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Profiling of prostanoids in zebrafish embryonic development
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Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 397–402 www.elsevier.com/locate/plefa
Profiling of prostanoids in zebrafish embryonic development Hui-Chun Yeh, Lee-Ho Wang Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA Received 3 January 2006; received in revised form 3 July 2006; accepted 2 August 2006
Abstract Prostanoids (PG) play important roles in vascular, pulmonary, reproductive and renal physiology. Little is known about their roles in the embryonic development. Using the oviparous zebrafish embryo as a model, we determined the temporal expression of PGs synthesized from exogenous prostaglandin H2. Prostaglandin E2 is the major PG throughout first 120 h post-fertilization (hpf), whereas prostaglandin F2a is at a lower but also a constant level. Reverse transcription-polymerase chain reaction (RT-PCR) showed that transcripts of cytosolic and membrane-bound PGE synthases were evident during the 120 hpf period. Compared with thromboxane A2, the level of prostacyclin (PGI2)is higher at first 24 hpf, the stage before the formation of blood vessel. RT-PCR showed that transcript of prostacyclin synthase appeared at 7 hpf whereas thromboxane synthase appeared at 48 hpf, suggesting that PGI2 has additional functions besides hemostasis. Interestingly, level of prostaglandin D2 (PGD2) followed an exponential decay over 120 hpf with a rate constant of 0.048 h1 and transcript of lipocalin-type PGD synthase was expressed at a higher level at early stage of development, suggesting that PGD2 is highly regulated during embryogenesis. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction Prostanoids (PGs) possess potent biological activities in vascular, pulmonary, reproductive and renal physiology as well as in pathophysiology of inflammation, thrombosis and cancer [1]. Their biosynthesis is initiated by the action of phospholipase to release arachidonic acid from membrane phospholipids, followed by the action of prostaglandin H synthase (also known as cyclooxygenase, COX). Two different COX isozymes, COX-1 and COX-2, encoded by separate genes, have been identified [2]. Their common product, prostaglandin H2 (PGH2), is an unstable compound with a half-life of 10 min in aqueous solution [3]. PGH2 is used by the Abbreviations: PG; prostanoid; COX; cyclooxygenase; PGD2; prostaglandin D2; PGE2; prostaglandin E2; PGF2a; prostaglandin F2a; PGI2; prostacyclin; TXA2; thromboxnae A2; hpf; hour postfertilization; dpf; day post-fertilization; RT-PCR reverse transcriptionpolymerase chain reaction Corresponding author. Tel.: +1 713 500 6794; fax: +1 713 500 6810. E-mail address: [email protected] (L.-H. Wang). 0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.08.001
specific enzymes for the formation of each PG. The type and quantity of the terminal PGs generated appear to be governed by the specific PG-synthesizing enzyme present in a given cell. PGs then exert their actions through specific receptors on plasma membranes [4]. In mammals, the major terminal PGs are prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a), prostaglandin I2 (PGI2, also known as prostacyclin (PGI2) and thromboxane A2 (TXA2) [5]. PGD2 is a major PG synthesized in the central nervous system and is involved in the regulation of sleep and pain responses [6]. It is also actively produced by mast cells, basophils and Th2 cells, acting as an allergic and inflammatory mediator [7]. Two distinctive types of PGD synthase have been identified: one is the lipocalintype enzyme and the other, the hematopoietic enzyme. PGE2 is the most common PG and is ubiquitously produced in the body. It has four subtypes of PGE receptors to carry out its diversified functions including pain, fever, inflammation, ovulation, fertilization and bone metabolism [8]. There are also two forms of PGE synthases derived from separate genes: cytosolic form
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and membrane-bound form. PGF2a is an inducer of leuteolysis and has been implicated in parturition [9]. PGI2 is a potent vasodilator and inhibits platelet aggregation [10]. In contrast, TXA2 is a potent inducer of vasoconstriction and platelet aggregation. The balance of PGI2 and TXA2 is therefore crucial for hemostasis. Both PGI2 and TXA2 are very unstable in water and are non-enzymatically converted to 6-keto PGF1a and TXB2, respectively. Their synthesizing enzymes, PGI synthase and TXA synthase, are members of cytochrome P450 [11]. PGI synthase is mainly present in endothelial and smooth muscle cells, whereas TXA synthase is present in platelets, monocytes and macrophages. The physiological roles of PGs were also explored from the studies of mice lacking the specific PGsynthesizing enzyme and/or specific PG receptor. In general, these knockout genes are non-lethal and the knockout mice reveal minor phenotypes [12]. For example, both TXA synthase- and TXA2 receptorknockout mice revealed mild bleeding disorders and altered vascular responses to arachidonic acid [13,14]. PGI2 receptor-knockout mice suffered obstructive thrombi upon FeCl3-induced endothelial injury [15], and 6-month-old PGI synthase-knockout mice showed vascular disorders with the thickening of vascular walls and interstitial fibrosis [16]. Lipocalin-type PGD synthase-knockout mice failed to respond to a touchevoked pain, but had a normal hyperalgesia response [17]. Interestingly, the knockout mice were also found to be glucose-intolerant and insulin-resistant on a low fat diet [18]. In addition, the lipocalin-type PGD synthaseknockout mice developed nephropathy and an aortic thickening on a high-fat diet, suggesting that PGD2 plays an important role in the vasculature, especially in atherosclerosis and diabetes. Zebrafish has been used as a model to study embryonic development of COX-1 and COX-2 [19,20]. COX-1 serves as a house-keeping function and is a constitutive enzyme, whereas COX-2 is highly regulated by growth factor, cytokine and mitogen. COX-1 is found ubiquitously early in the zebrafish embryos during blastula and gastrula stages. During somitogenesis, COX-1 is enriched in the posterior intermediate mesoderm, and at 24 h post-fertilization (hpf) it is confined to the distal end of nephric duct and developing vasculature. Knockdown of COX-1 by a morphonino oligonucleotide resulted in gastrulation arrest or defect in vascular tube formation. Conversely, knockdown of COX-2 caused no noticeable phenotype. These findings are in sharp contrast to those obtained with knockout mice. COX-1 knockout mice survived well and were fertile [21]. However, COX-2 knockout mice suffered higher mortality due to gastric peritonitis or kidney failure [22,23]. The COX-2 knockout females also revealed multiple reproductive defects including
ovulation, fertilization, implantation and decidualization. These controversial results can be explained by the possibility that PG knockout mice acquire maternal PG to allow normal development of embryos in uterus. Therefore, the physiological roles of PGs may not reflect the developmental roles of PGs in the knockout mice. In this regard, zebrafish as a vertebrate is well suited for studying the embryonic roles of PGs because the embryos develop externally from maternal body. In order to fully exploit the advantages of zebrafish for studying the developmental roles of PGs, it needs information of the specific PG-synthesizing enzymes and PG receptors in this species to enable detection of gene expression and examination of gene-perturbed phenotypes. Zebrafish PGE synthase, both cytosol and membrane-bound forms, is the only terminal PGsynthesizing enzyme that was reported [24,25]. As an onset to further understand the developmental roles of PGs, we report here the biosynthetic capacity of PGs in the zebrafish embryonic stages. PGE2 is the major PG throughout the embryonic stages. However, at the early developmental stages, PGI2 is synthesized at a higher level than TXA2, suggesting an important role of PGI2 besides hemostasis. While most PGs remain at the fairly constant levels during embryogenesis, levels of PGD2 biosynthesis decreases as embryo matures, suggesting an important role of PGD2 in the early stage of embryo development.
2. Materials and methods 2.1. Sample preparation and product analysis [1-14C]-PGH2 was synthesized by ovine COX-1 using 5 ml of [1-14C] arachidonic acid (Amersham Pharmacia; 55 mCi/mmol, 10 mCi/200 ml) mixed with 1 mg nonradiolabelled arachidonic acid (NuCheck) as the substrate. PGH2 was purified by a normal phase silica HPLC column as described previously [26]. About 50 zebrafish embryos were collected at different developmental stages. Each batch of embryos was washed three times with 0.2 ml PBS and resuspended in 0.2 ml of PBS. The embryos were lysed by sonication using a micro-tip cell disruptor (Model W225R, Heat System-Ultrosonics Inc.) with two cycles of 10-s burst/10-s cooling. After sonication, samples were centrifuged at 12,000g for 5 min at 4 1C. The supernatant was collected as embryo homogenate. A total of 10 ml of [1-14C]-PGH2 (1.4 mM) were added into embryo homogenate (200 ml). After incubation for 20 min at 23 1C, the reaction was stopped by adding 10 ml of 6 N HCl. Products were extracted three times each with three volumes of ether. Ether was evaporated under nitrogen. The products were then re-dissolved in 200 ml of methanol and separated on a Waters HPLC system
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equipped with a reverse phase C18 column (Waters, 5 mm, 3.9 150 mm). The column was run isocratically with acetonitrile/water/acetic acid (35:65:0.1, v/v/v) at a flow rate of 0.5 ml/min. Elutes were analyzed sequentially through a UV detector (monitored at 200 nm) and a b-RAM Model 2 b-detector (IN/US system). Nonradioactive primary eicosanoid standards (Cayman Chemical; 50 ml diluted with 150 ml of methanol) were resolved by the HPLC. The retention time of 6-keto PGF1a, TXB2, PGF2a, PGE2, and PGD2 was 3.9, 5.7, 7.0, 8.5, and 9.9 min, respectively. 2.2. Sequence analysis and reverse transcriptionpolymerase chain reaction (RT-PCR) The cDNAs for cytosolic and membrane-bound PGE synthases were published previously [24]. Search for zebrafish PGI synthase in the GenBank obtained four overlapping EST clones (fl20d03.X1, fl18d03.x1, fl20d03.y1 and fl18d03.y1). The sequence contig revealed an open reading frame corresponding to human PGI synthase from the 166th amino acid residue to the carboxyl terminus (500th amino acid residue). BLAST search using the EST clones of PGI synthase against zebrafish genomic sequence revealed a sequence contig on chromosome 6 (positions 28989758–28975989) that contains five exons. The genomic structures are conserved between the human and zebrafish PGI synthase genes [27], suggesting that the EST clones are authentic PGI synthase. Likewise, zebrafish TXA synthase cDNA (NM_205609) and gene (GeneID: 402902) were found in GenBank. Although the cDNA has not been expressed to demonstrate its enzymatic activity, the genomic structure of zebrafish TXA synthase is identical to the human counterpart [28], again suggesting an ortholog of TXA synthase. Similarly, the cDNA of lipocalin-type PGD synthase was obtained from GenBank (GeneID: AB059623). Total RNAs from different stages of embryos were prepared using a ToTALLY RNA kit (Ambion). RTPCR was carried out using a Superscript one-step RTPCR kit (Invitrogen). Briefly, 10 ml of the reaction buffer contained 23 ng of total RNA, 0.5 mM each of upstream and downstream primers, 5 ml of 2X Reaction Mix and 0.3 ml of RT/Platinum Tag Mix. Reaction was carried out at 46 1C for 40 min and 95 1C for 2 min, followed by 30 cycles (or 25 cycles for PGD synthase) of PCR (94 1C for 30 s, 56 1C for 30 s and 72 1C for 30 s). The final amplified product was extended at 72 1C for 7 min before loaded on a 2% agarose gel. The primers were designed based on the sequences deposited in the GenBank. For cytosolic PGE synthase (GeneID:AY724692), upstream primer, 50 -CAGAACCTGGCAAGTCTTGG-30 , and downstream primer, 50 CTCTTCTTCATCTGCACCATCC-30 , generated a 200-bp PCR product. For membrane-bound PGE
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synthase (GeneID:AY724691), upstream primer, 50 GCACTAAAAGCACCGACACG-30 , and downstream primer, 50 -ACTGGGTCATGCGAATGAGG-30 , generated a 108-bp PCR product. For lipocalin-type PGD synthase, upstream primer, 50 -GAAGATGGGAACTGCCATGC-30 , and downstream primer, 50 -CGACCATTTCTGGAGTGCGA-30 , generated a 284-bp PCR product. For PGI synthase, upstream primer, 50 -CTGAAGCTCTGAGGGCTGTG-30 , and downstream primer, 50 -ATCAGGTTTGGCATATTACC-30 , generated a 589bp PCR product. For TXA synthase, upstream primer, 50 -ATGAATTCTTCAGCAGACAC-30 , and downstream primer, 50 -ACTTAGAAGAACATTCCAAG30 , generated a 539-bp PCR product.
3. Results and discussion Zebrafish embryos at different embryonic stages were collected and subjected to sonication. Cell debris was removed by low-speed centrifugation, and supernatant was used as the embryo homogenate. Because arachidonic acid can also be used by lipoxygenase to generate leukotrienes, we thus used [1-14C]-PGH2 as the substrate and incubated it with embryo homogenates to obtain PGs. The PGs were extracted with ether and resolved by HPLC over a reverse phase C18 column. Extraction efficiency for each PG was independently examined using the eicosanoid mixture containing all five PGs (6keto PGF1a, TXB2, PGF2a, PGE2 and PGD2). About 90% of each PG was recovered, indicating that ether extraction is a suitable procedure for comparative studies. Radioactivity of individual PG synthesized from exogenous PGH2 at a given embryonic stage was calculated as percentage of the total PGs recovered. We examined embryonic stages over a period of five days at which time egg yolk is almost consumed, larva displays active movements and starts looking for food. Thus, it is considered that, at the day 5, morphogenesis is complete. PGD2 and PGE2 appear to be the major PGs (35%) at the first 10 h of embryos (Fig. 1). PGE2 remained at this level throughout 5 days post-fertilization (dpf). Similarly, PGF2a remained at 10–20% during the 5-day period. 6-Keto PGF1a, the hydrolyzed product of PGI2, also appeared at early stages of embryo and increased after 12 hpf. TXB2, the hydrolyzed product of TXA2, was produced as the least PG in the first 24 h of embryo. Compared with 6-keto PGF1a, TXB2 is produced at comparable levels only at later stages (440 hpf). All the five PGs were not found from the boiled cell extracts (data not shown), indicating the PGs we examined are indeed enzymatic products. To substantiate these findings, we carried out RTPCR to examine the temporal gene expression of zebrafish PG-synthesizing enzymes whose cDNAs are presently available in the GenBank. These cDNAs
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Fig. 1. Percentage of PGs synthesized by embryonic homogenates of different developmental stages. Each PG synthesized from exogenous PGH2 was quantitated, as described in ‘‘Section 2’’, and calculated as the percentage of total PGs. PGs are 6-keto PGF1a (filled circles), thromboxane B2 (open circles), PGF2a (inverted filled triangles), PGE2 (open triangles) and PGD2 (filled squares).
included cytosolic and membrane-bound PGE synthases, lipocalin-type PGD synthase, PGI synthase and TXA synthase. Equal amounts (23 ng) of total RNA from 7-, 24-, 48-, 72-, 96- and 120-hpf embryos were subjected to RT-PCR analyses. As shown in Fig. 2A, expression of PGD synthase is evident during the 5 days of embryogenesis but is markedly higher at 7 hpf. This result is consistent with the observation that enzymatic activities of PGD synthase are higher at early embryonic stages. For cytosolic PGE synthase, its expression could be readily detected through the developmental stages (Fig. 2B). Similarly, transcript of membrane-bound PGE synthase was detectable during the period of 5 dpf, yet, 1- and 2-dpf embryos gave significantly higher levels of the expression (Fig. 2C). It is unclear which type of PGE synthase plays a major role in synthesizing PGE2. However, considering that PGE2 is the most abundant PG produced during embryogenesis, it is anticipated that PGE synthase is critical for development. Indeed, knockdown of membrane-bound PGE synthase resulted in severe morphogenetic defects during gastrulation (5–10 hpf) [25]. Lower dosage of the knockdown embryos exhibited a mispositioned head and shortened anterioposterior axis, whereas higher dosage caused a complete gastrulation arrest. It should be noted that, in all cases, increasing PCR cycles enhanced the absolute intensities but retained the relative intensities of the amplified DNAs from different developmental stages (data not shown), indicating that our PCR conditions were within the range of linear amplification. We then examined PGI synthase and TXA synthase. Because the two respective enzymatic products carry out
opposite physiological functions, one would expect the two gene transcripts emerge at the same time. As shown in Fig. 2D and E, the expression of PGI synthase is clearly seen at 24 hpf, whereas the transcript of TXA synthase is seen at 48 hpf but became apparent at 72 hpf. In fact, the transcript of PGI synthase can be detected as early as 7 hpf. This is consistent with previous report in which PGI2 was produced about five-fold as much as TXA2 in the 7-hpf zebrafish embryos [20]. In view of the fact that the ratio of PGI2 and TXA2 plays an important role in hemostasis, our results suggested that PGI2 has additional functions because the ratio of PGI2/TXA2 is higher at the early stage of embryogenesis. Moreover, because blood vessels of zebrafish are formed at 24– 26 hpf [29], the advent of significant amounts of PGI2 produced within 24 hpf again suggested that PGI2 may play an important role in the earlier stage of embryogenesis. PGI2 has been demonstrated to activate peroximal proliferators-activated receptor d in vivo [30,31], eliciting its functions on embryo implantation, vascular angiogenesis and apoptosis [32]. It is intriguing to explore the role of PGI2 in the embryonic development. A surprising finding in this study is that the capability of embryo to synthesize PGD2 decreases as embryo matures (Fig. 3). The decay follows single-exponential kinetics with a rate constant of 0.048 h1. In contrast to our result, previous report by Cha et al showed that at 7 hpf PGD2 was undetectable [20]. It should be noted that Cha et al. extracted PGs from embryo for quantitative analysis, while we added PGH2 to the embryonic homogenates to synthesize PGs for analysis. It is plausible that PGD2 is short-lived whereas other PGs are accumulated in vivo, making PGD2 difficult to be detected from embryo extract. If this is the case, the accumulation of PGD2 cannot reflect the capability of PGD synthase at a given embryonic stage. Regardless, our results clearly support the notion that PGD synthase is highly regulated during embryogenesis and PGD2 plays an important role in the early embryonic development. The physiological roles of PGD2 are remarkably versatile. Two distinctive types of receptors, D type of PG receptor and CRTH2 receptor [33], are known to perform the signaling of PGD2. Furthermore, PGD2 can be metabolized to produce J series of PGs in vivo, such as PGJ2, D12-PGJ2 and 15-deoxy-D12,14-PGJ2. 15-Deoxy-D12, 14-PGJ2 is a potent ligand of peroximal proliferators-activated receptor g involving in differentiation of adipocytes and inhibition of NF-kB-dependent gene expression [34]. It is unclear at present the developmental roles of PGD2 in the mouse. Neither is known about what type of PGD2 receptors or synthases is participating in embryonic development. Unlike PGI2 or TXA2, PGD2 is stable in aqueous solution, making it possible to be transported from the mother. With the advantage of the oviparous embryos and availability of
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(E) Fig. 2. Appearance of the transcripts of (A) lipocalin-type PGD synthase, (B) cytosolic PGE synthase, (C) membrane-bound PGE synthase, (D) PGI synthase and (E) TXA synthase at different developmental stages. Reverse transcription was performed using 23 ng of total RNA from 7-(lane 2), 24(lane 3), 48-(lane 4), 72-(lane 5), 96-(lane 6) and 120-hpf (lane 7) embryos. Twenty-five cycles of PCR was carried out for PGD synthase and 30 cycles for the others. Amplified DNA fragments were separated on a 2% agarose gel. The sizes of markers (lane 1) in bp are indicated. 50
gene knockdown techniques, zebrafish will provide a definite role of PGD2 in the embryonic development of vertebrates.
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We thank Dr. Xinping Zhao at the Department of Ophthalmology for providing zebrafish embryos. This work is supported by Grant HL60625 from the National Institutes of Health
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Prostaglandins, Leukotrienes and Essential Fatty Acids 75 (2006) 397–402 www.elsevier.com/locate/plefa
Profiling of prostanoids in zebrafish embryonic development Hui-Chun Yeh, Lee-Ho Wang Division of Hematology, Department of Internal Medicine, University of Texas Health Science Center, Houston, Texas 77030, USA Received 3 January 2006; received in revised form 3 July 2006; accepted 2 August 2006
Abstract Prostanoids (PG) play important roles in vascular, pulmonary, reproductive and renal physiology. Little is known about their roles in the embryonic development. Using the oviparous zebrafish embryo as a model, we determined the temporal expression of PGs synthesized from exogenous prostaglandin H2. Prostaglandin E2 is the major PG throughout first 120 h post-fertilization (hpf), whereas prostaglandin F2a is at a lower but also a constant level. Reverse transcription-polymerase chain reaction (RT-PCR) showed that transcripts of cytosolic and membrane-bound PGE synthases were evident during the 120 hpf period. Compared with thromboxane A2, the level of prostacyclin (PGI2)is higher at first 24 hpf, the stage before the formation of blood vessel. RT-PCR showed that transcript of prostacyclin synthase appeared at 7 hpf whereas thromboxane synthase appeared at 48 hpf, suggesting that PGI2 has additional functions besides hemostasis. Interestingly, level of prostaglandin D2 (PGD2) followed an exponential decay over 120 hpf with a rate constant of 0.048 h1 and transcript of lipocalin-type PGD synthase was expressed at a higher level at early stage of development, suggesting that PGD2 is highly regulated during embryogenesis. r 2006 Elsevier Ltd. All rights reserved.
1. Introduction Prostanoids (PGs) possess potent biological activities in vascular, pulmonary, reproductive and renal physiology as well as in pathophysiology of inflammation, thrombosis and cancer [1]. Their biosynthesis is initiated by the action of phospholipase to release arachidonic acid from membrane phospholipids, followed by the action of prostaglandin H synthase (also known as cyclooxygenase, COX). Two different COX isozymes, COX-1 and COX-2, encoded by separate genes, have been identified [2]. Their common product, prostaglandin H2 (PGH2), is an unstable compound with a half-life of 10 min in aqueous solution [3]. PGH2 is used by the Abbreviations: PG; prostanoid; COX; cyclooxygenase; PGD2; prostaglandin D2; PGE2; prostaglandin E2; PGF2a; prostaglandin F2a; PGI2; prostacyclin; TXA2; thromboxnae A2; hpf; hour postfertilization; dpf; day post-fertilization; RT-PCR reverse transcriptionpolymerase chain reaction Corresponding author. Tel.: +1 713 500 6794; fax: +1 713 500 6810. E-mail address: [email protected] (L.-H. Wang). 0952-3278/$ - see front matter r 2006 Elsevier Ltd. All rights reserved. doi:10.1016/j.plefa.2006.08.001
specific enzymes for the formation of each PG. The type and quantity of the terminal PGs generated appear to be governed by the specific PG-synthesizing enzyme present in a given cell. PGs then exert their actions through specific receptors on plasma membranes [4]. In mammals, the major terminal PGs are prostaglandin D2 (PGD2), prostaglandin E2 (PGE2), prostaglandin F2a (PGF2a), prostaglandin I2 (PGI2, also known as prostacyclin (PGI2) and thromboxane A2 (TXA2) [5]. PGD2 is a major PG synthesized in the central nervous system and is involved in the regulation of sleep and pain responses [6]. It is also actively produced by mast cells, basophils and Th2 cells, acting as an allergic and inflammatory mediator [7]. Two distinctive types of PGD synthase have been identified: one is the lipocalintype enzyme and the other, the hematopoietic enzyme. PGE2 is the most common PG and is ubiquitously produced in the body. It has four subtypes of PGE receptors to carry out its diversified functions including pain, fever, inflammation, ovulation, fertilization and bone metabolism [8]. There are also two forms of PGE synthases derived from separate genes: cytosolic form
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and membrane-bound form. PGF2a is an inducer of leuteolysis and has been implicated in parturition [9]. PGI2 is a potent vasodilator and inhibits platelet aggregation [10]. In contrast, TXA2 is a potent inducer of vasoconstriction and platelet aggregation. The balance of PGI2 and TXA2 is therefore crucial for hemostasis. Both PGI2 and TXA2 are very unstable in water and are non-enzymatically converted to 6-keto PGF1a and TXB2, respectively. Their synthesizing enzymes, PGI synthase and TXA synthase, are members of cytochrome P450 [11]. PGI synthase is mainly present in endothelial and smooth muscle cells, whereas TXA synthase is present in platelets, monocytes and macrophages. The physiological roles of PGs were also explored from the studies of mice lacking the specific PGsynthesizing enzyme and/or specific PG receptor. In general, these knockout genes are non-lethal and the knockout mice reveal minor phenotypes [12]. For example, both TXA synthase- and TXA2 receptorknockout mice revealed mild bleeding disorders and altered vascular responses to arachidonic acid [13,14]. PGI2 receptor-knockout mice suffered obstructive thrombi upon FeCl3-induced endothelial injury [15], and 6-month-old PGI synthase-knockout mice showed vascular disorders with the thickening of vascular walls and interstitial fibrosis [16]. Lipocalin-type PGD synthase-knockout mice failed to respond to a touchevoked pain, but had a normal hyperalgesia response [17]. Interestingly, the knockout mice were also found to be glucose-intolerant and insulin-resistant on a low fat diet [18]. In addition, the lipocalin-type PGD synthaseknockout mice developed nephropathy and an aortic thickening on a high-fat diet, suggesting that PGD2 plays an important role in the vasculature, especially in atherosclerosis and diabetes. Zebrafish has been used as a model to study embryonic development of COX-1 and COX-2 [19,20]. COX-1 serves as a house-keeping function and is a constitutive enzyme, whereas COX-2 is highly regulated by growth factor, cytokine and mitogen. COX-1 is found ubiquitously early in the zebrafish embryos during blastula and gastrula stages. During somitogenesis, COX-1 is enriched in the posterior intermediate mesoderm, and at 24 h post-fertilization (hpf) it is confined to the distal end of nephric duct and developing vasculature. Knockdown of COX-1 by a morphonino oligonucleotide resulted in gastrulation arrest or defect in vascular tube formation. Conversely, knockdown of COX-2 caused no noticeable phenotype. These findings are in sharp contrast to those obtained with knockout mice. COX-1 knockout mice survived well and were fertile [21]. However, COX-2 knockout mice suffered higher mortality due to gastric peritonitis or kidney failure [22,23]. The COX-2 knockout females also revealed multiple reproductive defects including
ovulation, fertilization, implantation and decidualization. These controversial results can be explained by the possibility that PG knockout mice acquire maternal PG to allow normal development of embryos in uterus. Therefore, the physiological roles of PGs may not reflect the developmental roles of PGs in the knockout mice. In this regard, zebrafish as a vertebrate is well suited for studying the embryonic roles of PGs because the embryos develop externally from maternal body. In order to fully exploit the advantages of zebrafish for studying the developmental roles of PGs, it needs information of the specific PG-synthesizing enzymes and PG receptors in this species to enable detection of gene expression and examination of gene-perturbed phenotypes. Zebrafish PGE synthase, both cytosol and membrane-bound forms, is the only terminal PGsynthesizing enzyme that was reported [24,25]. As an onset to further understand the developmental roles of PGs, we report here the biosynthetic capacity of PGs in the zebrafish embryonic stages. PGE2 is the major PG throughout the embryonic stages. However, at the early developmental stages, PGI2 is synthesized at a higher level than TXA2, suggesting an important role of PGI2 besides hemostasis. While most PGs remain at the fairly constant levels during embryogenesis, levels of PGD2 biosynthesis decreases as embryo matures, suggesting an important role of PGD2 in the early stage of embryo development.
2. Materials and methods 2.1. Sample preparation and product analysis [1-14C]-PGH2 was synthesized by ovine COX-1 using 5 ml of [1-14C] arachidonic acid (Amersham Pharmacia; 55 mCi/mmol, 10 mCi/200 ml) mixed with 1 mg nonradiolabelled arachidonic acid (NuCheck) as the substrate. PGH2 was purified by a normal phase silica HPLC column as described previously [26]. About 50 zebrafish embryos were collected at different developmental stages. Each batch of embryos was washed three times with 0.2 ml PBS and resuspended in 0.2 ml of PBS. The embryos were lysed by sonication using a micro-tip cell disruptor (Model W225R, Heat System-Ultrosonics Inc.) with two cycles of 10-s burst/10-s cooling. After sonication, samples were centrifuged at 12,000g for 5 min at 4 1C. The supernatant was collected as embryo homogenate. A total of 10 ml of [1-14C]-PGH2 (1.4 mM) were added into embryo homogenate (200 ml). After incubation for 20 min at 23 1C, the reaction was stopped by adding 10 ml of 6 N HCl. Products were extracted three times each with three volumes of ether. Ether was evaporated under nitrogen. The products were then re-dissolved in 200 ml of methanol and separated on a Waters HPLC system
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equipped with a reverse phase C18 column (Waters, 5 mm, 3.9 150 mm). The column was run isocratically with acetonitrile/water/acetic acid (35:65:0.1, v/v/v) at a flow rate of 0.5 ml/min. Elutes were analyzed sequentially through a UV detector (monitored at 200 nm) and a b-RAM Model 2 b-detector (IN/US system). Nonradioactive primary eicosanoid standards (Cayman Chemical; 50 ml diluted with 150 ml of methanol) were resolved by the HPLC. The retention time of 6-keto PGF1a, TXB2, PGF2a, PGE2, and PGD2 was 3.9, 5.7, 7.0, 8.5, and 9.9 min, respectively. 2.2. Sequence analysis and reverse transcriptionpolymerase chain reaction (RT-PCR) The cDNAs for cytosolic and membrane-bound PGE synthases were published previously [24]. Search for zebrafish PGI synthase in the GenBank obtained four overlapping EST clones (fl20d03.X1, fl18d03.x1, fl20d03.y1 and fl18d03.y1). The sequence contig revealed an open reading frame corresponding to human PGI synthase from the 166th amino acid residue to the carboxyl terminus (500th amino acid residue). BLAST search using the EST clones of PGI synthase against zebrafish genomic sequence revealed a sequence contig on chromosome 6 (positions 28989758–28975989) that contains five exons. The genomic structures are conserved between the human and zebrafish PGI synthase genes [27], suggesting that the EST clones are authentic PGI synthase. Likewise, zebrafish TXA synthase cDNA (NM_205609) and gene (GeneID: 402902) were found in GenBank. Although the cDNA has not been expressed to demonstrate its enzymatic activity, the genomic structure of zebrafish TXA synthase is identical to the human counterpart [28], again suggesting an ortholog of TXA synthase. Similarly, the cDNA of lipocalin-type PGD synthase was obtained from GenBank (GeneID: AB059623). Total RNAs from different stages of embryos were prepared using a ToTALLY RNA kit (Ambion). RTPCR was carried out using a Superscript one-step RTPCR kit (Invitrogen). Briefly, 10 ml of the reaction buffer contained 23 ng of total RNA, 0.5 mM each of upstream and downstream primers, 5 ml of 2X Reaction Mix and 0.3 ml of RT/Platinum Tag Mix. Reaction was carried out at 46 1C for 40 min and 95 1C for 2 min, followed by 30 cycles (or 25 cycles for PGD synthase) of PCR (94 1C for 30 s, 56 1C for 30 s and 72 1C for 30 s). The final amplified product was extended at 72 1C for 7 min before loaded on a 2% agarose gel. The primers were designed based on the sequences deposited in the GenBank. For cytosolic PGE synthase (GeneID:AY724692), upstream primer, 50 -CAGAACCTGGCAAGTCTTGG-30 , and downstream primer, 50 CTCTTCTTCATCTGCACCATCC-30 , generated a 200-bp PCR product. For membrane-bound PGE
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synthase (GeneID:AY724691), upstream primer, 50 GCACTAAAAGCACCGACACG-30 , and downstream primer, 50 -ACTGGGTCATGCGAATGAGG-30 , generated a 108-bp PCR product. For lipocalin-type PGD synthase, upstream primer, 50 -GAAGATGGGAACTGCCATGC-30 , and downstream primer, 50 -CGACCATTTCTGGAGTGCGA-30 , generated a 284-bp PCR product. For PGI synthase, upstream primer, 50 -CTGAAGCTCTGAGGGCTGTG-30 , and downstream primer, 50 -ATCAGGTTTGGCATATTACC-30 , generated a 589bp PCR product. For TXA synthase, upstream primer, 50 -ATGAATTCTTCAGCAGACAC-30 , and downstream primer, 50 -ACTTAGAAGAACATTCCAAG30 , generated a 539-bp PCR product.
3. Results and discussion Zebrafish embryos at different embryonic stages were collected and subjected to sonication. Cell debris was removed by low-speed centrifugation, and supernatant was used as the embryo homogenate. Because arachidonic acid can also be used by lipoxygenase to generate leukotrienes, we thus used [1-14C]-PGH2 as the substrate and incubated it with embryo homogenates to obtain PGs. The PGs were extracted with ether and resolved by HPLC over a reverse phase C18 column. Extraction efficiency for each PG was independently examined using the eicosanoid mixture containing all five PGs (6keto PGF1a, TXB2, PGF2a, PGE2 and PGD2). About 90% of each PG was recovered, indicating that ether extraction is a suitable procedure for comparative studies. Radioactivity of individual PG synthesized from exogenous PGH2 at a given embryonic stage was calculated as percentage of the total PGs recovered. We examined embryonic stages over a period of five days at which time egg yolk is almost consumed, larva displays active movements and starts looking for food. Thus, it is considered that, at the day 5, morphogenesis is complete. PGD2 and PGE2 appear to be the major PGs (35%) at the first 10 h of embryos (Fig. 1). PGE2 remained at this level throughout 5 days post-fertilization (dpf). Similarly, PGF2a remained at 10–20% during the 5-day period. 6-Keto PGF1a, the hydrolyzed product of PGI2, also appeared at early stages of embryo and increased after 12 hpf. TXB2, the hydrolyzed product of TXA2, was produced as the least PG in the first 24 h of embryo. Compared with 6-keto PGF1a, TXB2 is produced at comparable levels only at later stages (440 hpf). All the five PGs were not found from the boiled cell extracts (data not shown), indicating the PGs we examined are indeed enzymatic products. To substantiate these findings, we carried out RTPCR to examine the temporal gene expression of zebrafish PG-synthesizing enzymes whose cDNAs are presently available in the GenBank. These cDNAs
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Fig. 1. Percentage of PGs synthesized by embryonic homogenates of different developmental stages. Each PG synthesized from exogenous PGH2 was quantitated, as described in ‘‘Section 2’’, and calculated as the percentage of total PGs. PGs are 6-keto PGF1a (filled circles), thromboxane B2 (open circles), PGF2a (inverted filled triangles), PGE2 (open triangles) and PGD2 (filled squares).
included cytosolic and membrane-bound PGE synthases, lipocalin-type PGD synthase, PGI synthase and TXA synthase. Equal amounts (23 ng) of total RNA from 7-, 24-, 48-, 72-, 96- and 120-hpf embryos were subjected to RT-PCR analyses. As shown in Fig. 2A, expression of PGD synthase is evident during the 5 days of embryogenesis but is markedly higher at 7 hpf. This result is consistent with the observation that enzymatic activities of PGD synthase are higher at early embryonic stages. For cytosolic PGE synthase, its expression could be readily detected through the developmental stages (Fig. 2B). Similarly, transcript of membrane-bound PGE synthase was detectable during the period of 5 dpf, yet, 1- and 2-dpf embryos gave significantly higher levels of the expression (Fig. 2C). It is unclear which type of PGE synthase plays a major role in synthesizing PGE2. However, considering that PGE2 is the most abundant PG produced during embryogenesis, it is anticipated that PGE synthase is critical for development. Indeed, knockdown of membrane-bound PGE synthase resulted in severe morphogenetic defects during gastrulation (5–10 hpf) [25]. Lower dosage of the knockdown embryos exhibited a mispositioned head and shortened anterioposterior axis, whereas higher dosage caused a complete gastrulation arrest. It should be noted that, in all cases, increasing PCR cycles enhanced the absolute intensities but retained the relative intensities of the amplified DNAs from different developmental stages (data not shown), indicating that our PCR conditions were within the range of linear amplification. We then examined PGI synthase and TXA synthase. Because the two respective enzymatic products carry out
opposite physiological functions, one would expect the two gene transcripts emerge at the same time. As shown in Fig. 2D and E, the expression of PGI synthase is clearly seen at 24 hpf, whereas the transcript of TXA synthase is seen at 48 hpf but became apparent at 72 hpf. In fact, the transcript of PGI synthase can be detected as early as 7 hpf. This is consistent with previous report in which PGI2 was produced about five-fold as much as TXA2 in the 7-hpf zebrafish embryos [20]. In view of the fact that the ratio of PGI2 and TXA2 plays an important role in hemostasis, our results suggested that PGI2 has additional functions because the ratio of PGI2/TXA2 is higher at the early stage of embryogenesis. Moreover, because blood vessels of zebrafish are formed at 24– 26 hpf [29], the advent of significant amounts of PGI2 produced within 24 hpf again suggested that PGI2 may play an important role in the earlier stage of embryogenesis. PGI2 has been demonstrated to activate peroximal proliferators-activated receptor d in vivo [30,31], eliciting its functions on embryo implantation, vascular angiogenesis and apoptosis [32]. It is intriguing to explore the role of PGI2 in the embryonic development. A surprising finding in this study is that the capability of embryo to synthesize PGD2 decreases as embryo matures (Fig. 3). The decay follows single-exponential kinetics with a rate constant of 0.048 h1. In contrast to our result, previous report by Cha et al showed that at 7 hpf PGD2 was undetectable [20]. It should be noted that Cha et al. extracted PGs from embryo for quantitative analysis, while we added PGH2 to the embryonic homogenates to synthesize PGs for analysis. It is plausible that PGD2 is short-lived whereas other PGs are accumulated in vivo, making PGD2 difficult to be detected from embryo extract. If this is the case, the accumulation of PGD2 cannot reflect the capability of PGD synthase at a given embryonic stage. Regardless, our results clearly support the notion that PGD synthase is highly regulated during embryogenesis and PGD2 plays an important role in the early embryonic development. The physiological roles of PGD2 are remarkably versatile. Two distinctive types of receptors, D type of PG receptor and CRTH2 receptor [33], are known to perform the signaling of PGD2. Furthermore, PGD2 can be metabolized to produce J series of PGs in vivo, such as PGJ2, D12-PGJ2 and 15-deoxy-D12,14-PGJ2. 15-Deoxy-D12, 14-PGJ2 is a potent ligand of peroximal proliferators-activated receptor g involving in differentiation of adipocytes and inhibition of NF-kB-dependent gene expression [34]. It is unclear at present the developmental roles of PGD2 in the mouse. Neither is known about what type of PGD2 receptors or synthases is participating in embryonic development. Unlike PGI2 or TXA2, PGD2 is stable in aqueous solution, making it possible to be transported from the mother. With the advantage of the oviparous embryos and availability of
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(E) Fig. 2. Appearance of the transcripts of (A) lipocalin-type PGD synthase, (B) cytosolic PGE synthase, (C) membrane-bound PGE synthase, (D) PGI synthase and (E) TXA synthase at different developmental stages. Reverse transcription was performed using 23 ng of total RNA from 7-(lane 2), 24(lane 3), 48-(lane 4), 72-(lane 5), 96-(lane 6) and 120-hpf (lane 7) embryos. Twenty-five cycles of PCR was carried out for PGD synthase and 30 cycles for the others. Amplified DNA fragments were separated on a 2% agarose gel. The sizes of markers (lane 1) in bp are indicated. 50
gene knockdown techniques, zebrafish will provide a definite role of PGD2 in the embryonic development of vertebrates.
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We thank Dr. Xinping Zhao at the Department of Ophthalmology for providing zebrafish embryos. This work is supported by Grant HL60625 from the National Institutes of Health
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