the alignments indicate the length of the omitted sequence. Arrows below the sequences indicate two amino acid positions critical for the lipid transfer activity of human MTP large subunit (Read et al., 2000). The first position corresponding to leucine-734 is conserved in the other vertebrate sequences, whereas the second position corresponding to valine-782 is not.

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(ratios ⬍ 1.3). These developmental mtp mRNA quantitative variations were confirmed using ␤-actin as an additional housekeeping control gene (data not shown).

Expression of mtp in Adult Zebrafish Tissues Northern blotting analysis revealed that mtp mRNA level was found to be more abundant in the anterior intestine than in the liver (data not shown). The relative amounts of mtp transcripts in adult zebrafish tissues were quantified by q-PCR, using ef1␣ transcript quantity as a standard (Fig. 3B). The ratio between mtp and ef1␣ transcript quantities was significantly higher in the anterior intestine (21.00 ⫾ 11.98, P ⬍ 0.05) than in the other tissues tested. A relatively high quantity of mtp mRNA was also found in liver (6.51 ⫾ 3.07) and posterior intestine (6.28 ⫾ 2.34) by comparison with brain (0.52 ⫾ 0.46), testis (0.65 ⫾ 0.28), kidney (0.74 ⫾ 0.82), heart (0.19 ⫾ 0.19), and ovary (0.007 ⫾ 0.002). Staining histological sections of the anterior intestine with toluidine blue revealed the presence of numerous large supranuclear inclusions in the enterocytes of fed adults (Fig. 4A). These large lipid droplets measuring up to 5 ␮m were stained with a black tint by the neutral lipid-specific dye Sudan black B (Fig. 4B). The presence of these large intracellular lipid droplets in the enterocytes might be correlated with the expression of mtp. The distribution of zebrafish mtp mRNA in the intestine was defined by in situ hybridization performed on sections. From mouth to anus, two intestinal loops were observed and transverse sections showed three different intestinal rings (Fig. 4C). No hybridization signal was detected in sections hybridized with the sense riboprobe (Fig. 4D). In situ hybridization detection of mtp expression revealed a very strong hybridization signal in the enterocytes of ring 1, corresponding to the proximal part of the anterior intestine (Fig. 4E). A lower hybridization signal was observed in ring 2, corresponding to the distal part of the anterior intestine, and in ring 3, corresponding to the posterior portion of the intestinal tube.

Fig. 3. Real-time quantitative polymerase chain reaction (q-PCR) analysis of mtp expression in zebrafish. A: At each developmental stage, three independent pools of more than 50 embryos were used to quantify in duplicate mtp transcript amount. mtp expression was significantly higher at 5 hours postfertilization (hpf; 40% epiboly), 6 hpf (shield stage), and 8 hpf (80% epiboly) in comparison to the other developmental stages tested. B: Liver, anterior intestine, and posterior intestine were collected from three fed females and pooled. Heart, brain, testis, ovary, and kidney tissues were collected from more than 10 different animals and pooled. The q-PCR was performed on three independent pools in duplicate. mtp expression was significantly higher in the anterior intestine in comparison with the other collected tissues. The indicated values are the mean ⫾ standard deviation of the ratio (microsomal triglyceride transfer protein large subunit copy number/elongation factor 1 ␣ transcript [MTP/EF1␣] copy number) ⫻ 100. Data were tested for statistical significance by analysis of variance followed by an evaluation using the Tukey–Kramer test. Asterisks indicate P ⬍ 0.05.

Regulation of mtp Expression by the Nutritional Status Whole-mount in situ hybridizations were performed before and after feeding at different nutritional developmental stages of the larva (Fig. 5). The investigated stages were after the first feeding (5 dpf), corresponding to the beginning of the mixed endotrophic– exotrophic period, at the end of this mixed nutritional phase (8 dpf), and during the exotrophic period (15 dpf), with nourishment of the larvae exclu-

sively from exogenous nutrients. No staining signal was revealed in either fasted (data not shown) or fed larvae hybridized with the sense riboprobe (Fig. 5A,D,G). As previously reported (Fig. 2G), mtp mRNA was clearly distinguished in the liver and anterior intestine of prefeeding larvae (Fig. 5B). Regardless of the developmental stage, feeding resulted in a marked increase in mtp hybridization signal in the anterior intestine and to a lessmarked increase in the liver (Fig. 5C,F,J) when compared with the fast-

Fig. 4. Intestinal mucosa of fed adult zebrafish. A,B: Histological semithin sections of the anterior intestine were stained with toluidine blue (A) or with the neutral lipid-specific stain Sudan black B (B). In both cases, the intestinal mucosa showed enterocytes (en) containing large lipid droplets (ld). C: Schema of adult zebrafish intestine. From the mouth to the anus, two intestinal loops are observed and transverse sections at loop levels give three different intestinal rings, numbered 1 to 3. D,E: The localization of mtp transcripts was performed in the intestine by in situ hybridizations with digoxigenin-labeled sense (D) and antisense (E) riboprobes on parallel transverse intestinal sections of an 8-month fed young adult female at the level indicated by a vertical line in C. Rings 1 and 2 are representative of the proximal and distal parts of the anterior intestine, respectively. Ring 3 could be attributed to the posterior part of the intestine due to the presence of a large amount of vacuoles in the supranuclear hyaloplasm (arrow with broken line) of enterocytes. No staining signal was observed by using the sense probe. A strong mtp hybridization signal was observed at ring 1 and, to a lesser extent, at rings 2 and 3. The staining signal was restricted to the enterocytes (arrow), whereas the mucous cells (arrowhead) were not labeled. lu, lumen; mc, mucous cells. Scale bars ⫽ 30 ␮m in A, 15 ␮m in B, 100 ␮m in D,E.

Fig. 5. The effect of feeding on mtp expression in zebrafish larvae as evaluated by whole-mount in situ hybridization. A–I: Larvae were fasted for 18 hr and fed (A,C,D,F,G,I) or not (B,E,H) with dried chicken egg yolk powder for 2 hr. A–I: The 5 days postfertilization (dpf; A–C), 8 dpf (D–F), and 15 dpf (G–I) larvae were then collected and hybridized with sense (A,D,G) or antisense riboprobes (B,C,E,F,H,I). Larvae are shown in lateral view with the anterior part to the left. B,C,E,F,H,I: At each developmental stage, mtp expression in the anterior intestine (ai) dramatically rose in fed larvae (C,F,I) compared with fasted larvae (B,E,H). mtp hybridization signal also appears to be increased in the liver (l). No transcripts could be detected in the posterior intestine (pi). A,D,G: No staining signal has been observed using the control sense probe. Scale bars ⫽ 500 ␮m.

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Fig. 6. The effect of feeding on mtp transcript level in zebrafish as evaluated by real-time quantitative polymerase chain reaction. A: Larvae were fasted for 18 hr and fed or not with dried chicken egg yolk powder for 2 hr. At each developmental stage (dpf, days postfertilization), seven independent pools of more than 30 larvae were used to quantify in duplicate mtp transcript amount. B: Male adult zebrafish were starved for 18 hr and fed or not for 2.5 hr. At each experimental sample, anterior intestine and liver were collected from two animals and pooled. mtp transcript amounts were determined on seven independent samples in duplicate. The indicated values are the mean ⫾ standard deviation of the ratio (microsomal triglyceride transfer protein large subunit copy number/elongation factor 1 ␣ transcript [MTP/EF1␣] copy number) ⫻ 100. Data were tested for statistical significance by performing a Mann–Whitney test. Asterisks indicate P ⬍ 0.05.

ing state (Fig. 5B,E,H). Along the digestive tract, mtp transcript signal was restricted to the anterior intestine, including the intestinal bulb, whereas no signal was detected in the pharynx and posterior intestine, regardless of the developmental stage and nutritional status of the larva. The effect of feeding on mtp transcript level in zebrafish larvae was evaluated by q-PCR using ef1␣ transcript level as a standard. Total RNAs were extracted from whole larvae at the three different nutritional developmental stages previously investigated by whole-mount in situ hybridization (Fig. 6A). mtp mRNA amounts significantly increased in response to feeding at 5 dpf (0.63 ⫾ 0.23 vs. 1.16 ⫾ 0.56, P ⫽ 0.037) and 8 dpf (0.97 ⫾ 0.16 vs. 2.23 ⫾ 1.39, P ⫽ 0.035). A nonsignificant increase in of mtp/ef1␣ transcript level ratio was observed in 15 dpf fed larvae (1.64 ⫾ 0.57 vs. 2.33 ⫾ 0.95), probably due to the reduction in the relative proportion of mtp-expressing cells in these fast-growing larvae. To test whether the nutritional state might play a role in the regulation of mtp expression in adults, we analyzed the effect of feeding on mtp expression in the anterior intestine and liver collected from adult males using q-PCR (Fig. 6B). mtp/ef1␣ transcript level ratio was increased approximately 2.6-fold by feeding in both anterior intestine (12.17 ⫾ 1.38 vs. 31.22 ⫾ 7.69, P ⫽ 0.028) and liver (5.61 ⫾ 1.48 vs. 14.9 ⫾ 5.3, P ⫽ 0.078).

DISCUSSION In this study, we performed the molecular cloning of a zebrafish protein homologous to mammalian MTP large subunit, determined its gene structure and expression pattern during development and its tissue-specific expression in adults, and studied in larvae and adults its nutritional gene expression regulation. The exon–intron organization of zebrafish mtp was similar to that of human Mttp (Sharp et al., 1994). The coding region had an overall amino acid identity of 54% with human MTP large subunit sequence, including conserved important functional domains. Similarly, Fugu and human MTP large subunit sequences shared 55% amino acid identity (Sellers et al., 2003). This finding suggests that fish and mammal proteins have similar properties for membrane binding and lipid transfer activity. A functional homolog of human MTP large subunit has been identified in Drosophila (Sellers et al., 2003) and shares 23% amino acid identity with both human and zebrafish sequences. In vitro, the insect protein was able to add lipids to the carboxyl-terminally truncated forms of human apoB to a similar extent as human MTP large subunit but displayed low vesicle-based TG transfer activity. A structural feature of human MTP large subunit is the presence of ␣A and ␣B, two A-sheet helices in its C-terminal part, which have been hypothesized to facilitate TG ac-

quisition by lipid interaction with the helical peptides (Read et al., 2000). The ␣A sequence is well conserved in zebrafish MTP large subunit but not in the Drosophila sequence (Sellers et al., 2003). This conservation includes leucine-734 of human MTP large subunit essential for the lipid transfer activity (Read et al., 2000). Valine-782 included in helix ␣B is a second critical amino acid for lipid transfer activity of human MTP large subunit. Valine-782 to alanine mutation reduces human MTP lipid transfer activity to 26% of the wild-type activity (Read et al., 2000). A glycine or an alanine is systematically found at the corresponding site in fish and Xenopus sequences. This difference could be associated with a reduced ␣B helix length and a lower MTP lipid transfer activity in fish compared with humans. It should be noted that arginine 540 and asparagine 780, two amino acids residues mutated in abetalipoproteinemic patients with impaired MTP activity (Rehberg et al., 1996; Ohashi et al., 2000), are conserved in the zebrafish MTP large subunit sequence. In mammals, Mttp is expressed during embryonic and fetal development (Terasawa et al., 1999; Shelton et al., 2000; Levy et al., 2001; Lu et al., 2002). Mouse Mttp mRNA has been detected in the YS at embryonic day E9.5 (Terasawa et al., 1999; Shelton et al., 2000) and MTP large subunit has been immunodetected in the YS of 9-day chicken embryos (Hermann et

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al., 2000). In birds and mammals, epithelial YS endodermal cells play a role in the nutrition of the developing embryo through apolipoproteins and lipoproteins synthesis and secretion that was correlated with Mttp expression (for reviews, see Poupard et al., 2000; Shelton et al., 2000; Lu et al., 2002). In addition, Mttp knockout mice have shown a deficiency in the lipoprotein formation in the YS endoderm associated with an embryonic lethality in homozygotes (Raabe et al., 1998). A high amount of mtp transcript was detected in zebrafish embryo during the gastrula period both by whole-mount in situ hybridization and q-PCR. These transcripts were restricted to the YSL. mtp expression was then down-regulated in this extraembryonic structure leading to a significant decrease in the total amount of mtp mRNA in the embryo. mtp expression down-regulation observed in zebrafish YSL after the gastrula stage and before the complete resorption of the endogenous reserves could be related to the absence of oil globules within zebrafish eggs (Selman et al., 1993) and, therefore, of large quantities of TG in the yolk cell of this species. In the absence of these oil globules rich in neutral lipids, the zebrafish could not maintain active YSL TG-rich lipoprotein synthesis during later stages of embryonic endotrophic nutrition. Following mtp expression in a fish species that has voluminous oil globules would be helpful to evaluate the role of MTP large subunit in the assembly of VLDL particles actively synthesized by the YSL (Vernier and Sire, 1977; Walzer and Scho¨nenberger, 1979; Poupard et al., 2000) during the mixed endotrophic– exotrophic period of development. Apolipoprotein gene expression and lipoprotein secretion by the YSL (Poupard et al., 2000) indicate that this extraembryonic temporary structure unique to teleosts fish could be the functional counterpart of the YS endoderm of higher vertebrates. The liver primordium and intestinal tube contain high amounts of mtp transcripts during late embryogenesis and in the prefeeding zebrafish larva. A similar Mttp expression pattern has been found in mouse and swine fetal intestine and liver (Raabe et al., 1998; Shelton et al., 2000; Lu et al., 2002).

Along the digestive tract, zebrafish mtp transcript signal was restricted to the anterior intestine, including the intestinal bulb, whereas no signal was detected in the pharynx or in the posterior intestine, regardless of the developmental stage and nutritional status of the larva. The functional cephalocaudal patterning of zebrafish intestinal tube appears to be established by 3 dpf, i.e., close to the larva hatching time, as revealed by the expression pattern of intestinal fatty acid-binding protein gene (fabp2; Andre´ et al., 2000; Her et al., 2004; Sharma et al., 2004), mtp (this study), and other genes implicated in the intestinal lipid metabolism (data not shown). The functional regionalization is maintained as the animal grows into adulthood with the anterior part of the fish intestine being devoted to dietary lipid absorption and transport (Sire et al., 1981; Honkanen et al., 1985). Similarly, the proximal third of the intestine is the main site of fat absorption in mammals (Borgstrom et al., 1957; Jersild and Clayton, 1971) and a proximal-to-distal gradient of Mttp expression in the intestine has been demonstrated (Lin et al., 1994; Lu et al., 2002). In human adult tissues, Mttp is expressed in liver, small intestine, testis, ovary, kidney, and heart (Shoulders et al., 1993; Nielsen et al., 1998). As previously reported in mammals (Lin et al., 1994; Raabe et al., 1998; Lu et al., 2002), the relative amount of mtp transcript was higher in zebrafish intestine and liver than in brain, testis, kidney, heart, and ovary. As MTP large subunit is a critical factor for chylomicron and VLDL formation, the tissue specificity of its gene expression during development and in adults could be correlated with the synthesis and secretion of apoB-containing lipoproteins (Raabe et al., 1998, 1999; Hussain et al., 2003). This lipoprotein synthesis has been demonstrated in mammalian liver, fetal and adult small intestine (Sabesin and Frase, 1977; Green and Glickman, 1981; Lin et al., 1994; Levy and Menard, 2000; Levy et al., 2001), and heart (Nielsen et al., 1998). Similarly, mtp high expression observed in zebrafish anterior intestine and liver could be associated with an intense lipoprotein particles synthesis and transfer in

these fish tissues (Iwai, 1969; Gauthier and Landis, 1972; Noaillac-Depeyre and Gas, 1974, 1976; Sire et al., 1981; Rombout et al., 1984; Wallaert and Babin, 1992; Andre´ et al., 2000). In addition to the future identification of functional promoter conserved elements mediating the expression of mtp in the gut epithelia as recently demonstrated by using fabp2 (Her et al., 2004), the expression pattern of zebrafish mtp appears to offer new opportunities for its use as a marker gene of the YSL and gut differentiation. While fabp2, encoding a protein with an important role in the intracellular binding and trafficking of fatty acids, the mtp-expressing sites could correlate with the capacity of the YSL or intestinal epithelia to package and transfer endogenous and exogenous lipid nutrients. In teleost fish, dietary fat absorption leads to significant accumulation of large lipid droplets in the enterocytes of the anterior intestine (Iwai, 1969; Bergot and Fle´chon, 1970; Gauthier and Landis, 1972; Noaillac-Depeyre and Gas, 1974, 1976; Sire et al., 1981; Rombout et al., 1984; Vetter et al., 1985; Diaz et al., 1997; Andre´ et al., 2000). After binding with the intracellular intestinal fatty acid binding protein (Andre´ et al., 2000; Her et al., 2004), short- and medium-chain dietary fatty acids can be delivered directly from the enterocytes to the circulatory system (Sire et al., 1981; Iijima et al., 1990). Long-chain dietary fatty acids can be oxidized, desaturated, or both (Bell et al., 2003). Dietary fatty acids can also be re-esterified to TG, which can be incorporated and secreted in the form of chylomicrons and VLDL. The presence after feeding of an enlarged intracellular pool of neutral lipids inside fish enterocytes may be due to a limited capacity for assembly and/or secretion of TG-rich lipoproteins during the absorption peak. The presence of large lipid droplets in fish enterocytes is similar to the intestinal steatosis observed in patients suffering genetic malabsorption syndromes, including abetalipoproteinemia (Samson-Bouma et al., 1996), a disease characterized by the absence or mutations of MTP large subunit (BerriotVaroqueaux et al., 2000; Hussain et al., 2003).

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In mammals, Mttp mRNA levels are not significantly modified in intestine and liver after fasting and a moderate pretranslational up-regulation in response to exposure to high fat (Lin et al., 1994; Bennett et al., 1995; Lu et al., 2002) or high cholesterol (Bennet et al., 1996) diets has been observed, whereas oxidized cholesterol supplemented diets lowered the hepatic expression of Mttp in rats (Ringseis and Eder, 2004). By using two different approaches, whole-mount in situ hybridization and q-PCR, a significant regulation by fasting/feeding of zebrafish mtp expression has been demonstrated in anterior intestine and to a lesser extent in liver. Intestinal mtp mRNA levels are up-regulated after feeding, starting at the first feeding of the larva. This up-regulation of mtp transcript level after food intake could not necessarily be correlated with a rapid significant increase of the lipoprotein synthesis capacity in the enterocytes. MTP mRNA levels may not directly correlate with protein levels or lipid transfer activity since MTP large subunit half-life has been determined to be approximately 4.4 days in HepG2 cells (Lin et al., 1995). The presence of large lipid droplets in fish enterocytes might be explained by the high efficiency and capacity of the anterior intestine for TG absorption, which under feeding conditions can exceed the capacity of the enterocytes to secrete TG in the form of TG-rich lipoproteins. In summary, our data provide the first detailed spatiotemporal expression pattern of a gene encoding a putative MTP large subunit in the developing fish system. The mtp-expressing sites could correlate with MTP action in the assembly and secretion of TGrich lipoproteins. Zebrafish mtp pretranslational up-regulation in the anterior intestine after feeding and the presence of large lipid droplets in fish enterocytes during the dietary absorption peak suggest differences between fish and mammals in the coupling between fat absorption and storage and the synthesis of intestinal TG-rich lipoprotein. Moreover, the effect of dietary fat components and diet composition on zebrafish mtp expression could provide important insights into the identification of particular constituents of the diet and the molecular

events involved in regulating lipoprotein synthesis and its relationship with intracellular accumulation of large lipid droplets in the zebrafish enterocyte. This nonmammalian model may be useful for further screening of chemicals, molecules, and physiological conditions that modulate mtp transcription, MTP activity, lipoprotein synthesis, and enterocyte lipid droplets accumulation and might provide a suitable model in which to study the human condition of malabsorption syndromes like abetalipoproteinemia and Anderson’s disease.

EXPERIMENTAL PROCEDURES Animals Adult zebrafish (Danio rerio) were purchased from local commercial sources. Embryos and larvae were obtained by natural mating and raised at 28.5°C, as previously described (http://zfin.org/zf_info/zfbook/cont. html). In some cases, larvae were raised in water containing 0.2 mM 1-phenyl-2-thio-urea (PTU) to prevent pigment formation. Developmental stages were recorded as hours or days postfertilization (hpf or dpf) as previously described (Kimmel et al., 1995).

Nutritional Experiments Larvae or adults used in nutritional experiments were fasted for approximately 18 hr and divided into two groups before feeding. One group of larvae or adults was fed for 2 hr or 2.5 hr, respectively, whereas the second group was starved. From the first feeding to 15 dpf, larvae were fed with dried chicken egg yolk powder. The animals were then fed as the adults with TetraMin Flakes (Tetra GmbH, Germany) containing 47% proteins, 7% fiber, 10% ash, and 6.2% lipids.

Extraction of Total RNA Total RNAs were extracted using a Nucleospin RNA II extraction kit (Macherey-Nagel, France) according to the manufacturer’s instructions. The integrity of the total RNA was checked by ethidium bromide staining in agarose borax gels.

Molecular Cloning of a Zebrafish Putative MTP Large Subunit RT-PCR was performed on 1.5 ␮g of total RNAs isolated from the anterior intestine of fed zebrafish females using the SMART by RACE, cDNA amplification kit (Clontech), and the M-MLV Superscript II Reverse Transcriptase RNase H Minus (Gibco BRL). cDNA aliquots were used to amplify the zebrafish MTP large subunit cDNA sequence by RT-PCR. The subsequent primer sequence positions were numbered from the ATG translation initiator codon. The sense oligonucleotide primer p2 (5⬘-TGGCCTGGAGGAACCCTG-3⬘; 193 bp–211 bp) was designed from a GenBank database (http://www.ncbi.nlm.nih.gov/) dbEST clone (gb/AA542502/). A degenerated antisense primer p4 (5⬘-ATCCACCAGCTGPGGBAG-3⬘; P ⫽ A or G, B ⫽ G, T or C; 1,059 bp–1,077 bp) was designed from a conserved nucleotide sequence region in human (gb/ X59657/; 1,066 bp–1,083 bp), bovine (gb/X78567/; 1,047 bp–1,061 bp), and Drosophila CG9342 (gb/NM_136231/; 1,089 bp–1,107 bp) cDNA sequences. These primers were used at 0.5 ␮M with 4 ␮l of cDNA, 5 units of Taq DNA polymerase (Promega, France), 2.5 mM MgCl2, and 2.5 mM dNTP to amplify an 884-bp zebrafish MTP large subunit cDNA fragment (clone MTPzeb884). The PCR profile contained one denaturating step at 94°C for 3 min followed by 30 cycles at 94°C for 20 sec, 56°C for 20 sec, 72°C for 30 sec; and one cycle at 72°C for 2 min. MTP large subunit cDNA 5⬘- (clone MTP-zeb5⬘) and 3⬘- (clone MTP-zeb3⬘) ends overlapping with clone MTPzeb884 were amplified by RACE-PCR with 4 ␮l of cDNA and the Advantage 2 PCR kit (Clontech) following the manufacturer’s instructions. The 5⬘end was obtained using the nested antisense primer p1 (5⬘-GCCTCTGAAGTGCCTCAAGCCTGACTT-3⬘; 331 bp–358 bp) and the 5⬘ universal primer adapter with the PCR program: one step at 94°C for 2 min; 5 cycles at 94°C for 30 sec, 72°C for 3 min; 5 cycles at 94°C for 30 sec, 70°C for 30 sec, 72°C for 3 min; 25 cycles at 94°C for 30 sec, 68°C for 30 sec, 72°C for 3 min, and one cycle at 72°C for 2 min. The 3⬘-end was amplified with

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the nested sense primer p6 (5⬘-CCGCGCAGCTTCCTGACGCTC-3⬘; 969 bp–990 bp) and an oligo-dT adapter. The PCR profile was one step at 94°C for 2 min; 5 cycles at 94°C for 20 sec, 69°C for 20 sec, 72°C for 2 min; 25 cycles at 94°C for 20 sec, 66°C for 20 sec, 72°C for 2 min, and 1 cycle at 72°C for 5 min. Each PCR product was subcloned into the EcoRI site of the pGEMT-Easy vector (Promega, France) and sequenced (Eurogentec, Belgium).

Sequence Sources and Analysis Sequences used for MTP large subunit or MTP large subunit-related sequences were Homo sapiens (human, Swiss-Prot (sp) sp/P55157/), Mus musculus (mouse, sp/O08601/), Sus scrofa (pig, gb/ AAO61497/), Gallus gallus (chicken, gb/ BU122717/ ⫹ contig 3.1104.11477. 928.13791, http://pre.ensembl.org/gallus_ gallus), Xenopus tropicalis (Western clawed frog, gb/AL868859/), Oncorhynchus mykiss (rainbow trout, gb/ BX860503/), Fugu rubripes (pufferfish, SINFRUP00000088005, http://genome. jgi-psf.org), and Danio rerio (zebrafish, gb/AJ428850/). H. sapiens and D. rerio MTP large subunit sequences were aligned with the Alignp program (Myers and Miller, 1988) using BLOSUM 62 matrix (http:// www.infobiogen.fr/services/analyseq/). Potential signal peptide cleavage sequence of zebrafish MTP large subunit was predicted with the SignalP program (http://www.cbs.dtu.dk/services/ SignalP/; Nielsen et al., 1997). Genomic sequences of zebrafish mtp (Zebrafish Nomenclature Guidelines, http://zfin.org/zf_info/nomen.html) large subunit were obtained in Sanger Institute Ensembl Genome Data Resources using the release 20.3b.1 (http://www.ensembl.org/Danio_rerio/) with accession no. ENSDARG 00000008637. Exon–intron junctions were identified using cDNA-genomic sequence alignment (Breathnach and Chambon, 1981).

Whole-Mount In Situ Hybridization and Sectioning Embryos and larvae were fixed in 4% paraformaldehyde overnight at 4°C and treated as previously described

(Babin et al., 1997). The MTP-zeb884 clone cDNA fragment was used as template to generate the RNA probes. Both antisense and sense digoxigeninlabeled RNA probes were obtained using T7 or SP6 RNA polymerase (Promega, France), and the digoxigenin RNA labeling mix (Roche, Germany) following manufacturer’s instructions. RNA probes were purified using the RNA purification Nucleospin RNA II kit (Macherey-Nagel) and checked for purity by denaturing agarose gel electrophoresis. Whole-mount in situ hybridizations were performed as previously described (Babin et al., 1997; http:// zfin.org/zf_info/zfbook/chapt9/9.82. html) with minor modifications. phosphate buffered saline (PBS) buffer used contained 0.04% (w/v) KCl, and 0.3% CHAPS was added to hybridization and rinsing buffers. Hybridization was performed at 62°C with a 30 sec to 15 min proteinase K treatment of 15 ␮g/ml for embryos and at 60°C with a 20 min to 35 min proteinase K treatment of 20 ␮g/ml for larvae. Embryos and larvae were mounted in 100% glycerol before being observed with an Eclipse E1000 Nikon microscope. Larvae sectioned after whole-mount in situ hybridization for light microscopy were rinsed twice in PBS, cryoprotected in 25% sucrose, embedded in gelatin, frozen in ornithine carbamyl transferase (OCT; Tissue-Tek, Sakura, Japan), and sectioned at 12 ␮m on a cryostat (Microm HM500M). Cryosections were then mounted in 100% glycerol.

Histological Study Samples were fixed with 1.5% glutaraldehyde in 0.15 M cacodylate buffer, pH 7.4, 1 hr at 4°C, washed in the same buffer, and post-fixed at 4°C for 30 min in 2% osmium tetroxide prepared in 0.2 M imidazole, pH 7.4. Samples were dehydrated in ethanol and propylene oxide and embedded in Epon 812. Semithin sections (2.5 ␮m) were stained with toluidine blue. Lipid detection was carried out on semithin sections with Sudan black B (Sire and Vernier, 1980).

Tissue Sectioning and In Situ Hybridization An 8-month fed female intestinal cavity was fixed in 4% paraformaldehyde overnight at 4°C, washed in PBS, embedded in paraffin, and sectioned at 7 ␮m with a Leica RM 2125RT microtome. In situ hybridizations were carried out on these sections as previously described (Bogerd et al., 2000) with minor modifications. Hybridizations were performed at 60°C in a buffer containing 2% sheep serum and 2 mg/ml bovine serum albumin (BSA). Sections were incubated with 5 ng/␮l of denatured sense or antisense riboprobe synthesized from clone MTPzeb884. Sense and antisense hybridized sections were mounted in 100% glycerol before being observed.

q-PCR Reverse transcriptions were performed with 1 ␮g of total RNAs using M-MLV Reverse Transcriptase RNase H Minus (Promega, France), 500 ng/␮l oligodT, and 500 ng/␮l hexamer random primers (Promega, France) following manufacturer’s instructions. qPCR amplifications were performed with the iCycler iQ Real-Time PCR Detection System (Bio-Rad). The q-PCR amplifications were carried out with 4 ␮l of diluted (1/20) cDNA, 2⫻ iQ SYBR Green Supermix (Bio-Rad) and oligodeoxyribonucleotide primers (0.3 ␮M). One primer pair for zebrafish mtp transcript (sense primer MTP-S 5⬘-GAGGCCACGCTGGATTTCAT-3⬘, 2,415 bp–2,435 bp, and antisense primer MTP-R 5⬘-TTGGACACCGTCTCTCTGAAG-3⬘, 2,501 bp–2,522 bp) and another one for elongation factor 1 ␣ (ef1␣) transcript (EF1␣, gb/X77689/; sense primer EF1␣-2 5⬘-CGTCTGCCACTTCAGGATGTG-3⬘, 798 bp– 819 bp, and antisense primer EF1␣-3 5⬘-ACTTGCAGGCGATGTGAGCAG-3⬘, 1,153 bp–1,174 bp) were used to amplify a 107-bp fragment (clone MTP-zeb1073⬘) and a 376-bp fragment (clone EF1␣-zeb376), respectively. MTP-S and MTP-R were designed from exon boundaries of intron number 17 and in exon number 18, respectively. EF1␣-2 and EF1␣-3 were designed from exons 5 and 7, respectively (Gao et al., 1997). The PCR profiles contained an initial

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denaturation step at 95°C for 1 min 30 sec followed by 35 cycles of 30 sec at 95°C and 30 sec at 62.3°C for mtp transcript amplification or by 25 cycles of 30 sec at 95°C, 30 sec at 63.7°C, and 30 sec at 72°C for ef1␣ transcript amplification. After amplification and 1 min at 95°C, a melting curve was obtained by using 80 cycles, decreasing the temperature by 0.5°C every 2 cycles. The q-PCR product sizes were checked on a 2% agarose gel. No amplification was observed in reversetranscription negative controls performed without RT and no primer– dimer formation occurred in the nontemplate controls. By using serial dilutions from 2.107 to 2.102 molecules per reaction of quantified MTPzeb107-3⬘ and EF1␣-zeb376 clones, standard curves representing cycle threshold (CT) value as a function of the logarithm of vector copy number were generated. The efficiency of qPCR was higher than 95% and the correlation coefficient was ⬎ 0.95 for each target. Quantitative analyses interassay reproducibility was assessed on more than 10 runs. The CT variation coefficients for 2.105 EF1␣-zeb376 and MTP-zeb107-3⬘ plasmid molecules per reaction were 0.04 and 0.07, respectively. ef1␣ transcript quantity was used to standardize the results by eliminating variations in mRNA and cDNA quantity and quality among the samples. At least three independent samples, representing three independent batches of embryo or larval pools, or adult tissues, were tested in duplicate.

Statistical Analysis Data are presented as mean ⫾ standard deviation (SD). mtp transcript level variations among developmental stages or among different adult tissues were tested for statistical significance by one-way analysis of variance followed by an evaluation using a Tukey–Kramer multiple range test. The feeding impact on mtp transcript level was tested for statistical significance by using a Mann–Whitney test (GraphPad InStat v 3.5, GraphPad Software). The P value chosen for statistical significance was 0.05.

ACKNOWLEDGMENTS E.M. is supported by a PhD fellowship from the French Ministry of Research and Education.

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