Molecular characterization of the prostaglandin E receptor subtypes 2a and 4b and their expression patterns during embryogenesis in zebrafish

The molecular expression profiles of zebrafish ep2a and ep4b have not been defined to-date. Phylogenetic trees of EP2a and EP4b in zebrafish and other species revealed that human EP4 and zebrafish EP4b were more closely related than EP2a. Zebrafish EP2a is a 281 amino acid protein with high identity to that of human (43%), mouse (44%), rat (43%), dog (44%), cattle (41%), and chicken (41%). Zebrafish EP4b encoded a precursor of 497 amino acids with high amino acid identity to that of mammals, including human (57%), mouse (54%), rat (55%), dog (55%), cattle (56%), and chicken (54%). Whole-mount in situ hybridization revealed that ep2a was robustly expressed in the anterior four somites at the 10-somites stages, but was absent in the somites at 19 hpf. It was observed again in the pronephric duct at 24 hpf, in the intermediate cell mass located in the trunk, and in the rostral blood island at 30 hpf. Ep2a was also expressed in the notochord at 48 hpf. During somitogenesis, ep4b was highly expressed in the eyes, somites, and the trunk neural crest. From 30 to 48 hpf, ep4b could be detected in the posterior cardinal vein and the neighboring ICM. From these data, we conclude that ep2a and ep4b are conserved in vertebrates and that the presence of ep2a and ep4b transcripts during developmental stages infers their role during early zebrafish larval development. In addition, the variable expression of the two receptor isoforms was strongly suggestive of divergent roles of molecular regulation.


Introduction
Prostaglandin E2 (PGE2) is an important arachidonate metabolite that regulates an array of physiological processes in vertebrates, including immune responses (Kutyrev et al. 2017), gastrointestinal function (Takeuchi and Amagase 2018), testicular homeostasis (Rey-Ares et al. 2018), and ovulation (Baker and Van Der Kraak 2019). PGE2 binds to four specific Gprotein-coupled receptors (EP1, EP2, EP3, and EP4) in humans and mice (Ball et al. 2013;Kimple et al. 2013;Zhu et al. 2018). EP1 is coupled to Gq to elevate intracellular Ca 2+ , EP3 couples to Gi to inhibit adenylyl cyclase (AC) and cyclic AMP (cAMP), and both EP2 and EP4 couple to Gs to stimulate AC and increase cAMP (Samuchiwal et al. 2017;Wang et al. 2010), and have been identified as crucial mediators of PGE2 in both physiological and pathophysiological processes, including closure of the ductus arteriosus (Sakuma et al. 2018;Yokoyama et al. 2014), bone formation (Graham et al. 2009), ovulation and fertilization (Niringiyumukiza et al. 2018) and salt sensitive hypertension (Kennedy et al. 1999). Knockdown of EP2a results in smaller embryonic livers in zebrafish (Nissim et al. 2014). In medaka, impairment of the EP4b receptor in vitro using a competitive antagonist resulted in anovulation (Fujimori et al. 2012).
Though both EP2 and EP4 were identified in mammals (Kershaw-Young et al. 2009;Kowalewski et al. 2008;Reitmair et al. 2010) and chickens (Kwok et al. 2008), their expression has not been investigated in fish. Zebrafish (Danio rerio) are a vertebrate model organism that is widely used for genetic and pharmacological analysis of embryogenesis due to their high fecundity and translucency (Akhter et al. 2016;Ellertsdottir et al. 2010). In addition, a number of disease models have been developed in zebrafish that can be combined with in vivo imaging approaches to monitor specific pathological processes, including cardiovascular disease and cancer metastasis (Bournele and Beis 2016;Tulotta et al. 2016). In this regard, it has been shown that prostaglandin receptors play a key role in zebrafish development, including ovulation and T cell precursor development (Baker and Van Der Kraak 2019;Villablanca et al. 2007). However, the expression of zebrafish ep2a and ep4b has not been systematically defined in the literature.
Here, we report the expression patterns of ep2a and ep4b during zebrafish embryogenesis. We show that ep2a is robustly expressed in the somites, the pronephric duct, ICM, and notochord, whilst ep4b is highly expressed in the somites, the trunk neural crest, the posterior cardinal vein and the neighboring ICM. These data reveal important information regarding the expressional characteristics of zebrafish ep2a and ep4b during developmental stages. Differences in the expression of these transcripts during development indicate differential molecular regulatory patterns.

Zebrafish models
Embryos of AB wild type zebrafish were raised and staged as described by Kimmel et al (Kimmel et al. 1995). Embryos were maintained in E3 solution at 28.5°C with 0.003% 1phenyl-2-thiourea (Sigma) to inhibit pigmentation. Embryos were staged according to somite number or hours post-fertilization (hpf).

Whole mount in situ hybridization
Antisense probes for ep2a and ep4b were synthesized using DIG RNA labeling kit (Roche, US). Standard procedures were performed as per the manufacturer's recommendations. Whole mount in situ hybridizations was performed as described by Thisse et al (Thisse and Thisse 2014). Embryos were imaged on a Zeiss confocal microscope. In situ experiments were performed on a minimum of three independent occasions using 20 embryos.

Phylogenetic tree of EP2a in different species
As shown in Figure 2, a close distance between chicken EP2 and zebrafish EP2a was observed. Interestingly, the distance between human EP2 and zebrafish EP2a was less close. Analysis of the phylogenetic tree indicated that the zebrafish EP2a receptor branched from its ancestor. Zebrafish EP2a receptors similarly diverged from their ancestors. This branching may have occurred due to the different ligand recognition properties of the EP2 receptors. (Homo sapiens EP2: NM_000956.1), mouse (Mus musculus EP2: NM_008964.1), rat (Rat EP2: NM_031088.1), dog (Canis lupus EP2: NM_001003170.1), cattle (Bos taurus EP2: AF539402.1) and chicken (Gallus gallus EP2: EF200120.1). The seven putative transmembrane domains are shaded and labeled. Sequences underlined and in bold represent the third intracellular loop (IC3). Three highly variable regions (I, II, and III) between zebrafish and other species are boxed and labeled. Arrow heads indicate the conserved threonine. Two cysteine residues forming disulphide bonds are boxed. Dots indicate amino acids that are not present within the sequence.

Fig 2.
Phylogenetic tree of EP2 across different species constructed using the Neighborjoining method. An evolutionary relationship of EP2 in different species, including human, mouse, rat, dog, cattle, chicken and zebrafish were observed. Numbers adjacent to the branch points indicate bootstrap values.

Fig 4.
Phylogenetic tree of EP4 across the indicated species. The phylogenetic tree was constructed using the Neighbor-joining method. Evolutionary relationships of EP4 in different species, including human, mouse, rat, dog, cattle, chicken and zebrafish. Numbers adjacent to the branch points indicate bootstrap values.

Expression of zebrafish ep2a during early embryogenesis
Whole mount in situ hybridization was used to determine the temporal and spatial expression patterns of ep2a. At the 10-somites stage (14 hpf), robust ep2a expression was observed in the anterior four somites ( Figure 5A). Interestingly, at 19 hpf, ep2a could no longer be detected in the somites, although its expression was diffuse in the anterior region of the embryo ( Figure 5B). At 24 hpf, ep2a expression was observed in the pronephric duct, and diffusely in the posterior region of the trunk ( Figure 5C). At 30 hpf, ep2a was maintained in the ICM of the trunk and in the rostral blood island ( Figure 5D). At 48 hpf, ep2a was strongly expressed in the notochord, but weakly expressed in the blood ( Figure 5E).

Zebrafish ep4b expression during early embryogenesis
Whole mount in situ hybridization was used to investigate the temporal and spatial expression pattern of ep4b. During the shield stage, ep4b was present in the germ ring, showing peak expression in the embryonic shield ( Figure 6A). At the end of gastrulation, ep4b became more localized to the posterior region of the embryo ( Figure 6B). The dorsal view showed that the expression was arranged in two sides (ellipse) of the prechordal plate hypoblast ( Figure 6C), in which the cells contribute to the posterior trunk, somites, and neural (Kimmel et al. 1995). During somitogenesis, ep4b was robustly expressed in the eyes, somites, and the trunk neural crest ( Figure 6D and E). At 24 hpf, ep4b remained highly expressed in the somites and the trunk neural crest ( Figure 6F). Ep4b expression in the trunk neural crest continued to the 30 hpf stage ( Figure 6G). From 30 to 48 hpf, ep4b could be detected in the posterior cardinal vein and the neighboring ICM ( Figure 6G and H), as reported for ptger4a (Baker and Van Der Kraak 2019).

Discussion
In this study, we show that ep2a is robustly expressed in the anterior four somites at 14 hpf, but is absent in the somites at 19 hpf. At later stages, ep2a could be observed in the pronephric duct, ICM, and in the rostral blood island between 24 to 30 hpf. Robust expression was observed in the notochord at 48 hpf. Interestingly, the expression of ep2a and ep4b differed during zebrafish development. During somitogenesis, ep4b was present in the eyes, somites, and the trunk neural crest. From 30 to 48 hpf, ep4b was present in the posterior cardinal vein and the neighboring ICM. Variations in the expression of these transcripts during developmental stages suggested different modes of molecular regulation.
Species comparison of EP2 and EP4 showed that EP2a and EP4b showed high homology across mammalian and non-mammalian vertebrate species, including human (43%; 57%, respectively), mouse (44%; 54%, respectively), rat (43%; 55%, respectively), dog (44%; 55%, respectively), cattle (41%; 56%, respectively), and chicken (41%; 54%, respectively), as shown in Figure 1 and 3. Of note, the distance between human EP4 and zebrafish EP4b was close compared to human and zebrafish IP receptors. Interestingly, the distance between human EP4 and zebrafish EP4c was less close (Tsuge et al. 2013). Consistent with these data, ONO-AE1-329, an agonist specifically designed for human EP4, showed high activity against zebrafish EP4b but low activity against the EP4c receptor (Stillman et al. 1998). Thus, zebrafish EP4 receptors and their interaction with these compounds were closely related to the structural conservation of the human receptor. Compared with EP4 and IP clusters, EP2 receptors showed higher differentiation. The distance between human EP2 and zebrafish EP2a was relatively large.
EP2 and EP4 belong to the GPCR superfamily and can be subdivided into rhodopsin receptor members. Both share common characteristics. Phylogenetic trees with bootstrap values for EP2 and EP4 were constructed using the Neighbor-joining method (Figure 2 and  4), and indicated that both EP2 and EP4 are closely related to their corresponding counterparts in mammalian species and chicken. This suggested that the cloned receptors are potential EP2 and EP4 receptors in zebrafish. Two highly conserved cysteines present amongst GPCRs (Stillman et al. 1998) with proposed roles in disulphide bond formation were located as Cys110 and Cys188 in EP2, and Cys122 and Cys200 in EP4 (Figure 1 and  3). A threonine residue that is conserved amongst EP2 and EP4 has also been suggested to be important for ligand binding (Stillman et al. 1998), and was identified in the cloned receptors (Thr186 in EP2 and Thr198 in EP4) (Figure 1 and 3). Despite these similarities, EP2 and EP4 showed several distinct differences. As in mammals (Desai et al. 2000), EP4 was longer than EP2 (497 a.a. vs. 281 a.a.), with the major differences observed in the lengths of the 3rd intracellular loops and intracellular C-termini (Figure 1 and 3). The longer C-terminus of EP4 possesses a number of putative phosphorylation sites that undergo short term ligand-induced receptor internalization and desensitization. This was suggestive of potential functional differences in signal transduction between EP2 and EP4 in response to their ligands (Davidson and Zon 2004;Sugimoto and Narumiya 2007).
Using whole mount in situ hybridization, ep2a and ep4b expression were analyzed in the zebrafish embryos. Robust expression of ep2a was observed in the anterior four somites at 14 hpf ( Figure 5A), indicating its relationship to the development stages of somatic cells. At 24 hpf, ep2a expression was observed in the pronephric duct, mainly and in the posterior region of the trunk with diffuse but ubiquitous expression ( Figure 5C). At 30 hpf, ep2a is expressed in the ICM and is located in the trunk and in the rostral blood island ( Figure 5D), whilst definitive hemopoiesis occurred in the DA-PCV joint region, which is equivalent to the AGM region (Villablanca et al. 2007). Similarly, pges was expressed in the endothelium, and cox-2 was expressed in the wall of the aortic arch (Pini et al. 2005). The expression patterns of ep2a and cox-2 and pges-1 in zebrafish highlight their potential during vascular development and ability to modulate key components of these developmental stages, including VEGF. During somitogenesis, ep4b was expressed in the eyes, somites, and the trunk neural crest ( Figure 6D and E). At 24 hpf, ep4b remained highly expressed in the somites and the trunk neural crest ( Figure 6F). In addition, at 30 hpf, ep4b was expressed in the trunk neural crest ( Figure 6G). These data suggest that ep4b plays an important role in the development of somites and the trunk neural crest. From 30 to 48 hpf, ep4b expression could be detected in the posterior cardinal vein and the neighboring ICM ( Figure 6G and H), and was similar to that observed for ep4b (Baker and Van Der Kraak 2019). In addition, cox-1 and cox-2 were expressed in the carotid arteries and the vasculature of the pharyngeal arches at 96 hpf (Ishikawa et al. 2007;Pini et al. 2005). This highlights a novel role for the PGE2 / EP4 axis during vascular development.

Conclusion
In this study, full-length cDNAs for zebrafish ep2a and ep4b were cloned and their expression patterns were characterized during zebrafish development. Ep2a was mainly expressed in the somites, pronephric duct, ICM, and notochord, while ep4b was expressed in the somites, the trunk neural crest, and ICM. The differences in expression between ep2a and ep4b transcripts during these developmental stages highlight divergent modes of molecular regulation during developmental stages.

Conflicts of interest
No conflict of interest to declare.