Terebratalia transversa has a conserved repertoire of Wnt genes

Metazoans have a large Wnt repertoire with 13 subfamilies [24, 48, 65]. To characterize the Wnt complement of the brachiopod Terebratalia transversa, we surveyed a reference transcriptome of this species for Wnt genes using similarity searches with known Wnt genes from other animals. We identified 13 Wnt genes with representatives of 12 of the 13 Wnt subfamilies (Fig. 1). T. transversa is missing wnt3, a gene known to have been lost in Protostomia [27, 48], and has two copies of wnt1. One of the wnt1 paralogs—named hereafter wnt1t—has a conserved Wnt domain, but is highly divergent at the sequence level compared to other wnt1 orthologs across bilaterians (Additional file 1: Fig. S1). Our phylogenetic analysis suggests that this paralog originated via a lineage-specific duplication within T. transversa or rhynchonelliform brachiopods (Additional file 2: Fig. S2). Besides the loss of wnt3 and duplication of wnt1, T. transversa shows a single representative ortholog for the remaining subfamilies, suggesting that the ancestral repertoire of metazoan Wnt genes remained largely conserved.

Fig. 1

Orthology assignment of Terebratalia transversa Wnt genes. Best-scoring tree of a maximum likelihood phylogenetic analysis using the amino acid sequences of known metazoan Wnt genes. The color-coding represents different Wnt subfamilies and the numbers show the support values of individual branches. Terebratalia transversa (Tt) orthologs are outlined by a box. The other species are Branchiostoma floridae (Bf), Capitella teleta (Ct), Drosophila melanogaster (Dm), Homo sapiens (Hs), Lingula anatina (La), Lottia gigantea (Lg), Platynereis dumerilii (Pd), Saccoglossus kowalevskii (Sk), and Tribolium castaneum (Tc)

Wnt genes are upregulated in concert during axial elongation

To uncover the developmental dynamics of Wnt expression in T. transversa, we analyzed stage-specific RNA-Seq data from the unfertilized egg to the post-metamorphic juveniles (Fig. 2 and Table 1). We detect no Wnt transcripts expressed in the oocyte or mid blastula stages (the high levels of wnt4 and wntA in early stages is due to a bias in the expression quantification, see “Methods” for a detailed explanation). Wnt expression shifts significantly at the late blastula stage (19 h), when a concerted upregulation of wnt1, wnt1t, wnt8, wnt10, and wnt16 occurs (Fig. 2). Throughout gastrulation, Wnt genes continue to be upregulated with wnt1 and wnt5 in the early gastrula (26 h); wnt6, wnt7, and wnt11 in the mid gastrula (37 h); and wnt2, wnt9, and wnt10 in the late gastrula (51 h). Between the late gastrula and early larva, all Wnt genes are expressed, but some are downregulated after gastrulation (wnt6 and wnt10) and after metamorphosis (wnt7 and wnt16) (Fig. 2). Therefore, Wnt expression is dynamic throughout development but peaks late in gastrulation, when the body elongates along the anteroposterior axis, and at the onset of the morphological differentiation of the larval lobes in T. transversa.

Fig. 2

Expression of Wnt signaling components during Terebratalia transversa development. The heatmap represents the log-normalized transcript counts for ligands, receptors, and antagonists calculated from stage-specific RNA-Seq data. Each cell shows the average value between two replicates. Asterisks in wnt4 and wntA denote samples where the expression levels were overestimated due to the expression of an antisense gene present in the same transcript (see “Methods” for details). The black outline marks the late gastrula stage (51 h), when all Wnt genes are expressed. The illustrations depict T. transversa developmental stages from the oocyte until the post-metamorphic juvenile. The stages we analyzed using in situ hybridization (early gastrula to late larva) are highlighted in magenta

Table 1 Developmental stages sampled for the stage-specific transcriptome of Terebratalia transversaWnt expression domains partially overlap along the anteroposterior axis

To uncover the spatial localization of Wnt ligands during brachiopod development, we performed in situ hybridization in T. transversa embryos from late blastula to competent larva (Figs. 3, 4, 5, Additional file 3: Fig. S3, Additional file 4: Fig. S4, and Additional file 5: Fig. S5).

Fig. 3

Whole-mount colorimetric in situ hybridization of Terebratalia transversa wnt1, wnt1t, wnt2, wnt4, wnt5, wnt6, and wnt7. The panels show representative expression patterns for the developmental stages between late blastula and late larva. The samples are oriented with a blastoporal/ventral view and anterior end to the top. Black arrowheads indicate the apical–mantle boundary. White arrowheads indicate the mantle–pedicle boundary. Panels for wnt1 originally published under a Creative Commons Attribution License in [64] and reprinted here for completion. Scale bars = 20 µm

Fig. 4

Whole-mount colorimetric in situ hybridization of Terebratalia transversa wnt8, wnt9, wnt10, wnt11, wnt16, and wntA. The panels show representative expression patterns for the developmental stages between late blastula and late larva. The samples are oriented with a blastoporal/ventral view and anterior end to the top. Black arrowheads indicate the apical–mantle boundary. White arrowheads demarcate the mantle–pedicle boundary. Scale bars = 20 µm

Fig. 5

Whole-mount double-fluorescent in situ hybridization of Terebratalia transversa Wnt genes. A–D Expression of wnt1 (magenta) and wnt5 (green) in the early gastrula (A), late gastrula (B) and early larva in ventral (C) and dorsal (D) views. E–F Expression of wnt5 (green) and fz5/8 (magenta) in the mid gastrula (E) and late gastrula (F) in ventral views. G,H Expression of wnt7 (green) engrailed (en) (magenta) in the early larva in ventral (G) and dorsal (H) views. I–L Expression of wnt1 (magenta) and wnt8 (green) in the mid gastrula (I) and late gastrula (J) in ventral views, late gastrula in an midbody optical section (K), and early larva in dorsal view (L). Green and magenta lines highlight the extension and overlap between domains. Areas in the tissue where the expression overlaps appear in white. Samples oriented with anterior end to the top. Black arrowheads indicate the apical–mantle boundary. White arrowheads demarcate the mantle–pedicle boundary. The panels show representative expression patterns for each sample. Scale bars = 20 µm

wnt1 is expressed in the ectoderm and invaginating endomesoderm of the posterior blastopore lip in the late blastula (Fig. 3, Additional file 3: Fig. S3, and [64]). This domain expands laterally, forming a ventral ectodermal band at the anterior most portion of the pedicle lobe in the early larva, a region that gives rise to the ventral shell primordium in the late larva. From the late gastrula stage, wnt1 is also expressed in a narrow ectodermal stripe around the posterior region of the apical lobe and in the dorsal shell primordium. The apical lobe expression fades, and a new wnt1 domain appears encircling the posterior subdivision of the pedicle lobe in the late larva.

wnt1t expression domains differ significantly from its paralog wnt1. We first detect wnt1t transcripts in a single ectodermal spot at the animal pole of the early gastrula (Fig. 3 and Additional file 3: Fig. S3). This domain localizes to the anterior end of the embryo and is expressed until the early larva, when only subsets of cells continue expressing wnt1t. At this stage, a central patch of ventral ectoderm posterior to the mouth also begins expressing wnt1t. Finally, in the late larva, wnt1t is upregulated in an ectodermal ring beneath the mantle lobe, and at the terminal tip of the pedicle lobe ectoderm.

wnt2 is only expressed in the late gastrula and early larva stages in bilateral ectodermal bands that encircle the posterior portion of the apical lobe almost entirely, except for the ventral and dorsal midlines (Fig. 3 and Additional file 3: Fig. S3).

wnt4 is expressed at the posterodorsal ectoderm from late blastula to late gastrula (Fig. 3 and Additional file 3: Fig. S3). The pattern is similar to wnt1, but wnt4 transcripts are localized more dorsally (Additional file 5: Fig. S5A–C). In the early larva, the expression at the posterior end and dorsal portion fades, the domain becomes narrower, and acquires a subterminal position within the ventral ectoderm of the pedicle lobe. This domain is still present in the late larva, when wnt4 begins to be expressed in the posterior endoderm.

wnt5 is expressed in three distinct ectodermal domains—in the apical, mantle, and pedicle lobes, respectively. We first detect expression in the early gastrula with transcripts at the posterior blastopore lip and anterolateral ectoderm (Fig. 3 and Additional file 3: Fig. S3). The posterior ectodermal domain is narrower than the wnt1 domain (Figs. 3 and 5A) and maintains a terminal position until the late larva stage, when the tip of the pedicle lobe is cleared from expression (Fig. 3 and Additional file 3: Fig. S3). The anterolateral domains expand in the mid gastrula to encircle the posterior portion of the apical lobe ectoderm, and fade in the late larva. wnt5 is also expressed in the leading edge of the growing mantle lobe ectoderm from mid gastrula to late larva. The ectodermal expression domains of wnt5 and wnt1 occupy distinct regions along the anteroposterior axis that coincide with the subdivisions of the larval lobes (Fig. 5B,C and Additional file 5: Fig. S5G).

wnt6 transcripts localize to the posterior blastopore lip, similarly to wnt1 and wnt4, from the early to the late gastrula (Fig. 3 and Additional file 3: Fig. S3). This ectodermal domain is cleared in the early larva, when wnt6 is activated in a midbody section of the endoderm. In the late larva, we also detect wnt6 domains in the ectoderm of the apical and pedicle lobes.

wnt7 initiates as a lateral pair of anterior ectodermal stripes that progressively extend around the entire embryo circumference until the early larva (Fig. 3 and Additional file 3: Fig. S3). This wnt7 stripe demarcates the apical–mantle boundary, partially overlapping with wnt1- and engrailed-expressing cells at the anteriormost region of the mantle lobe (Fig. 5G,H and Additional file 5: Fig. S5H; see also [64]). In the early larva, the anterior wnt7 stripe disappears, and a posterior ectodermal stripe appears demarcating the boundary between the subterminal and terminal regions of the pedicle lobe, between the posterior territories of wnt1 and wnt5.

wnt8 is expressed in a ring of cells in the invaginating endomesoderm and in a broad ectodermal band encircling the late blastula (Fig. 4 and Additional file 4: Fig. S4). In the early and mid gastrula, wnt8 transcripts are cleared from the endomesoderm and from the anterior ectoderm, and two distinct ectodermal domains remain: a pair of broad lateral territories in the apical lobe, and a wide posterodorsal domain in the pedicle lobe. The lateral territories expand ventrally and dorsally, encircling the apical lobe ectoderm, while the posterior ectodermal domain fades in the late gastrula. The anterior wnt8 territories partially overlap with the anterior expression of wnt1 in the apical lobe ectoderm (Fig. 5I–L and Additional file 5: Fig. S5G).

wnt9 transcripts are first expressed in the invaginated endomesoderm of late gastrula embryos at a subterminal position (Fig. 4 and Additional file 4: Fig. S4). The domain forms a contiguous patch of mesodermal and endodermal cells expressing wnt9 in the early larva, which differentiates into two distinct territories, one endodermal in the central portion of the gut and another mesodermal in a bilateral pair of anterior domains near the apical–mantle boundary.

wnt10 is expressed from the mid gastrula stage in a posterior ectodermal domain, which acquires a subterminal position within the pedicle lobe in the early larva (Fig. 4 and Additional file 4: Fig. S4). Additionally, we detect wnt10 transcripts in the late gastrula at a dorsal ectodermal patch of the apical lobe, similar to the dorsal domain of wnt1t, and in the late larva at the posterior mesoderm.

wnt11 is expressed in a posterodorsal ectodermal domain in the early gastrula, similar to wnt4 (Fig. 4 and Additional file 4: Fig. S4). The domain encircles the pedicle lobe ectoderm in the early larva and becomes reduced to a narrow ectodermal stripe on the ventral portion of the pedicle lobe in the late larva. In the early larva, wnt11 is also expressed in the ventral ectoderm at the mantle–pedicle boundary, and in the posterior endoderm of the larval gut (Fig. 4 and Additional file 4: Fig. S4).

wnt16 is expressed in the invaginating endomesoderm and vegetal ectoderm around the blastopore in the late blastula (Fig. 4 and Additional file 4: Fig. S4). During gastrulation, the endomesodermal expression clears, and only the ectodermal domain remains as lateral patches near the blastopore lip. With the blastopore closure, wnt16 forms a heart-shaped domain in the ectoderm and presumably mesoderm at the ventral midline of the mantle lobe in the early larva.

wntA appears in the mid gastrula as paired, ventral ectodermal domains located at the posterior portion of the apical lobe (Fig. 4 and Additional file 4: Fig. S4). In the late larva, these anterior ectodermal domains are cleared, and wntA expression is activated in paired, ventral mesodermal bands adjacent to the mouth.

Overall, Wnt genes are primarily expressed in the ectoderm, in diverse partially overlapping domains along the anteroposterior axis (Additional file 6: Fig. S6 and Additional file 7: Fig. S7).

Frizzled genes exhibit gradual expression changes throughout embryogenesis

Frizzled genes encode seven-pass transmembrane proteins with an extracellular cystein-rich domain and act as receptors in Wnt signaling pathways [66]. There are five Frizzled subfamilies in metazoans [67], but the subfamily fz3/6 is only found in tunicates and vertebrates [68]. In the brachiopod T. transversa, we identified a total of four Frizzled genes with a single ortholog for the fz1/2/7, fz5/8, fz9/10, and fz4 subfamilies, respectively (Additional file 8: Fig. S8).

Frizzled receptors are expressed throughout T. transversa development. In the unfertilized oocyte, fz1/2/7 and fz5/8 are highly expressed (Fig. 2). The expression of fz1/2/7 remains high from the oocyte to juvenile stages, while the expression of fz5/8 peaks before gastrulation and decays over time. fz4, which is initially expressed at lower levels, peaks late in development, at the larval stages, an expression profile complementary to the one of fz5/8 (Fig. 2). In contrast, fz9/10 expression increases during gastrulation and remains relatively constant in subsequent stages.

Overall, each Frizzled shows a unique expression profile but in contrast to Wnt dynamic changes, the levels of Frizzled transcripts change more gradually during development.

Frizzled expression domains occupy broad but distinct body regions

We carried out in situ hybridization for all Frizzled genes of T. transversa to reveal their domains of expression during axial elongation (Fig. 6).

Fig. 6

Whole-mount colorimetric in situ hybridization of Terebratalia transversa Frizzled and Wnt antagonist genes. Developmental stages between late blastula and late larva for fz1/2/7, fz4, fz5/8, fz9/10, sfrp1/2/5, dkk5, and wif. The panels show representative expression patterns for each sample. The samples are oriented with a blastoporal/ventral view and anterior end to the top, except for sfrp1/2/5 early gastrula showing the animal pole. Black arrowheads indicate the apical–mantle boundary. White arrowheads demarcate the mantle–pedicle boundary. Scale bars = 20 µm

fz1/2/7 expression is mostly ubiquitous (Fig. 6 and Additional file 9: Fig. S9). It is expressed in all tissues of the late blastula, with strong signal in the animal pole ectoderm and invaginating endomesoderm. During gastrulation, the anterior and middle portions of the body continue to express fz1/2/7 across all germ layers, but the posterior transcripts get progressively cleared from the pedicle lobe tissues. In larval stages, fz1/2/7 is upregulated in the terminal portion of the pedicle lobe ectoderm, and becomes nearly ubiquitous again in the late larva.

fz4 is first expressed in the animal pole ectoderm of the late blastula (Fig. 6 and Additional file 9: Fig. S9). These anterior transcripts form a subapical ectodermal ring encircling the animal pole of the early gastrula that localizes to the posterior portion of the apical lobe in subsequent stages. fz4 is also expressed in the anterior mesoderm from the early gastrula. In the late gastrula, we detect fz4 transcripts in the dorsal ectoderm between the mantle and pedicle lobes, a domain that becomes stronger in the late larva as it expands around the dorsal ectoderm of the pedicle lobe as well as in the infolded ectodermal and mesodermal tissues of the growing mantle lobe. Additionally, a fz4 domain appears at the posterior tip of the pedicle lobe ectoderm in the late larva.

fz5/8 is mainly expressed at the anteriormost region of the embryo’s ectoderm and mesoderm (Fig. 6 and Additional file 9: Fig. S9). We first detect transcripts in the animal pole ectoderm of the late blastula, these transcripts become restricted to the anterodorsal portion of the ectoderm in the early gastrula, and finally expand to the anteroventral ectoderm from mid gastrula to early larva. This ectodermal territory of fz5/8 is complementary to the ectodermal domain of fz4 in the apical lobe without overlapping with the apical lobe domain of wnt5 (Fig. 5E,F). fz5/8 is also expressed in the anterior mesoderm from the early gastrula and in the chaetae sacs of the late larva.

fz9/10 transcripts are limited to the middle portion of the body throughout development (Fig. 6 and Additional file 9: Fig. S9). In the late blastula, fz9/10 is initially expressed in the ectoderm posterior to the blastopore, but this domain expands to cover almost the entire ectoderm around the blastopore of the early gastrula; it is only absent from a narrow anterior portion. With gastrulation, fz9/10 begins to be expressed in the entire mesoderm, as well as in the ectoderm of the apical–mantle boundary, and in the anterior portion of the pedicle lobe ectoderm. Expression in the lateral mantle lobe ectoderm is weaker, and the terminal portion of the pedicle lobe is cleared from fz9/10 transcripts. Interestingly, the anterior limit of fz9/10 expression abuts the posterior limit of fz4 expression in the apical lobe. fz9/10 expression in the late larva fades, except in the posterior apical lobe ectoderm, and in the anterior and posterior region of the mesoderm.

Taken together, the expression of most Frizzled genes extend over broad but distinct domains along the body. Except for fz1/2/7, which is expressed ubiquitously, the ectodermal territories of fz5/8, fz4, and fz9/10 are sequentially arranged from anterior to posterior, respectively, without overlap until the late gastrula stage and the onset of larval morphogenesis.

Wnt antagonist expression is mostly limited to the anterior end

To obtain a more comprehensive picture of the Wnt signaling landscape in T. transversa, we also analyzed the expression of three Wnt antagonist genes: a Secreted Frizzled-Related Protein (sfrp), a Dickkopf protein (dkk), and a Wnt Inhibitory Factor (wif).

sFRP is a soluble protein that antagonizes Wnt activity by directly binding to Wnt ligands or to Frizzled receptors [69]. It has a cysteine-rich domain with high affinity to Wnt proteins. The sFRP family can be divided into two subfamilies, sfrp1/2/5 and sfrp3/4 [69, 70]. In T. transversa, we only identified a sfrp1/2/5 ortholog (Additional file 10: Fig. S10), which is highly expressed throughout development (Fig. 2). The transcripts locate to the animal pole ectoderm in the late blastula and forms a strong anterior ectodermal domain in subsequent stages, in a pattern similar to the expression of fz5/8 (Fig. 6 and Additional file 11: Fig. S11). sfrp1/2/5 is also expressed in a narrow domain at the anterior mesoderm throughout development, and in a paired domain in the mantle lobe mesoderm restricted to the early larva stage. In the late larva, the anterior domain becomes limited to dorsal patches on the dorsal ectoderm of the apical lobe.

Dkk is a secreted glycoprotein containing two cysteine-rich domains that antagonizes Wnt signaling by inhibiting lrp5/6 co-receptors [71, 72]. These proteins are generally divided into two subfamilies, dkk1/2/4 and dkk3 [71]. In T. transversa, however, we identified a single dkk ortholog that groups with a previously unidentified Dkk subfamily, named hereafter dkk5 (Additional file 12: Fig. S12). Our phylogenetic analysis reveals that non-vertebrate deuterostomes, such as hemichordates and cephalochordates, have orthologs for dkk1/2/4, dkk3, and dkk5, suggesting this was the ancestral Dkk repertoire of bilaterians, and that dkk1/2/4 and dkk5 were subsequently lost in protostomes and vertebrates, respectively (Additional file 12: Fig. S12). The expression of dkk5 in T. transversa is upregulated in the late blastula and downregulated in the juvenile (Fig. 2). It localizes to the animal pole ectoderm in the late blastula, and anterior ectoderm in subsequent stages similar to the expression of sfrp1/2/5, except that dkk5 becomes limited to the ventral ectoderm and is not expressed in the mesoderm (Fig. 6 and Additional file 11: Fig. S11).

Wif is another protein that inhibits Wnt activity by direct binding to Wnt proteins [73]. The protein has five EGF repeats and a typical WIF domain which is shared with RYK receptor tyrosine kinases [72, 73]. In T. transversa, we identified one wif ortholog (Additional file 13: Fig. S13). The expression levels are relatively low and somewhat stable throughout development, with a peak in the late gastrula (Fig. 2). Unlike sfrp1/2/5 and dkk5, wif is mainly expressed in mesodermal tissues throughout the analyzed developmental stages; it is also broadly but faintly expressed in the ectoderm until the early larva, and it is not expressed in the endoderm (Fig. 6 and Additional file 11: Fig. S11).

Overall, the expression of the analyzed Wnt antagonist genes is restricted to the anterior portion of the ectoderm (sfrp1/2/5 and dkk5), and to the mesoderm (wif), regions which coincide with the absence or limited expression of Wnt ligands.

Planar cell polarity genes show patched expression during axial elongation

Proper regulation of planar cell polarity (PCP) is crucial to guide morphogenetic processes, such as convergent extension, and to orient the formation of structures during development [74, 75]. We identified several core components of the PCP pathway in T. transversa. These include orthologs for dishevelled (dsh, also known as dvl), diego (dgo, also known as ankrd6 or diversin), prickle (pk), flamingo (fmi, also known as stan or celsr), strabismus (stbm, also known as vang or vangl), and the downstream transducer c-jun n-terminal kinase (jnk, also known as mapk8). Then, we analyzed their expression between the early and late gastrula stages.

Dsh is a central regulator of the Wnt/beta-catenin, Wnt/PCP, and Wnt/calcium pathways [76]. The protein has three conserved domains (DIX, PDZ, and DEP domains), and two conserved regions before and after the PDZ domain [77]. In T. transversa, we identified a single copy of dsh (Additional file 14: Fig. S14) which is highly expressed in every developmental stage (Additional file 15: Fig. S15). The expression is stronger in a narrow dorsal domain of the anterior ectoderm and in the anterior portion of the mesoderm (Fig. 7), but dsh transcripts are also expressed at lower levels in all embryonic tissues (Additional file 16: Fig. S16).

Fig. 7

Whole-mount colorimetric in situ hybridization of Terebratalia transversa Wnt/PCP pathway components. Developmental stages between early gastrula and late gastrula for dsh, dgo, pk, fmi, stbm, and jnk. The panels show representative expression patterns for each sample. The stainings for dsh are underdeveloped (see Additional file 16: Fig. S16). The samples are oriented in a blastoporal/ventral view (left) and in a lateral view (right). Black arrowheads indicate the apical–mantle boundary. White arrowheads demarcate the mantle–pedicle boundary. Scale bars = 20 µm

Dgo is a cytoplasmic protein containing 6–8 ankyrin repeat domains that suppresses Wnt/beta-catenin signaling and activates the Wnt/PCP pathway [78, 79]. We found a single dgo ortholog in T. transversa with six ankyrin repeats (Additional file 17: Fig. S17). dgo transcripts are deposited maternally, quickly degrade, and only recover higher levels of expression in the late larva (Additional file 15: Fig. S15). However, we still detect two pairs of dorsal ectodermal domains in the apical and pedicle lobes of the late gastrula (Fig. 7).

Pk is a protein that contains a PET domain and three LIM domains [80] and competes with Dgo for Dsh binding [81]. We identified a single pk ortholog in T. transversa (Additional file 18: Fig. S18), which is highly expressed throughout development (Additional file 15: Fig. S15). pk transcripts are present in a small patch of ectoderm posterior to the blastopore in the early gastrula (Fig. 7). In the mid gastrula, pk is upregulated in the mesoderm and forms paired ventral domains within the mantle lobe mesoderm of the late gastrula, when paired ventral domains also appear in the apical lobe ectoderm. Given the high expression levels of pk in our RNA-Seq data, we cannot exclude the possibility that it is more broadly expressed than we could detect in our in situ hybridization.

Fmi is a seven-pass transmembrane cadherin that regulates cell polarity [82, 83]. In T. transversa, we identified one ortholog of fmi (Additional file 19: Fig. S19). In contrast to other polarity genes, it is not expressed maternally; fmi expression peaks around the late gastrula (Additional file 15: Fig. S15). fmi transcripts are present in most ectodermal tissues but show stronger signal on bilateral patches present in the apical lobe ectoderm of the late gastrula (Fig. 7).

Stbm is a four-pass transmembrane protein that forms a signaling complex with FMI [84, 85]. Terebratalia transversa stbm ortholog (Additional file 20: Fig. S20) is initially expressed in high levels, which gradually decay during development (Additional file 15: Fig. S15). Accordingly, stbm is ubiquitously expressed in embryonic tissues during gastrulation (Fig. 7).

Jnk is a kinase that regulates epithelial metamorphosis and is a downstream transducer of the PCP pathway [86]. The jnk ortholog in T. transversa (Additional file 21: Fig. S21) is highly expressed throughout the development (Additional file 15: Fig. S15) and ubiquitously expressed in the late gastrula, except for broad bilateral regions in the apical lobe ectoderm (Fig. 7).

In conclusion, while fmi, stbm, and dsh are expressed ubiquitously, the other cell polarity genes dgo, pk, and jnk are expressed in non-overlapping patches at different regions of the late gastrula.

Distinct Wnt subregions coincide with larval body subdivisions

Given the importance of specific ligand–receptor contexts for the outcome of Wnt signaling [