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Wnt8a is one of the candidate genes that play essential roles in the elongation of the seahorse prehensile tail

  • Corresponding author: Qiang Lin, linqiang@scsio.ac.cn
  • Received Date: 2020-09-06
    Accepted Date: 2021-01-08
    Published online: 2021-06-29
  • Edited by Jiamei Li.
  • Seahorses are a hallmark of specialized morphological features due to their elongated prehensile tail. However, the underlying genomic grounds of seahorse tail development remain elusive. Herein, we evaluated the roles of essential genes from the Wnt gene family for the tail developmental process in the lined seahorse (Hippocampus erectus). Comparative genomic analysis revealed that the Wnt gene family is conserved in seahorses. The expression profiles and in situ hybridization suggested that Wnt5a, Wnt8a, and Wnt11 may participate in seahorse tail development. Like in other teleosts, Wnt5a and Wnt11 were found to regulate the development of the tail axial mesoderm and tail somitic mesoderm, respectively. However, a significantly extended expression period of Wnt8a during seahorse tail development was observed. Signaling pathway analysis further showed that Wnt8a up-regulated the expression of the tail axial mesoderm gene (Shh), while interaction analysis indicated that Wnt8a could promote the expression of Wnt11. In summary, our results indicate that the special extended expression period of Wnt8a might promote caudal tail axis formation, which contributes to the formation of the elongated tail of the seahorse.
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Wnt8a is one of the candidate genes that play essential roles in the elongation of the seahorse prehensile tail

    Corresponding author: Qiang Lin, linqiang@scsio.ac.cn
  • 1. CAS Key Laboratory of Tropical Marine Bio-Resources and Ecology, South China Sea Institute of Oceanology, Chinese Academy of Sciences, Guangzhou 510275, China
  • 2. Institution of South China Sea Ecology and Environmental Engineering, Chinese Academy of Sciences, Guangzhou 510275, China
  • 3. Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou 511458, China
  • 4. School of Life Sciences, University of Science and Technology of China, Hefei 230026, China
  • 5. University of Chinese Academy of Sciences, Beijing 100049, China

Abstract: Seahorses are a hallmark of specialized morphological features due to their elongated prehensile tail. However, the underlying genomic grounds of seahorse tail development remain elusive. Herein, we evaluated the roles of essential genes from the Wnt gene family for the tail developmental process in the lined seahorse (Hippocampus erectus). Comparative genomic analysis revealed that the Wnt gene family is conserved in seahorses. The expression profiles and in situ hybridization suggested that Wnt5a, Wnt8a, and Wnt11 may participate in seahorse tail development. Like in other teleosts, Wnt5a and Wnt11 were found to regulate the development of the tail axial mesoderm and tail somitic mesoderm, respectively. However, a significantly extended expression period of Wnt8a during seahorse tail development was observed. Signaling pathway analysis further showed that Wnt8a up-regulated the expression of the tail axial mesoderm gene (Shh), while interaction analysis indicated that Wnt8a could promote the expression of Wnt11. In summary, our results indicate that the special extended expression period of Wnt8a might promote caudal tail axis formation, which contributes to the formation of the elongated tail of the seahorse.

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Introduction
  • Seahorses (genus Hippocampus) are known for their unique morphological features, such as an elongated and prehensile tail, and ovoviviparous reproductive strategies (Li et al. 2020; Lin et al. 2016; Neutens et al. 2014; Zhang et al. 2020). The seahorse tail is regarded as a remarkable evolutionary innovation, as it is not used for rapid swimming but, instead, as a flexible appendage for grasping (Ashley-Ross 2002; Porter et al. 2015). The seahorse tail is finless, square-prism shaped, elongated, flexible, and prehensile, enabling it to hold on to objects such as sea grasses, coral reefs, and mangrove roots (Hale 1996; Neutens et al. 2014). In the lined seahorse (Hippocampus erectus), the number of tail somites (34–39, most 36) is significantly higher than that of the trunk (11) (Lourie et al. 2004). The ratio of tail to trunk somites (36/11) is significantly higher than that of common fish, such as zebrafish (14/18) (Kimmel et al. 1995) and medaka (11/24) (Iwamatsu 2004). The length of the seahorse tail accounts for almost half of the whole body length, and the tail shows obviously elongated tail segments compared with those of other teleosts (Neutens et al. 2014; Porter et al. 2015; Praet et al. 2012). Embryology studies have shown that the specialized tail morphological feature of seahorses is established during embryogenesis. According to standardized classification, seahorse embryogenesis can be divided into four sequential stages: early-embryogenesis, mid-embryogenesis (or eye development), late-embryogenesis (or snout formation), and juvenile stages (Sommer et al. 2012; Whittington et al. 2015). The seahorse tail developmental process occurs throughout these four stages, such that the tail bud primordium forms in the early-embryogenesis stage and tail somite extension mainly occurs during the following two developmental stages (Novelli et al. 2018; Sommer et al. 2012).

    Vertebrate tails are formed with an anterior–posterior (A–P) progression that is driven by a population of multipotent precursors at the tail bud (Cambray and Wilson 2002; Charrier et al. 1999; Gont et al. 1993). Data from diverse deuterostomes (amphioxus, fish, frog, chicken, and mouse) suggest that the fate of the stem cells at the tail bud is orchestrated by a tightly regulated interplay of several signaling pathways (Aires et al. 2018; Benazeraf and Pourquie 2013; Thorpe et al. 2005). The crosstalk among these signaling pathways provides positional information and induces cell differentiation (Huelsken and Birchmeier 2001; Nelson and Nusse 2004; Nusse 2008; Ryan and Baxevanis 2007; Saito-Diaz et al. 2013). Crucial mediators of this cell-to-cell communication process are the Wnt ligands (Huelsken and Birchmeier 2001; Kestler and Kuhl 2008; Krausova and Korinek 2014; Moon et al. 1997; Ramel et al. 2005). Wnt ligands regulate the expression of several genes that are involved in several developmental processes, from axis formation to tissue generation (Klaus and Birchmeier 2008). They function via different Wnt signaling pathways, which are categorized into the canonical Wnt signaling pathway (or Wnt/β-catenin signaling pathway) and non-canonical Wnt signaling pathway (including the Wnt/Ca2+ and the Wnt/PCP signaling pathways) groups (Reya and Clevers 2005). Wnt signaling pathways form an ancient system that has been highly conserved during evolution (Angers and Moon 2009; Wiese et al. 2018).

    In vertebrates, tail development has been under investigation for decades, but the mechanism underlying tail diversification among species is largely unknown. The unique elongated and prehensile tail of the seahorse makes it the perfect model to elucidate the molecular mechanisms underlying the diverse tail development in vertebrates. The present study focused on elucidating the vital role of the Wnt gene family in the elongated tail formation in seahorses using a comparative genomic analysis approach by identifying potential key Wnt elements and molecular regulatory mechanisms involved in this process.

Results

    Wnt genes in lined seahorse

  • A total of 22 Wnt gene family members were identified from the whole-genome data of the lined seahorse (Supplementary Table S1). Similar to other teleosts, gene synteny analysis revealed that the genomic arrangements of Wnts were non-clustered in the seahorse and that most Wnts shared a genomic organization that was conserved during seahorse evolution (Supplementary Fig. S1). The Wnt locus length was variable. The exon numbers ranged from three to seven (Supplementary Fig. S2), which is comparable to those in other teleosts.

    Similar to other teleosts, the lengths of Wnt proteins in the seahorse ranged from 290 to 422 amino acids (Supplementary Table S1). The domain architecture of homologous Wnt proteins was also highly conserved in the seahorse. Although the N terminus was variable, a highly conserved "Wnt1" domain was observed at the C terminal of each Wnt protein (Supplementary Figs. S3, S4).

  • Evolution of Wnt genes

  • Wnt protein sequences from other eight representative vertebrate species available in the NCBI database were used to construct the Wnt phylogenetic tree. Similar to other vertebrates, the Wnt proteins of the seahorse could be subdivided into 12 subfamilies, including Wnt1–11 and Wnt16 (Fig. 1, Supplementary Fig. S5), but no seahorse-specific subfamily was identified. The subfamilies of Wnt1, Wnt11, and Wnt16 were single orthologs, a finding that was consistent among various vertebrates. The other Wnt subfamilies were multiple orthologs that contained two or three copies. Wnt orthologs from two seahorses showed the most distant evolutionary relationship within teleosts. Compared to other teleosts, the Wnt gene families in the seahorses H. erectus and H. comes were conserved, and no special expansion or contraction of Wnt gene families was observed.

    Figure 1.  The Wnt gene repertoire of several representative vertebrates. The tree on the left summarizes the phylogenetic relationships of the seahorse and seven additional vertebrates. Different Wnt subfamilies are delineated by gray background. A box indicates the presence of a member of a given Wnt subfamily in a particular group

  • Temporal expression of Wnt genes during seahorse embryo development

  • To screen the potential Wnts involved in seahorse tail development at the embryonic stages, the Wnt family expression profiles at four typical tail development stages were evaluated. The data revealed 22 Wnts that showed dynamic changes during seahorse embryo development. Interestingly, four Wnts—Wnt5a, Wnt8a, Wnt9b, and Wnt11—showed particular fluctuations during the developmental process (Fig. 2). Wnt8a was prominently expressed during the early- and mid-embryogenesis stages, and its expression gradually decreased during the following stages. In contrast, the expression of Wnt9b kept increasing throughout the embryo developmental process. Both Wnt5a and Wnt11 were prominently expressed during the mid- and late-embryogenesis stages, during which rapid seahorse tail axial growth and somite formation took place. The expression of two other Wnt genes, namely Wnt2bb and Wnt4b, also changed significantly during the embryo developmental stages, however, these two genes exclusively regulate liver specification and central nervous system development (Inohaya et al. 2010; Negishi et al. 2009; Ober et al. 2006). Therefore, we speculated that among members of the Wnt gene family, Wnt5a, Wnt8a, Wnt9b, and Wnt11 may be important in regulating seahorse tail development.

    Figure 2.  Expression profiles of Wnts in seahorse embryos at four typical tail developmental stages. qRT-PCR experiments were performed in triplicate for each developmental stage. The development stages are indicated at the left, and the relative expression levels of the 22 Wnts at each stage are shown at the right

  • In situ hybridization analysis of the key candidate Wnt genes

  • To test this hypothesis, the expression of the Wnts identified in specific morphological regions was evaluated. Whole-mount in situ hybridization results showed that three (Wnt5a, Wnt8a, and Wnt11) of the four candidate Wnts were highly expressed in the developing tails.

    Given that Wnt8a is a maternal gene (Hino et al. 2018), it was first observed at the animal pole of the early cleavage at the early-embryogenesis stage (Fig. 3a1), and it gradually accumulated in the primitive streak (Fig. 3b1). In the mid-embryogenesis stage, in which the tail bud primordium arises, a Wnt8a buildup was observed in the tail bud primordium, and Wnt8a continued to accumulate during tail segment outgrowth (Fig. 3c1, d1). In the late-embryogenesis stage, as the new tail somite formation gradually ended, the accumulation of Wnt8a in the tail bud gradually decreased (Fig. 3e1), until it was undetectable by the end of the new tail somite formation (Fig. 3f1, g1). Unlike in zebrafish, in which the expression of Wnt8a is limited to the blastula and gastrula (Lu et al. 2011; Shimizu et al. 2005), the expression of Wnt8a was significantly extended during tail development in the seahorse.

    Figure 3.  Whole-mount in situ hybridization analysis of Wnt5a, Wnt8a, and Wnt11 in lined seahorse embryos at different developmental stages. Arrowheads indicate Wnt accumulation in the tail. The boxes in the upper right corners of panels show the higher magnification of the tail. Embryos in a, a1, and a2 are shown with the animal pole up; embryos in b, b1, and b2 are shown with the primitive streak up; the dorsal view is shown for embryos in c2, with the head to the left; the remaining images show a lateral view, with the head to the left and dorsal side up. These images are representative of at least five individual specimens. Ear early-embryogenesis stage, Mid mid-embryogenesis stage, Lat late-embryogenesis stage, Juv Juvenile. Bar = 200 μm

    Interestingly, the expression pattern of Wnt5a partially overlapped with that of Wnt8a. During the early-embryogenesis stage, the accumulation of Wnt5a was detected at the animal pole of the blastomere, and later, it was detected in the primitive streak (Fig. 3a, (b). In the mid-embryogenesis stage, Wnt5a continuously accumulated in the tail bud primordium in a graded manner along the A–P axis, with the highest accumulation observed in the distal tail bud (Fig. 3c, d). During the late-embryogenesis stage, the accumulation of Wnt5a in the tail bud primordium gradually decreased (Fig. 3e, f). However, the accumulation of Wnt5a in the tail bud was maintained for longer than that of Wnt8a (Fig. 3eg). Moreover, Wnt5a also accumulated in abundance in other distal tissues that outgrow from the body axis, such as the dorsal fin bud, pectoral fin buds, and caudal end of the gut (Fig. 3eg, Supplementary Fig. S6).

    Wnt11 showed a strikingly different expression pattern. Initially, it accumulated in the animal pole, and later, it accumulated at the primitive streak in the early-embryogenesis stage (Fig. 3a2, b2) like Wnt5a and Wnt8a. However, with the formation of the body axis, Wnt11 began to accumulate in the whole somites and notochord (Fig. 3c2, d2). By the late-embryogenesis stage, with the anterior trunk somitogenesis completed, the accumulation of Wnt11 at the anterior trunks gradually decreased and was mostly limited to the tail somites and notochord (Fig. 3e2). When the tail somitogenesis ended, Wnt11 was no longer detectable by in situ hybridization (Fig. 3f2, g2).

    In contrast to the Wnts described above, Wnt9b showed quite a different expression pattern. From the early-embryogenesis to mid-embryogenesis stage, Wnt9b was undetectable by in situ hybridization. By the late-embryogenesis and juvenile stages, a significant Wnt9b signal had accumulated in the primary digestive tract (Supplementary Fig. S7).

    Overall, these results indicate that three Wnts (Wnt5a, Wnt8a, and Wnt11) could be involved in seahorse tail development. Wnt5a and Wnt8a may function in the tail bud in a partially redundant or complementary manner, whereas Wnt11 may contribute to the regulation of the tail axial somite development.

  • Signaling pathway analysis of the key candidate Wnt genes

  • To explore the potential regulatory functions of Wnt5a, Wnt8a, and Wnt11 in seahorse tail development, the signaling pathway of downstream tail component marker genes was evaluated after Wnts overexpression in seahorse primary embryonic cell lines. The results showed that the expression of tail component genes were up-regulated after the overexpression of each of the three Wnts (Fig. 4a–(c). During this process, different Wnt signaling pathways were activated (Supplementary Fig. S8). Cells overexpressing Wnt5a or Wnt8a showed enhanced expression of Shh (Fig. 4a, b), which indicated the potential of Wnt5a and Wnt8a in promoting the formation of the seahorse tail axial mesoderm and/or primitive streak. Cells overexpressing Wnt11 showed a higher expression of MyoD (Fig. 4c), indicating the potential for promoting the formation of the seahorse tail somitic mesoderm. Nevertheless, the expression patterns of both tail marker genes in vivo showed significant correlation with the three Wnts during seahorse embryo tail development. The axial mesoderm marker gene, Shh, was highly expressed during rapid tail outgrowth (in the mid- and late-embryogenesis stage), whereas it was reduced in the juvenile stage, when tail somitogenesis ended (Fig. 4d). In turn, the somitic mesoderm marker gene, MyoD, was highly expressed during the mid-embryogenesis stage and remained elevated throughout the following stages (Fig. 4e).

    Figure 4.  Potential functional analysis of the three key Wnts in seahorse. ac Shh and MyoD tail tissue marker genes were assessed after overexpression of each Wnt in seahorse primary embryonic cell lines. Wnt5a, Wnt8a, and Wnt11 promote the expression of different tail mesoderm marker genes. de Shh and MyoD expression at different embryo development stages. f Summary of hypothesized Wnt roles during seahorse tail development. Statistical analyses, the results of which are shown in panel ac were performed using Student's t test, and of which are shown in panel d and e were performed using one-way analysis of variance. Different letters above the bars indicate a significant difference (P < 0.05)

    Therefore, Wnt5a and Wnt8a may regulate seahorse tail development by promoting tail bud axial mesoderm formation, while Wnt11 may contribute to seahorse tail development by promoting tail somitogenesis (Fig. 4f).

  • Interaction analysis of the key candidate Wnt genes

  • Given the partially overlapped expression pattern of the three Wnts in the seahorse tail, determining their potential interplays would be helpful to better understand the underlying mechanisms during seahorse tail development. Significant interactions among the three Wnts were observed. Overexpression of Wnt5a resulted in an up-regulation of Wnt11, but down-regulation of Wnt8a (Fig. 5a), whereas overexpression of Wnt8a only impacted Wnt11, leading to its up-regulation (Fig. 5b). However, the expression of neither Wnt5a nor Wnt8a was influenced by the overexpression of Wnt11 (Fig. 5c). Thus, a potential regulation loop could be inferred to exist among the three key Wnts involved in seahorse tail development (Fig. 5d).

    Figure 5.  Analysis of the potential interactive relationship among the three key Wnts in seahorse tail development. ac Relative mRNA levels of the different Wnts after overexpression of each Wnt separately in seahorse primary embryonic cell lines. d Predicted regulatory interaction among Wnt5a, Wnt8a, and Wnt11. *P < 0.05, **P < 0.01

Discussion
  • How novel traits arise in organisms has long been a fundamental problem in biology (Wagner and Lynch 2010). Currently, there exist two appealing hypotheses regarding the molecular events leading to the development of novel traits. One hypothesis suggests that novel traits usually evolve through the co-option of preexisting genes, mainly via gene duplication and functional specialization of proteins. An alternative hypothesis is that novel traits can evolve without requiring gene duplication, but rather through changes in spatiotemporal gene expression patterns or functional modifications of the proteins encoded by genes or a combination of both (Harlin-Cognato et al. 2006). The Wnt gene family encodes a class of signaling molecules that play vital roles in the regulation of vertebrate posterior body development (Huelsken and Birchmeier 2001; Kestler and Kuhl 2008; Krausova and Korinek 2014; Martin and Kimelman 2008; Moon et al. 1997; Ramel et al. 2005). As in many other gene families, the expansion and contraction of the Wnt gene family, by gene duplication or loss, is associated with developmental diversification in the animal kingdom (Cho et al. 2010). In vertebrates, the Wnt gene family is subdivided into 12 subfamilies, of which only six have counterparts in Ecdysozoa (Prud'homme et al. 2002). Studies showed that the Wnt gene family in mammals (mouse and human) is composed of 19 Wnts (Garriock et al. 2007; Nusse 2001). However, the Wnt gene family is expanded in frog and teleost genomes (Garriock et al. 2007), which is suggestive of duplication events. Whole-genome analysis revealed 22 Wnts in the lined seahorse and the genomic organization of the Wnt loci was conserved when compared with that in other teleosts. Our results were consistent with data from other bony fishes (Duncan et al. 2015; Garriock et al. 2007). Moreover, our data indicated that no specific gene duplication of the Wnt gene family occurred in the seahorse when compared with other teleosts. The 22 Wnts in the seahorse could be subdivided into 12 Wnt subfamilies, and sequence alignment analysis showed that the Wnt proteins were evolutionarily conserved. Furthermore, no special functional specialization or modifications were noted in Wnt proteins of the seahorse. These findings indicate that the Wnt gene family was not subjected to genomic changes in the seahorse, and the development of the special seahorse tail was not associated with the gene duplication or functional specialization of Wnt gene family.

    The mRNA expression profile and in situ hybridization analyses further revealed that three Wnts (Wnt5a, Wnt8a, and Wnt11) may be involved in the seahorse tail developmental process. Among them, Wnt5a and Wnt8a may be involved in tail bud development, whereas Wnt11 may be involved in tail somite development. Previous studies reported that the Wnts were involved in tail development, but they only mentioned Wnt5 or Wnt8a individually or the combination of Wnt5a and Wnt11 (Andre et al. 2015; Cha et al. 2008, 2009; Song et al. 2008; Yamaguchi et al. 1999). During mouse embryogenesis, Wnt5a is expressed in multiple outgrowing structures and is important for their outgrowth. Mutation of Wnt5a results in severe shortening of the A–P axis and limb truncation (Yamaguchi et al. 1999). In zebrafish, Wnt8a is involved in the convergent extension of the primal body axis (Erter et al. 2001) and is a crucial determinant of the posterior body axis during gastrulation (Erter et al. 2001; Lekven et al. 2001; Lu et al. 2011). At later stages of zebrafish embryonic development, Wnt11 is expressed in several mesoderm-induced organs, such as somites, and the developing notochord (Makita et al. 1998). Wnt11 is also involved in posterior mesoderm and notochord differentiation in all vertebrates (Makita et al. 1998; Tada and Smith 2000). Expression of dominant-negative Wnt11 leads to convergent extension and neural tube defects (Tada and Smith 2000). In the present study, signaling pathway analysis suggested that both Wnt5a and Wnt8a promote the expression of the tail axial mesoderm marker gene and Wnt11 promotes the expression of the tail somitic mesoderm marker gene. Taken together, these findings suggest that Wnt5a and Wnt8a may promote seahorse tail axial mesoderm formation, and Wnt11 may promote seahorse tail somitogenesis. These data were consistent with those of previous studies and confirmed the functional conservation of the three Wnts in vertebrate tail development.

    A current view of animal evolution posits that the differential expression of conserved sets of regulatory genes contributes to body plan diversification (Carroll et al. 2001). Although the function of the three Wnts was conserved among vertebrates, the expression pattern of Wnt8a during seahorse tail development showed some differences. Compared to other teleosts, the expression period of Wnt8a was significantly extended during the seahorse tail development process until the late stage of tail somitogenesis. In vertebrates, Wnt8a is critical for the generation of the axial mesoderm and maintenance of the axial stem cell pool (Cunningham et al. 2015) and is required for dorsal axis determination in the early-embryogenesis stage (Lu et al. 2011; Shimizu et al. 2005). In zebrafish and medaka, Wnt8a is transiently expressed in the tail bud in the early-embryogenesis stage, including blastula and gastrula (Cunningham et al. 2015; Ramel et al. 2005). Additional stimulation of grafted naive cells of zebrafish with BMP, Nodal, and Wnt8a was found to induce a second tail formation (Agathon et al. 2003). Moreover, inhibition of Wnt8a function in zebrafish causes the loss of all but the first 8–12 somites, as the mesodermal progenitors fail to facilitate posterior somite development (Agathon et al. 2003; Lekven et al. 2001 Martin and Kimelman 2008; Thorpe et al. 2005). In Xenopus, the dominant-negative overexpression of Wnt8a disrupts posterior development (Hoppler et al. 1996). Given the conservative function of homologous genes, as well as the fact that Wnt8a may promote seahorse tail axial mesoderm formation, the extended expression of Wnt8a during seahorse tail development might promote caudal tail bud formation for a longer time. Moreover, both Wnt5a and Wnt8a promote the expression of Wnt11, which regulates tail somitogenesis. In turn, the particularly extended expression of Wnt8a, coordinated with the commonly expressed Wnt5a, may cause a dual-positive regulation of Wnt11, which might further support the elongated outgrowth of the seahorse tail somite (Fig. 6).

    Figure 6.  Schematic showing the hypothesized roles of Wnt family members in the seahorse tail extended outgrowth. The development of the specialized seahorse tail is regulated by the combined activity of Wnt5a, Wnt8a, and Wnt11; Wnt5a and Wnt8a strongly promote tail bud extension and Wnt11 promotes tail somite formation

Conclusions
  • In summary, by comprehensively analyzing critical body plan genes, i.e., the Wnt gene family, in lined seahorse, we found that this gene family is evolutionarily conserved and that three specific Wnts, Wnt5a, Wnt8a, and Wnt11, are involved in tail development in the seahorse, as in other vertebrates. However, a specific expression pattern and significantly extended expression of Wnt8a during seahorse tail development may mediate tail bud formation, as well as tail somitogenesis, contributing to the development of the elongated seahorse tail.

Materials and methods

    Experimental animals

  • Pregnant male lined seahorses (H. erectus) were obtained from the Shenzhen Seahorse Aquamarine Culture Center of the South China Sea Institute of Oceanology, Chinese Academy of Sciences. Embryos at different tail development stages were collected from the pregnant seahorses. Seahorse embryo staging was based on morphological features, as described previously (Sommer et al. 2012; Whittington et al. 2015). Briefly, lined seahorse embryos were divided into early-embryogenesis (1–3 days, from zygote to early tail bud formation), mid-embryogenesis (3–7 days, tail segment extension), late-embryogenesis (14–17 days, further tail segment extension and tail somite formation), and juvenile (within 12 h after embryo release, larva fish, end of tail somite formation and tail functionality) stages (Supplementary Fig. S9). This study was approved by the Chinese Academy of Sciences (IACUC #160413).

  • Wnt gene family in seahorse

  • To obtain a comprehensive scenario of the Wnt gene family in seahorse, Wnt sequences from publicly available whole-genome data for the lined seahorse were obtained (NCBI Sequence Read Archive, accession number SRA392578; Lin et al. 2016). The sequences were verified by polymerase chain reaction (PCR), and the Wnt genes were identified by sequence alignment and domain analysis. The Wnt amino acid sequences associated with the obtained gene sequences were predicted using the ORF finder software (https://www.ncbi.nlm.nih.gov/orffinder/). Signal peptides and other conserved Wnt domains were analyzed via the SMRT Server (http://smart.embl-heidelberg.de/smart/set_mode.cgi?NORMAL=1). To analyze the conservation of the Wnt locus in seahorse genome evolution, syntenic analyses of Wnt genes were performed using data for five representative teleosts in publicly accessible databases (http://www.genomicus.biologie.ens.fr/genomicus). To determine the phylogenetic relationship of Wnt family members across species, the publicly available amino acid sequences (NCBI database) encoded by the Wnt genes from tiger-tail seahorse (H. comes), zebrafish (Danio rerio), medaka (Oryzias latipes), fugu (Takifugu rubripes), spotted gar (Lepisosteus oculatus), frog (Xenopus laevis), chicken (Gallus gallus), and mouse (Mus musculus) were used (Supplementary Table S2). A Wnt phylogenetic tree was generated using "Wnt1" domains. The maximum likelihood method was used for analysis with MEGA 5.1 software (https://www.megasoftware.net), with 1000 bootstrap replicates.

  • Expression profile of the Wnt gene family

  • To identify the key candidate Wnts involved in the regulation of the seahorse tail development, quantitative real-time PCR (qRT-PCR) with specific primers (Supplementary Table S3) was performed to analyze the expression of Wnt genes at four typical tail developmental stages. βactin was used as the internal reference for each sample. qRT-PCR was performed in 10 μl volumes, using SYBR Premix Ex Taq (Takara, Japan). The thermal cycling protocol was as follows: denaturation for 3 min at 95 ℃; 40 cycles of 20 s at 95 ℃, 20 s at the primer-specific annealing temperature, and 20 s at 72 ℃; followed by 30 s at 95 ℃ and 1 min at 60 ℃. Fluorescence signals were acquired at the end of each cycle and melting curve analysis was performed at the end of the reaction to validate the specificity of the PCR amplification.

  • In situ hybridization

  • To further validate the involvement of Wnts in seahorse tail development, whole-mount in situ hybridization was performed with embryos at different developmental stages. The embryos were dissected in RNase-free phosphate-buffered saline solution and fixed in 4% paraformaldehyde at 4 ℃ overnight. The embryos were dehydrated and rehydrated with a graded methanol series. Gene-specific antisense RNA probes corresponding to the candidate Wnts were labeled with digoxigenin-UTP for hybridization (Roche, Switzerland). Hybridization was performed as reported previously (Correia and Conlon 2001).

  • Seahorse embryo cell line establish and overexpression analysis

  • Given that Wnt signals can modulate the expression of several genes, critical genes known to be involved in tail formation were evaluated after ectopic Wnt overexpression. In this study, the tail-related genes analyzed were Shh and MyoD, which were indicative of the tail axial mesoderm and somitic mesoderm, respectively (Cambray and Wilson 2002; Hopwood et al. 1989; Martin and Kimelman 2008). Shh, a morphogenetic signal produced by the polarizing region (Welscher et al. 2002), is a marker gene of primitive streak and axial mesoderm and is widely used to examine axial body plan in embryo development (Cambray and Wilson 2002; Zhang et al. 2020). MyoD is a skeletal muscle-specific gene, the expression of which is restricted to the somites (Hopwood et al. 1989). It is a marker gene of the somite mesoderm and is used to examine somitogenesis in embryo development (Martin and Kimelman 2008; Martin and Kimelman 2012). The in vitro overexpression analysis was performed in embryonic cell line of the lined seahorse (Chinese patent: ZL 2017 1 1050527.5). Briefly, gastrula-stage seahorse embryos were harvested and dissociated into single cells with trypsin/EDTA solution. The cells were transferred into 6-well cell culture plates containing 5 ml per well Dulbecco's modified eagle medium: Ham's nutrient mixture F-12 (1:1) medium (DMEM/F12) supplemented with fetal bovine serum (FBS, 20%) and antibiotics (penicillin, 400 U/ml; streptomycin, 400 µg/ml; Gibco BRL). Cells were maintained at 28 ℃ in an ambient air incubator. The cells at passage 30 were transfected using Lipofectamine 2000 (Life Technologies, USA) according to the manufacturer's instructions. Each candidate Wnt was cloned into the pcDNA 3.1 plasmid (Wnt5a: : pcDNA 3.1, Wnt8a: : pcDNA 3.1, Wnt11:: pcDNA 3.1) and transfected. The empty vector pcDNA 3.1 was transfected as a control. Wnt expression assays were performed 48 h post-transfection.

    To evaluate the relationship between Wnt in vitro expression and the exact embryo development in vivo, the tail-related genes were assessed by qRT-PCR in seahorse embryos at different developmental stages.

  • Interaction analysis

  • As the spatiotemporal expression patterns of the key candidate Wnts partly overlapped, we evaluated the potential interactions of the key candidate Wnts during seahorse tail development. This analysis was conducted using the Wnt-overexpressing seahorse cell lines described above.

  • Statistical analysis

  • For data analysis of qRT-PCR, the 2-ΔΔCt method (Livak and Schmittgen 2001) was used to calculate the relative mRNA expression of each gene. All data in this study were expressed as the mean ± standard error of the mean of three independent experiments.

Supplementary Information
Acknowledgements
  • This work was supported by the K.C.Wong Education Foundation, the National Natural Science Foundation of China (nos. 41825013, 41706178, 41576145, 41806189, 32000350), the China postdoctoral science foundation grant (no. 2019M663151), the Guangdong Special Support Program of Youth Scientific and Technological Innovation (2017TQ04Z269). We are also grateful to Wenqi Hu for help with cell culture.

Author contributions
  • QL and BZ designed the study, performed laboratory protocols, and analyzed bioinformatics. BZ, GQ and YHZ prepared the tables and figures. BZ and CYL carried out the qRT-PCR and in situ hybridization analysis. LLQ and CLC helped with the experiments. All authors read and approved the final manuscript.

Declarations

    Conflict of interest

  • The authors declare no conflicts of interest.

  • Animal and human rights statement

  • This article does not contain any studies with human participants. All animal experiments were conducted in accordance with established guidelines and approved by the Chinese Academy of Sciences (IACUC #160413).

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