Essential role of prostaglandin E2 and the EP3 receptor in lymphatic vessel development during zebrafish embryogenesis

Lymphatic endothelial cells arise from the venous endothelial cells in embryonic lymphatic development. However, the molecular mechanisms remain to be elucidated. We here report that prostaglandin (PG) E2 plays essential roles in the embryonic lymphatic development through the EP3 receptor, one of the PGE2 receptors. Knockdown of the EP3 receptor or inhibition of cyclooxygenases (COX; rate-limiting enzymes for PG synthesis) impaired lymphatic development by perturbing lymphatic specification during zebrafish development. These impairments by COX inhibition were recovered by treatment with sulprostone (EP1/3 agonist). Knockdown of the EP3 receptor further demonstrated its requirement in the expression of sex determining region Y-box 18 (sox18) and nuclear receptor subfamily 2, group F, member 2 (nr2f2), essential factors of the lymphatic specification. The EP3 receptor was expressed in the posterior cardinal vein (region of embryonic lymphatic development) and the adjacent intermediate cell mass (ICM) during the lymphatic specification. COX1 was expressed in the region more upstream of the posterior cardinal vein relative to the EP3 receptor, and the COX1-selective inhibitor impaired the lymphatic specification. On the other hand, two COX2 subtypes did not show distinct sites of expression around the region of expression of the EP3 receptor. Finally, we generated EP3-deficient zebrafish, which also showed defect in lymphatic specification and development. Thus, we demonstrated that COX1-derived PGE2-EP3 pathway is required for embryonic lymphatic development by upregulating the expression of key factors for the lymphatic specification.

Prostaglandin (PG) E 2 is an arachidonate metabolite that is synthesized via a pathway with cyclooxygenase (COX) as the rate-limiting enzyme. PGE 2 has been shown to exert a variety of actions by binding to four specific G-protein-coupled receptors (EP1, EP2, EP3, and EP4) on the plasma membrane of neighboring cells in humans and mice 18,19 . The EP1 receptor is coupled to Gq to increase intracellular Ca 2+ concentrations. The EP2 and EP4 receptors are coupled to Gs and increase intracellular cAMP concentrations via the activation of adenylyl cyclase. The EP3 receptor is coupled to Gi, and mediates the inhibition of adenylyl cyclase and the upregulation of intracellular Ca 2+ concentrations 20,21 . The formation of lymphatic vessels is known to occur during not only embryogenesis but also the progression of various cancers 22 , and COX2-derived PGE 2 was found to facilitate lymphangiogenesis during tumor development through the EP3 receptor in mice 23,24 . In addition, PGE 2 was shown to accelerate the formation of lymphatic vessels through the EP3 receptor in granulation tissues 25,26 . These data suggest the possibility that the PGE 2 -EP3 pathway plays important roles in the formation of lymphatic vessels. However, it remains unknown whether the PGE 2 -EP3 pathway is involved in the development of lymphatic vessels during embryogenesis.
To identify the role of the PGE 2 -EP3 pathway in lymphatic vessel development during embryogenesis, we used zebrafish as a model organism, because zebrafish embryos are optically clear and undergo rapid early development outside the maternal body 27 . In addition, zebrafish share many similarities in their molecular mechanisms of lymphatic vessel formation with other vertebrates, and express three COX subtypes (COX1, COX2a, and COX2b) and eight PGE 2 receptor subtypes (EP1a, EP1b, EP2a, EP2b, EP3, EP4a, EP4b, and EP4c) [28][29][30][31] . Here, we report novel functions of the PGE 2 -EP3 pathway in the formation of lymphatic vessels during early development.

the pGe 2 -EP3 pathway is involved in lymphatic vessel formation during early development.
To investigate whether the PGE 2 -EP3 pathway regulates lymphatic vessel formation in embryogenesis, we analyzed the effects of EP3 receptor knockdown on zebrafish lymphatic development using Tg(fli1a:egfp) embryos, in which both blood and lymphatic vessels are labeled 27,32 . We used two splice-blocking morpholino antisense oligos (MOs), EP3 MO1 and MO2 ( Supplementary Fig. S1A, Supplementary Table S1). Quantitative analysis showed that injection of EP3 MO1 or MO2 both markedly decreased the mature mRNA expression level of the EP3 receptor at 24 hours post fertilization (hpf) (Supplementary Fig. S1B). To evaluate lymphatic vessel formation, we analyzed the parachordal lymphangioblast (PL) at the horizontal myoseptum, which is commonly used to study lymphatic development in zebrafish 9,27 . In control (Cont) MO-injected embryos, the PL was fully formed in most of the segments at 52 hpf (Fig. 1A,B). On the other hand, PL formation was severely impaired in morphants injected with EP3 MO1 or MO2 (Fig. 1A), and the ratio of PL-positive segments was also significantly reduced in these morphants (Fig. 1B). We then investigated whether indomethacin, a non-selective COX inhibitor 28 , induces lymphatic vessel defects. In vehicle-treated embryos, PL was normally formed in the horizontal myoseptum at 52 hpf. On the other hand, treatment with indomethacin inhibited PL formation and significantly reduced the ratio of PL-positive segments (Fig. 1C,D). This effect of indomethacin was substantially recovered by cotreatment with sulprostone, an EP1/3 agonist 30 (Fig. 1C,D). Furthermore, we investigated the formation of thoracic duct (TD), which is located immediately ventral to the dorsal aorta in the trunk, at 5 days post fertilization (dpf) (Fig. 1E,F). In Cont MO-injected embryos, the TD was fully formed in most of the segments. On the other hand, TD formation was severely impaired in morphants injected with EP3 MO1 and MO2 (Fig. 1E), and the ratio of TD-positive segments was significantly reduced in the EP3 MO1-injected morphants (Fig. 1F). These results indicated that the PGE 2 -EP3 pathway plays an important role in lymphatic vessel formation during early development. the pGe 2 -EP3 pathway is required for lymphatic specification from venous to lymphatic endothelial cells. Lymphatic endothelial cells are generated from pre-existing endothelial cells of the posterior cardinal vein from approximately 24 to 36 hpf, and subsequently sprout from the posterior cardinal vein after 36 hpf and migrate to colonize embryonic tissues. To identify the lymphatic development process in which the PGE 2 -EP3 pathway is involved, we performed time-dependent inhibition of PG synthesis by treatment with indomethacin for a limited time ( Fig. 2A). There was no significant difference in the ratios of PL-positive segments between vehicle treatment and indomethacin treatment from 12 to 24 hpf, when the posterior cardinal vein develops. On the other hand, indomethacin treatment from 24 to 36 hpf significantly reduced the ratio of PL-positive segments. The degree of reduction by the treatment from 24 to 36 hpf was equivalent to that by the treatment from 24 to 60 hpf. These data suggest the importance of the PGE 2 -EP3 pathway in the lymphatic specification process. Therefore, to determine whether the PGE 2 -EP3 pathway acts during the first steps of lymphatic development, we analyzed the expression levels of lyve1b, which is a lymphatic marker 13 . Compared with control morphants, EP3 receptor morphants had significantly lower expression levels of lyve1b at both 24 and 36 hpf (Fig. 2B). Whole-mount in situ hybridization (WISH) analysis demonstrated that lyve1b was expressed around the posterior cardinal vein in Cont MO-injected embryos at both 24 and 36 hpf (Fig. 2C,D). By contrast, embryos injected with EP3 MO1 showed substantial decreases in lyve1b-derived signals around the posterior cardinal vein at both time points (Fig. 2C,D). Embryos treated with indomethacin also showed decreased expression levels of lyve1b at 24 hpf (Fig. 2E). This effect of indomethacin was significantly recovered by cotreatment with sulprostone but not ONO-AE1-259 (an EP2 agonist) 31 or ONO-AE1-329 (an EP4 agonist) 31 (Fig. 2E). WISH analysis of embryos at 24 hpf demonstrated that indomethacin markedly reduced lyve1b-derived signals around the posterior cardinal vein, where lyve1b-derived signals were observed in vehicle-treated embryos (Fig. 2F). Lyve1b-derived signals that were decreased by indomethacin were recovered to the levels similar to that of vehicle-treated embryos by the cotreatment of sulprostone (Fig. 2F). These results indicated that the PGE 2 -EP3 pathway contributes to the formation of lymphatic vessels by regulating the lymphatic specification, which is the first step of lymphatic development during embryogenesis.
www.nature.com/scientificreports www.nature.com/scientificreports/  www.nature.com/scientificreports www.nature.com/scientificreports/ Expression levels of genes involved in the lymphatic specification. To analyze the function of the PGE 2 -EP3 pathway in the lymphatic specification, we investigated the mRNA expression level of various genes (vegfc, flt4, sox18, nr2f2, apln, ccbe1, wnt5b, and bmp2b) crucially involved in the lymphatic specification 4-10,14-17 . By reverse transcription-quantitative PCR (RT-qPCR), we analyzed the expression levels of these genes in EP3 receptor morphants at 24 and 36 hpf (Fig. 3A-H), which are early and late phases of lymphatic specification, respectively. At both 24 and 36 hpf, expression levels of sox18 were significantly decreased in EP3 receptor morphants compared with control morphants (Fig. 3C). Expression levels of nr2f2 at 36 hpf were also significantly decreased in EP3 receptor morphants, although expression levels of nr2f2 at 24 hpf did not change (Fig. 3D). There was no significant difference in the expression levels of vegfc, flt4 (also known as a vein marker at 24 hpf), apln, ccbe1, wnt5b, and bmp2b (Fig. 3A,B,E-H). Because nr2f2 is expressed in not only the posterior cardinal vein but also the cranial and spinal cord 10 , we then examined the expression of nr2f2 around the posterior cardinal vein at 24 and 36 hpf by WISH analysis (Fig. 3I,J). Signals of nr2f2 were detected in the posterior cardinal vein and spinal cord in the trunk of Cont MO-injected embryos. In the trunk of EP3 MO1-injected embryos, nr2f2-derived signals were decreased only in the posterior cardinal vein but not the spinal cord, specifically in 36 hpf but not 24 hpf. These data indicated that the EP3 receptor plays important roles in the expression of sox18 and nr2f2 in the lymphatic specification. www.nature.com/scientificreports www.nature.com/scientificreports/ COX1-derived PGE 2 regulates the lymphatic specification through the EP3 receptor. We performed WISH analysis to identify the sites of EP3 receptor expression during the lymphatic specification. As previously reported 33 , EP3 receptor-derived signals were detected around blood vessels of the trunk at 24 hpf (Fig. 4A). To analyze these expression sites in more detail, we compared the signals of the EP3 receptor with those of several tissue markers at 24 hpf, namely, hes-related family bHLH transcription factor with YRPW motif 2 (hey2; an aorta marker) 34 , flt4 (a vein marker) 35 and GATA binding protein 1a (gata1a; an erythrocyte progenitor marker) 36 , which is reported to be exclusively expressed in the intermediate cell mass (ICM) between the dorsal aorta and the posterior cardinal vein at this developmental stage 36 . The signals of the EP3 receptor were located ventral to those of hey2 and around those of flt4 and gata1a (Fig. 4A). Furthermore, we made transverse sections of these embryos that were subjected to WISH analysis (Fig. 4B). As previously reported 34 , hey2-derived signals were detected under the notochord in transverse sections. Signals of flt4 were located in a more ventral region than signals of hey2, and signals of gata1a were observed in a region between those of hey2 and flt4. EP3 receptor-derived signals appeared to be located in the region with both flt4-derived and gata1a-derived signals. These results indicated that the EP3 receptor is expressed in the ICM and the posterior cardinal vein, where lymphatic endothelial cells are generated.
COX are rate-limiting enzymes for the biosynthesis of PGE 2 . As zebrafish have three subtypes of COX (COX1, COX2a, and COX2b), we tried to identify the specific COX subtypes involved in the lymphatic specification. We first performed WISH analysis at 24 hpf to identify the region of expression of each COX subtype during the lymphatic specification. COX1-derived signals were detected in the region more upstream of the posterior cardinal vein relative to EP3 receptor-derived signals (Fig. 5A). On the other hand, COX2a-and COX2b-derived distinctive signals were not detected in the trunk of zebrafish embryos (Fig. 5A). To clarify whether COX1 is involved in the lymphatic specification, we treated zebrafish embryos with a COX1-selective inhibitor, SC-560 37 and investigated the expression levels of lyve1b at 24 hpf using WISH analysis. SC-560 diminished lyve1b-derived signals around the posterior cardinal vein (Fig. 5B). The decrease in lyve1b-derived signals was recovered by cotreatment with sulprostone (Fig. 5B). These results suggested that COX1-derived PGE 2 plays important roles in the lymphatic specification from venous to lymphatic endothelial cells during early development.

Impairment of lymphatic specification and development by EP3 receptor deficiency.
To confirm the role of the EP3 receptor in the lymphatic specification, we finally generated EP3 receptor-deficient (EP3 −/− ) zebrafish using transcription activator-like effector nucleases (TALEN) 38 . Sequencing analysis showed that a stop codon was introduced into the transmembrane II region of the wild-type EP3 receptor by the deletion of seven base-pairs in transmembrane I (Fig. 6A). EP3 +/− and EP3 −/− zebrafish were born at approximately expected Mendelian ratio and were viable (Fig. 6B). We then performed the Ca 2+ mobilization assay to confirm whether this deletion actually results in a loss of the function of the EP3 receptor. Although HeLa cells transfected with a construct encoding the wild-type EP3 receptor dose-dependently induced Ca 2+ mobilization by sulprostone, HeLa cells transfected with a construct encoding the mutant EP3 receptor did not respond to sulprostone (Fig. 6C). Subsequently, we investigated the expression levels of lyve1b in EP3 +/− and EP3 −/− zebrafish to evaluate the effects of EP3 receptor deficiency on the lymphatic specification. Compared with EP3 +/+ zebrafish, EP3 −/− zebrafish but not EP3 +/− zebrafish had significantly lower expression levels of lyve1b at 24 hpf (Fig. 6D). Additionally, WISH analysis showed that the expression levels of lyve1b around the posterior cardinal vein were remarkably decreased in EP3 −/− zebrafish but not EP3 +/− zebrafish at 36 hpf (Fig. 6E). Finally, we investigated the expression of lyve1b at 52 hpf using WISH analysis to analyze PL formation in EP3 −/− zebrafish (Fig. 6F). In EP3 +/+ zebrafish, lyve1b signals were detected in the horizontal myoseptum, indicating adequate PL formation. On the other hand, signals of lyve1b were not detected in EP3 −/− zebrafish, indicating failure of PL formation. These results indicated the importance of EP3 receptor expression in lymphatic specification and development. Thus, the defects of lymphatic specification and development observed upon EP3 receptor knockdown and inhibition of PG synthesis were also observed in EP3 receptor gene-deficient mutants. www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
In this study, we showed for the first time that the PGE 2 -EP3 pathway plays an essential role in embryonic lymphatic development, by regulating the lymphatic specification, which is the first step of embryonic lymphatic development (Figs 1, 2, 5, 6). We found that the EP3 receptor is important for the expression of sox18 and nr2f2 but not for vegfc, flt4, apln, ccbe1, wnt5b, and bmp2b (Fig. 3). The EP3 receptor was expressed in the posterior cardinal vein and the neighboring ICM (Fig. 4). On the other hand, COX1, but not two COX2 subtypes, was expressed in the region more upstream of the posterior cardinal vein relative to the region of expression of the EP3 receptor, and contributed to lymphatic specification (Fig. 5). Lymphatic vessels develop from endothelial cells in the posterior cardinal vein. Therefore, normal development of the posterior cardinal vein is important for lymphatic vessel development 2,3 . Our data using RT-qPCR and WISH analyses indicated that the expression levels of flt4, used as a maturation marker of venous endothelial cells, were not altered in EP3 receptor morphants at 24 hpf, when the posterior cardinal vein is fully developed (Fig. 3 and data not shown). Our observation in Tg(fli1a:egfp) zebrafish also showed that the posterior cardinal vein of EP3 receptor morphants was similar to that of control morphants at both 24 and 36 hpf (data not shown). Additionally, lymphatic vessel development was not impaired by the inhibition of PG synthesis from 12 to 24 hpf, when the posterior cardinal vein develops ( Fig. 2A). These data indicated that the EP3 receptor does not affect the development of PCV, at least on evaluation of lymphatic development. However, further detailed studies are needed to precisely determine whether the PGE 2 -EP3 pathway is involved in the differentiation and/or maturation of the posterior cardinal vein, at a level at which there is no effect on lymphatic development.
We found that the EP3 receptor plays important roles in the expression levels of sox18 and nr2f2 (Fig. 3). These genes are transcriptional factors that facilitate the lymphatic specification [8][9][10] . It was reported that sox18 genetically interacts with vegfc in the early phase of lymphatic development 9 , and that sox18 is required for the expression of nr2f2 in zebrafish at 24 hpf 11 . Therefore, most of the effects of the EP3 receptor on the lymphatic specification might be exerted by the regulation of sox18 expression. These two transcriptional factors are expressed in the posterior cardinal vein 10,39 , where embryonic lymphatic development begins. Interestingly, EP3 receptors were found to be expressed in the posterior cardinal vein and the neighboring ICM (Fig. 4). These results suggested that the functions of the EP3 receptor in lymphatic specification were exerted directly (in the posterior cardinal vein) or indirectly (by certain secreted or plasma membrane-associated factors supplied from the adjacent ICM through a paracrine or juxtacrine route). Then, we investigated in vitro whether the EP3 receptor expressed in venous endothelial cells accelerates differentiation toward lymphatic endothelial cells as a direct consequence of endothelial cell-autonomous activation of the EP3 receptor. However, stimulation by the selective human EP3 receptor agonist ONO-AE-248 did not upregulate the expression levels of the lymphatic marker LYVE1 in human umbilical vein endothelial cells (HUVECs), even when the human EP3 receptor was overexpressed in HUVECs (data not shown). On the other hand, expression levels of secreted regulatory factors such as apln, ccbe1, wnt5b, and bmp2b were not affected by knockdown of the EP3 receptor at both 24 and 36 hpf (Fig. 3). Additionally, there have been no reports to our knowledge regarding plasma membrane-localized molecules that are involved in the lymphatic specification. Further studies are required to fully understand the molecular mechanism of the lymphatic specification promoted by the PGE 2 -EP3 pathway.
In this study, we found that COX1-derived PGE 2 accelerated the lymphatic specification during embryonic lymphatic development (Fig. 5), and the EP3 receptor had no effect on the expression levels of vegfc and flt4 (Fig. 3). In contrast to our study, COX2-derived PGE 2 was reported to accelerate lymphangiogenesis through the EP3 receptor in tumor implantation and granulation formation models 23,25,26 . Mice and human cells were used in these models and PGE 2 upregulated the expression of Vegfc and Flt4 through the EP3 receptor. Although the www.nature.com/scientificreports www.nature.com/scientificreports/ www.nature.com/scientificreports www.nature.com/scientificreports/ reason for these differences is unclear, a likely explanation is that these discrepancies may be owing to differences of venous endothelial cell types among species and tissues, and differences between embryos and adults. Additionally, the different results might be explained by the absence or presence of inflammation. In tumor implantation and granulation formation models, inflammation was induced and immune cells invaded the tissues. It has been reported that migrated macrophages produce Vegfc through the PGE 2 -EP3 pathway in granulation formation models 25,26 . On the other hand, there have been no reports stating that immune cells are involved in embryonic lymphatic development, particularly in lymphatic specification. Sox18 was reported to be critical for tumor-induced lymphangiogenesis 40 . It is therefore possible that the PGE 2 -EP3 pathway might promote lymphangiogenesis in tumors and granulation tissues by the upregulation of not only Vegfc and Flt4 expression but also Sox18 expression.
In summary, we found that the PGE 2 -EP3 pathway plays crucial roles in the lymphatic specification from venous to lymphatic endothelial cells through the upregulation of sox18 and nr2f2. These data strongly suggest a novel function of COX1-PGE 2 -EP3 pathway in the formation of the lymphatic system during early development.

Materials and Methods
Materials. The following materials were obtained from the sources indicated; PGE 2 , sulprostone, and SC-560 Zebrafish line and maintenance. A wild-type zebrafish strain was obtained from National BioResource Project Zebrafish (RIKEN, Japan). The transgenic zebrafish line Tg(fli1a:egfp) has been described previously 27,32 , and was used to monitor lymphatic development. Zebrafish were maintained at 28.5 °C under a 14 h-light/10 h-dark cycle. Embryos were maintained in 1/3 ringer solution buffer at 28.5 °C. All experimental protocols were approved by Kumamoto University (24-060, F29-195). All experiments with zebrafish were performed in accordance with the guidelines of Kumamoto University.
Morpholino antisense oligos. EP3 MO1 (targeting the splice site between exon 1 and intron 1), EP3 MO2 (targeting the splice site between intron 1 and exon 2), and Cont MO were purchased from Gene Tools, LLC (Philomath, OR). Each MO (10 ng) was injected into the yolk of 1-2 cell stage embryos. The sequence of each MO is shown in Supplementary Table S1. RNA extraction and RT-qPCR. Total RNA was extracted from zebrafish embryos at the indicated stages using Sepasol RNA I Super G (Nacalai Tesque, Kyoto, Japan), and was subjected to RT with PrimeScript RT Master Mix (Takara Bio, Shiga, Japan). Synthesized cDNA was subjected to qPCR using a LightCycler (Roche Applied Science, Penzberg, Germany) and Fast Start DNA Master SYBR Green I according to the manufacturer's instructions. Crossing point values were acquired by the second derivative maximum method. The expression level of each gene was quantified using external standardized dilutions. Relative expression levels among samples were normalized by the value of gapdh. Sequences of the used primers are shown in Supplementary Table S2. The specificity of qPCR was confirmed by the lengths and melting temperatures of the amplified products.
Whole-mount in situ hybridization. Total RNA was isolated from zebrafish embryos, and cDNA was synthesized using SuperScript III (Invitrogen, San Diego, CA) and oligo (dT) primers. The coding sequence of each gene (lyve1b, nr2f2, hey2, and flt4,) were amplified from the cDNA by PCR and cloned into the pTA2 vector (Toyobo, Osaka, Japan). Primer sequences used in the PCR are shown in Supplementary Table S3. Cloning of the coding sequences of gata1a, the EP3 receptor, COX1, COX2a, and COX2b was performed as previously described 20,41 . These plasmids were linearized by restriction enzymes. Digoxigenin (DIG)-labeled anti-sense RNA was transcribed from each linearized vector by in vitro transcription using DIG mix and transcription buffer (Roche Diagnostics). These RNA was purified by ethanol precipitation, dissolved in hybridization buffer (50% formamide, 5× SSC, 5 mM ethylenediaminetetraacetic acid, 0.1% Tween-20, and 1 mg/mL torula RNA), and used as hybridization probes. WISH was performed as previously described 41 . After fixation by 4% paraformaldehyde, zebrafish embryos at 52 hpf were incubated in 3% hydrogen peroxide solution to remove dark pigments. Embryos stained with WISH were embedded in OCT Compound (Sakura, Tokyo, Japan) and transverse sections (10-20 μm) were prepared using a cryostat (Leica Microsystems, Wetzlar, Germany). Images were taken with a fluorescence microscope (BZ-X700; KEYENCE, Osaka, Japan) and were processed using the attached software or Adobe Photoshop (Adobe, San Jose, CA).
Generation of EP3 receptor-mutant zebrafish. TALEN plasmids were constructed using a two-step assembly system, as described previously 42 . Briefly, six or fewer TAL effector repeat domains were ligated into the pFUS vector 43 . Subsequently, each pFUS vector and last TAL effector repeat were ligated into the pCS2 vector as a TALEN plasmid 44 . The TALEN plasmids were linearized by Not I digestion, and TALEN mRNA was transcribed using the mMESSAGE mMACHINE SP6 kit (Life Technologies, Gaithersburg, MD) and purified using the RNeasy Mini Kit (QIAGEN, Hilden, Germany). Forward and reverse TALEN mRNA (400 pg each) was simultaneously injected into zebrafish blastomeres at the one-cell stage. To detect genome modification in zebrafish, we utilized the heteroduplex mobility assay as reported previously 45 . The sequences of the primers used in the heteroduplex mobility assay are shown in Supplementary Table S4. To check the functional ability of the TALEN-induced EP3 receptor mutant, the Ca 2+ mobilization assay was performed using the FLIPR Calcium 5 Assay Kit (Molecular Devices, Sunnyvale, CA) according to the manufacturer's instructions. HeLa cells were