Divergent Wnt8a Gene Expression in Teleosts

The analysis of genes in evolutionarily distant but morphologically similar species is of major importance to unravel the changes in genomes over millions of years, which led to gene silencing and functional diversification. We report the analysis of Wnt8a gene expression in the medakafish and provide a detailed comparison to other vertebrates. In all teleosts analyzed there are two paralogous Wnt8a copies. These show largely overlapping expression in the early developing zebrafish embryo, an evolutionarily distant relative of medaka. In contrast to zebrafish, we find that both maternal and zygotic expression of particularly one Wnt8a paralog has diverged in medaka. While Wnt8a1 expression is mostly conserved at early embryonic stages, the expression of Wnt8a2 differs markedly. In addition, both genes are distinctly expressed during organogenesis unlike the zebrafish homologs, which may hint at the emergence of functional diversification of Wnt8a ligands during evolution.


Introduction
Teleosts are particularly suitable for comparative studies to elucidate evolutionary events resulting in genomic diversity. With as many as 25.000 species arising within the last 250 million years, it is the most diverse group of vertebrates and with the increasing number of genome sequences available an invaluable resource for genome comparisons. Amongst the sequenced fish genomes are the zebrafish (Danio rerio) [1] and the medakafish (Oryzias latipes) [2], which share their last common ancestor about 110 million years ago [3]. Due to an ancient genome duplication, teleosts frequently have two copies of genes represented only once in the mammalian genome [4][5][6]. The Duplication-Degeneration-Complementation (DDC) model of gene evolution has helped framing ideas about how genomes and gene functions evolve following duplication events [7]. For instance, gene paralogs can lose their function during evolution or the coding sequence of one duplicate can change resulting in novel protein functions (neo-functionalisation). Past research has also shown that mutations in gene regulatory regions can result in the acquisition of a new site or time of expression and a paralog's function is changing (sub-functionalisation) [7][8][9][10]. Frequently, the expression domain and function of the single mammalian gene homolog is equivalent to the combination of the expression domains and functions of the two fish paralogs. This sub-functionalization of genes in fish can be used to dissect the whole range of gene functions of their nonduplicated mammalian homologs [11,12]. Thus, it is of fundamental importance to study gene expression and function in related and morphologically similar but evolutionarily distant species to gain insights into the functional complexity of a given gene and to address questions regarding gene/genome evolution also compared to mammalian systems.
The Wnt signaling pathway is highly conserved and amongst the most intriguing signaling cascades studied to date. Its exceptionally wide range of functional properties ranging from cell proliferation and tissue homeostasis to cell differentiation and cellular diversity in development and disease has inspired many researchers investigating Wnt signaling [13,14]. However, by today, 28 different Wnt ligands have been identified in zebrafish [15] and only a very small subset of ligands has been analyzed in medaka [16]. Wnt8 genes are amongst the most prominent ligands in Wnt signaling. In vertebrates, two Wnt8 genes are present in the genome, Wnt8a and Wnt8b. In teleosts, the genome duplication resulted in the generation of an additional Wnt8a paralog in close genomic proximity and in tandem to the first [17]. This arrangement appears very conserved amongst teleosts [17][18][19]. Interestingly, besides a transcript for the second Wnt8a paralog, a bicistronic Wnt8a transcript encoding both Wnt8a proteins has been identified in zebrafish [17].
The two Wnt8a paralogs are largely overlappingly expressed during early zebrafish development and appear to exert similar but distinct functions [17,18]. However, it is not known whether these features are conserved within the teleost lineage. Moreover, an expression or functional analysis at later stages has not been carried out.
Here we report the expression analysis of the two medaka Wnt8a paralogs during embryonic development. Like for other teleosts, the two genes are arranged in close proximity to each other in the genome. However, unlike in zebrafish, both maternal and zygotic expression of the medaka Wnt8a genes differs in various developing tissues. Our data indicate that Wnt8a gene expression has diverged between distantly related teleost species and implies that they may have acquired different functions during evolution. Moreover, we find sites of medaka Wnt8a expression during organogenesis stages, which we did not detect in zebrafish but are found also in mammals with the exception of the caudal hematopoietic system and gall bladder.

Ethics statement
The approval for all animal work carried out was obtained from the Regierungsprä sidium Karlsruhe (35-9185.64).

Fish maintenance
Wildtype Oryzias latipes from a closed stock at the University Heidelberg and the Danio rerio AB x TL line were kept as described [20][21][22].

Whole mount in situ labeling
Whole mount in situ hybridization using digoxigenin labeled RNA riboprobes for medaka Wnt8a1 and Wnt8a2 and zebrafish Wnt8a ORF1 and Wnt8a ORF2 were perform as described [23,24].
Cloning of the full length medaka Wnt8a1 and Wnt8a2 genes Total RNA was isolated from 1, 2 and 3 dpf medaka embryos using TrizolH (Invitrogen, Darmstadt, Germany). First strand cDNA was prepared according to the manufacturer's protocol (Invitrogen SuperScriptHIII First-Strand kit, Darmstadt, Germany). To clone both medaka full-length Wnt8a genes from the synthesized cDNA, PCR was performed using the Taq DNA polymerase kit (QIAGEN, Hilden, Germany) with the following primers.
Wnt8a1 forward 59 AGCGTGGAGGGAGGCTGCAT 39, Wnt8a1 reverse 59 TGGAGTGCCCCGTGTTCTGT 39, Wnt8a2 forward. 59 AGGAAAATTGAAGAAGCGAACCAGGA 39 and Wnt8a2 reverse. 59 AGCCGTAATCTTTCATCTGGGGGC 39. PCR program: Denaturation for 30 sec at 95uC, annealing for 30 sec at 70uC for Wnt8a1 and 62uC for Wnt8a2 followed by extension for 1 min at 72uC for a total of 35 cycles. The first cycle was preceded with initial denaturation for 3 min at 95uC and the last cycle was followed by additional extension for 3 min at 72uC and cooling at 25uC for 30 sec. The purified full-length Wnt8a cDNAs (Wnt8a1: 1349 bp and Wnt8a2: 1110 bp) were cloned into the pCRII-TOPO vector (Invitrogen, Darmstadt, Germany) according to the manufacturer's instructions and sequenced.

RT-PCR detection of Wnt8a transcripts in medaka and zebrafish
To detect maternal and zygotic expression of medaka Wnt8a genes, we isolated total RNA from medaka embryos at 2-cells, 4cells, 16-cells and 40% epiboly. The synthesized first strand cDNAs were used as templates for PCR carried out as above to obtain the Wnt8a2 cDNA. To obtain a 530 bp long Wnt8a1 fragment, we used the same PCR conditions as above with an annealing temperature of 60uC and for Wnt8 the following primers: Wnt8a1 forward 59 AGCGTGCAAGTGTCACGGCG 39, Wnt8a1 reverse 59 CACGGTCCCTGCGCTTCGTT 39. Total RNA from zebrafish was isolated from embryos at 80% epiboly and from heads dissected posterior to the hindbrain and the remaining tails at 48 hpf. To obtain a 217 bp long Wnt8a ORF1 cDNA fragment and a 264 bp long Wnt8a ORF2 fragment (see supporting information Figure S1I), the PCR conditions were used as above with an annealing temperature of 62uC with the following primers: Wnt8a ORF1 forward 59 GCTGTCAAAG-CAACGCTCAA 39, Wnt8a ORF1 reverse 59 AGCT- GAACGTGTCCGCTATT  The Wnt8a cDNA sequence alignments and the phylogenetic trees were built using Geneious software. The sequences were aligned using the global multiple alignment method with free end gaps. The tree was built using the Phylogenetic Maximum Likelihood method (PhML) [25] and the Hasegaw-Kishino-Yano (HKY85) substitution model with 10.000 bootstrap replicates using Ciona intestinalis as outgroup. To disentangle nodes in the tree, MrBayes analysis was applied [26]. The MCMC (Markov chain Monte Carlo) was used to calculate the posterior probabilities of phylogenetic trees.

Results and Discussion
Wnt8a ligands are conserved amongst vertebrates Like in other teleosts [17][18][19], the two medaka Wnt8a paralogs are organized in a tandem arrangement in the genome. We isolated the full length coding sequences of medaka Wnt8a1 (formerly named Wnt8-like) [16] and Wnt8a2. Phylogenetic analysis shows that the medaka Wnt8a paralogs cluster well with the respective Wnt8a paralogs of the evolutionarily close Fugu and Tetraodon ( Figure 1). In contrast, the zebrafish Wnt8a genes cluster together on the Wnt8a1 branch of the tree. Consistent with its evolutionary distance, the medaka Wnt8a1 protein shares higher homology with its Fugu homolog (78% identical amino acids (aa)) rather than with zebrafish Wnt8a ORF1 (also named Wnt8.1, Wnt8a or Wnt8a.1) (69% identical aa). However, the second Wnt8a protein, Wnt8a2, appears similarly conserved (Fugu Wnt8.2 shares 71% identical aa with medaka Wnt8a2 and 72% with zebrafish Wnt8a ORF2; also named Wnt8.2 [17]).
This indicates that the medaka genome contains two paralogous Wnt8a copies, which are well conserved within closely related teleosts and that Wnt8a1 is the gene more similar to the single mammalian Wnt8a.

Differential initiation of medaka Wnt8a gene paralog expression
Zebrafish Wnt8a ORF1 is expressed maternally [27] and the earliest Wnt8a expression in other animal models has been reported in the posterior epiblast prior to gastrulation in mice and chick [28,29]. Early zygotic zebrafish Wnt8a expression is found in lateral and ventral marginal cells of the blastoderm  [17,27] (Figure S1A and B). Its expression in the embryonic shield remains controversial. This gene was initially described to be expressed in the shield and downregulated subsequently [27]. A later study mentioned that Wnt8a ORF1 (and Wnt8a ORF2) is not expressed in the shield until about 75% epiboly [17]. At subsequent stages, Wnt8a ORF1 is expressed in the paraxial mesoderm [27] and Wnt8a ORF2 remains expressed in marginal cells similar to its paralog [17].
We find that medaka Wnt8a1 is maternally expressed (Figure 2A,C,E), while we did not detect any Wnt8a2 transcripts by whole mount in situ hybridization or RT-PCR ( Figure 2B,D,F). The early zygotic medaka Wnt8a1 expression is well conserved amongst vertebrates and is seen in lateral and ventral marginal cells at 40% epiboly with the exception of the shield ( Figure 2G) [16,17,27,28,[30][31][32]. At this stage, Wnt8a2 is expressed weakly in a slightly broader region of the blastoderm margin including cells of the shield (Figure 2H). At late gastrulation, medaka Wnt8a1 is also found in cells of the dorsal blastoderm margin with lower expression in the ventral part similar to its homologs in other vertebrates [17] (Figure 2I and inset). However, no Wnt8a2 transcripts are found in any of the marginal cells. Therefore the temporal expression of medaka Wnt8a2 in the shield and the blastoderm margin markedly differs from medaka Wnt8a1 and zebrafish Wnt8a ORF2. This may imply functional differences of the medaka Wnt8a genes during early embryonic development, which may be more prominent than in zebrafish [17].
Shortly before the end of gastrulation, we find Wnt8a2 transcripts specifically in the paraxial mesoderm ( Figure 2J). This transient Wnt8a2 expression is similar to the reported expression of zebrafish Wnt8a ORF1 and the single Wnt8a genes of Xenopus and mouse [17,28,33,34] (Table 1).

Medaka Wnt8a expression during somitogenesis and organogenesis
During zebrafish somitogenesis, both Wnt8a genes are expressed in cells of the tailbud [17,35] (Figure S1C and D). At 2 and 4 days of development, we find ubiquitous staining for both genes in the developing brain and no staining in the trunk and tail ( Figure S1E-H). However, the same holds true for the genes' sense probes suggesting that the antisense probes give rise to background color. Indeed, using RT-PCR on RNA extracted from dissected heads and tails of 2 dpf embryos, we observed low amounts of cDNA products for both genes in both regions ( Figure S1I). This suggests that also during stages following somitogenesis both zebrafish Wnt8a genes are expressed at low levels, which, however, is not detectable by in situ hybridization. In mouse and chick embryos Wnt8a expression is confined to the fore-and midbrain as well as limbs and branchial arches [36]. Furthermore, expression in the gut, heart and otic vesicles has been reported for mouse embryos [36][37][38]. Thus, many of the later Wnt8a expression domains may not be conserved between zebrafish and mouse and chick (Table 1).
We find that medaka Wnt8a1 is expressed in the axial part of the presomitic mesoderm in the tailbud ( Figure 3A) [16] including cells of the most posterior notochord. Conversely, Wnt8a2 expression is seen broadly throughout the posterior somatic mesoderm reaching into the forming somites ( Figure 3B). While Wnt8a1 remains expressed in a decreasing number of tailbud cells up to 3,5 dpf ( Figure 3C,E) similar to zebrafish, Wnt8a2 transcripts disappear in this part of the embryo. Instead, Wnt8a2 starts to be expressed in cells of the caudal hematopoietic system at 2,5 days, where it is strongly expressed in the caudal vein from 3,5 dpf onwards ( Figure 3D,F and inset) [39]. Moreover, expression is seen in the otic vesicles at 4 dpf ( Figure 3H). On the other hand Wnt8a1 transcripts appear in the entire gut by 3.5 dpf extending anteriorly into the oesophagus subsequently ( Figure 3G,I). In addition, Wnt8a1 is expressed in the outflow tract of the heart, the swim bladder and the gall bladder ( Figure 3G,I and inset). These observations represent evolutionary diversification of gene expression within the teleost lineage and amongst vertebrate species in general. Unlike other model systems analyzed, we were not able to detect expression of zebrafish Wnt8a genes at stages after somitogenesis by in situ hybridization. Conversely, Wnt8a gene expression in cells of the developing hindbrain rhombomeres seen in various species including zebrafish appears not to be conserved in medaka [17,28,29,33]. Moreover, Wnt8a expression in limbs and branchial arches of mouse and chick embryos is not seen in fish [36]. Intriguingly, expression in the gut, heart and otic vesicles appears to be conserved between medaka and mouse [36][37][38], while only medaka Wnt8a gene expression is found in the caudal hematopoietic tissue, gall-and swim bladder (Table 1). In summary, the medaka Wnt8a paralogs have acquired very different sites of expression when compared to each other, but the sum resembles much of Wnt8a expression in animals with a single Wnt8a gene in their genome.
The early medaka Wnt8a1 gene expression is similar to Wnt8a expression of other vertebrate species. However, the temporalspatial dynamics of Wnt8a2 expression appears to have diverged during teleost evolution. It is tempting to speculate that this may be due to differences on the transcriptional level such that for instance unlike zebrafish and Fugu Wnt8a, medaka may not have a bicistronic Wnt8a transcript [17,18].
Our phylogenetic analysis shows that the zebrafish Wnt8a genes do not separate into the two Wnt8a gene clusters of other teleosts, which may explain their similar expression and semi-redundant function described in zebrafish [17]. Conversely, the separation of medaka Wn8a paralogous copies together with the divergence of their expression suggests that the two genes may have acquired different functions during evolution. Low levels of blue color is ubiquitously distributed in the brain of 2 dpf and 4 dpf old embryos, which is however also seen using the sense probe (insets in E and F). (I) RT-PCR analysis to detect Wnt8a gene transcripts reveals low levels of Wnt8a gene expression in both heads and tails of zebrafish embryos. Therefore the color visible in the head is likely to be background. 80%, 80% epiboly; n.c., negative control; st., standard; tb, tailbud. (TIF)