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. 2026 Apr 8;43(4):msag089. doi: 10.1093/molbev/msag089

Wnt signaling patterns a central–peripheral body axis in the early-diverging metazoan Trichoplax adhaerens

Guotao Di 1,2,#, Minjun Jin 3,4,#, Wanqing Li 5,6, Meng Yuan 7,8, Yifan Zhang 9,10, Chengtian Zhao 11,12,13,✉,c
Editor: Yichen Dai
PMCID: PMC13128682  PMID: 41964527

Abstract

Body axis formation was a pivotal innovation in animal evolution, providing the spatial framework necessary for organized multicellularity. While the genetic basis of axis specification is well established in bilaterians, how axial organization emerged in early-diverging metazoans remains unresolved. Here, we address this question in Trichoplax adhaerens, a placozoan representing one of the simplest extant animal body plans. We show that Wnt signaling, a conserved regulator of axial patterning, exhibits polarized expression enriched in the peripheral region of this morphologically simple organism. Functional perturbations demonstrate that Wnt activity promotes peripheral cell proliferation and maintains central–peripheral tissue balance. Transcriptomic profiling further reveals distinct molecular identities along this axis, resembling the oral–aboral polarity of cnidarians. Together, our findings uncover a Wnt-dependent axial system in placozoans and support the view that core components of metazoan body axis patterning were already established in early animal evolution.

Keywords: placozoa, Trichoplax adhaerens, Wnt, body plan, central–peripheral axis, proliferation

Introduction

Body plan formation is a crucial event for bilaterians that serves as the foundation for the spatial organization of tissues and organs in a developing organism. The establishment of the body plan involves three body axes—anterior–posterior (head-to-tail), dorsal–ventral (back-to-belly), and left–right asymmetry—each of which is essential for the proper positioning and function of the body structures. Of these, the anterior–posterior (AP) axis is particularly significant as it was the first embryonic axis to arise in evolution. In bilaterians, the AP axis commonly corresponds to the head–tail axis that is defined during embryonic development. Disruptions in the AP axis formation can result in profound developmental abnormalities, including misplacement of body parts and failure of organ development (Kimelman and Martin 2012). However, in prebilaterian animals, such as poriferans (sponges), cnidarians (e.g. jellyfish, corals), and ctenophores (comb jellies), the concept of AP axis is less straightforward due to their simpler body plans and radial or asymmetric symmetry. Despite this, the study of body plan formation in prebilaterians can offer valuable insights into the evolutionary origins of body axis specification and the molecular mechanisms that predate the evolution of bilaterality (Genikhovich and Technau 2017).

Wnt signaling is a highly conserved pathway with pivotal roles in embryonic development and tissue homeostasis. It ensures proper cell fate determination, tissue patterning during embryogenesis, and maintains a balance in adult tissues by regulating processes like cell proliferation, differentiation, and apoptosis (Steinhart and Angers 2018; Liu et al. 2022). During embryonic development, Wnt signaling plays a critical role in the establishment of the AP axis in bilaterians (Petersen and Reddien 2009; Niehrs 2010; Holstein 2012; Holzem et al. 2024). Wnt signaling functions through both canonical (β-catenin-dependent) and noncanonical (β-catenin-independent) pathways to influence axis specification (Loh et al. 2016). In most species, a gradient of Wnt activity along the embryo's length helps establish regional identities, with high Wnt signaling promoting posterior development and low or inhibited Wnt activity defining the anterior regions (Martin and Kimelman 2009; Niehrs 2010). The precise regulation of Wnt signaling is critical for proper AP axis formation, and disruptions in this pathway can lead to severe developmental abnormalities, such as axis duplication or defective body plan specification (Petersen and Reddien 2009; Holzem et al. 2024). While the role of Wnt signaling in AP axis formation is highly conserved, its specific mechanisms and timing differ across species. In Drosophila melanogaster, the formation of AP axis mainly relies on maternally provided factors such as bicoid and nanos, while Wnt signaling seems only functions in the specification of the polarity of individual segments (Driever and Nusslein-Volhard 1989; Swarup and Verheyen 2012). In contrast, vertebrates like amphibians, birds, and mammals, establish the AP axis during gastrulation through Wnt signaling gradients (Petersen and Reddien 2009; Hikasa and Sokol 2013).

The mechanisms of how the AP axis is determined in nonbilaterians are much less investigated. Cnidarians exhibit some degree of primary body axis formation, such as the oral–aboral (OA) axis, which can be compared to the AP axis in bilaterians (Schaffer et al. 2016; Lebedeva et al. 2021). Interestingly, Wnt signaling was also suggested to be involved in the formation of such an axis, suggesting the role of Wnt signaling during the establishment of the primary body axis evolved before the divergence of bilaterians and nonbilaterians (Hobmayer et al. 2000; Kusserow et al. 2005; Lee et al. 2006, 2007; Nakamura et al. 2011; Leclère et al. 2016; Wang et al. 2020). In contrast, in the earlier diverging groups such as ctenophores and sponges, the role of Wnt signaling during body axis formation remains controversial. In the ctenophore Mnemiopsis leidyi, all of the four Wnt genes are expressed at the aboral pole in a striking pattern (Pang et al. 2010). However, activating Wnt signaling through GSK-3 inhibition in M. leidyi does not yield significant phenotypic changes (Pang et al. 2010). The ctenophore Pleurobrachia pileus also has four Wnt genes, all of which are expressed around the mouth in adults, while the role of these Wnt genes remains elusive (Jager et al. 2013). The role of Wnt signals is far more complex in the phylum Porifera. Although Wnt genes showed polarized expression patterns in some sponges, including the demosponge Amphimedon queenslandica and the calcareous sponge Sycon ciliatum, it remains inconclusive whether Wnt signaling can regulate embryonic polarization in sponges (Adamska et al. 2010; Leininger et al. 2014; Reid et al. 2018). It is more likely that Wnt is only expressed in certain cells that only direct a localized region of cells for a particular role (Reid et al. 2018). Moreover, the absence of Wnt genes in the glass sponge Aphrocallistes vastus may further complicate this (Riesgo et al. 2014).

In contrast to the complicated structure of ctenophores and sponges, Placozoa are probably the simplest known free-living metazoans that branched after sponges during evolution, sharing a common ancestor with cnidarians and bilaterians (Laumer et al. 2018) (Fig. 1a). Trichoplax adhaerens, the representative species of the phylum Placozoa, has a flattened, disk-like body that typically measures 0.2 to 2 mm in diameter (Srivastava et al. 2008; Leria et al. 2024) (Fig. 1b). Trichoplax adhaerens usually reproduce asexually during culture, primarily by binary fission or budding (Zuccolotto-Arellano and Cuervo-Gonzalez 2020). Despite the presence of tens of thousands of cells within each individual, T. adhaerens mainly contains six major somatic cell types forming two cellular layers (Smith et al. 2014; Najle et al. 2023). Due to the simplest body structure, it has a long thought that AP or dorsal–ventral (DV) body axes are ambiguous in Placozoa. Interestingly, gene expression of several Chordin/Tgf-β signaling genes suggests the presence of potential “dorsal–ventral” patterning in T. adhaerens (DuBuc et al. 2019). Moreover, T. adhaerens also exhibits polarized movement and organized feeding behavior suggests that a rudimentary form of body polarity may exist (Fortunato and Aktipis 2019; Jin et al. 2024).

Figure 1.

For image description, please refer to the figure legend and surrounding text.

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Identification of Wnt signaling homologs in Trichoplax adhaerens. a) Phylogenetic tree of metazoans, with choanoflagellates serving as the outgroup. Placozoa is depicted as the sister group of a clade of Cnidaria + Bilateria. b) Dorsal view of a live T. adhaerens. c) Schematic representation of the canonical Wnt signaling pathway in T. adhaerens. Protein names are color-coded to denote Wnt pathway components in the T. adhaerens genome: red, present; green, incomplete. d) Predicted conserved domains of Wnt/β-catenin signaling components identified in T. adhaerens. e) Bayesian phylogenetic tree of Wnt homologs in representative metazoan taxa, with posterior probability support shown next to the nodes. Different subfamilies were highlighted in various colors, and Wnt homologs from T. adhaerens (TaWntA, TaWntB, TaWnt8) were highlighted in red. Species codes are as follows: Ml = Mnemiopsis leidyi; Aq = Amphimedon queenslandica; Ta = Trichoplax adhaerens; Nv = Nematostella vectensis; Pd = Platynereis dumerilii; Pv = Patella vulgata; Tc = Tribolium castaneum; St = Strongylocentrotus purpuratus; Bf = Branchiostoma floridae; Cr = Ciona robusta; Dr = Danio rerio. f) Distribution of Wnt gene subfamilies across representative metazoan species, with the number of Wnt genes shown in different colors. Scale bar = 100 μm. See also Figure S1.

Here, we focused on the body polarity pattern of T. adhaerens. By investigating the expression of Wnt signaling, we discovered an amazing spatial expression pattern of Wnt genes in T. adhaerens. Moreover, further functional studies suggest the critical role of Wnt signals during cell proliferation and body plan formation in T. adhaerens. Our data provide functional studies for Wnt genes in basal metazoans, which suggest that, although still ambiguous in Porifera and Ctenophora, the role of Wnt signals during body plan establishment has already been well established in Placozoa.

Results

Trichoplax adhaerens contains a complete canonical Wnt signaling pathway

By analyzing the T. adhaerens genome (Srivastava et al. 2008), we identified all key components of the canonical Wnt signaling pathway, each of which contains conserved domains characteristic of their respective roles (Fig. 1c and d). Our analysis revealed three Wnt genes in the genome, and phylogenetic analyses using Bayesian and maximum likelihood (ML) methods placed one of these Wnt proteins in the Wnt8 subfamily (Fig. 1e, Figure S1). However, the other two Wnt proteins exhibited weak phylogenetic affinity with established Wnt subfamilies. We designated these proteins as WntA and WntB, which occupy distinct positions in the phylogenetic tree, akin to “WntX” proteins observed in ctenophores and sponges (Fig. 1e and f, Figure S1) (Pang et al. 2010; Holstein 2012; Holzem et al. 2024). Notably, the number and distribution of Wnt proteins in cnidarians are similar to those in bilaterians, whereas ctenophores, sponges, and placozoans possess fewer Wnt ligands. This distribution pattern supports the basal evolutionary positions of these groups (Fig. 1e and f).

Spatiotemporal expression of Wnt signaling genes in T. adhaerens

We performed whole-mount fluorescence in situ hybridization (FISH) to investigate the expression patterns of Wnt signaling genes in T. adhaerens. All three Wnt genes were predominantly expressed in the peripheral regions of the animal (Fig. 2a). While WntA and WntB expression was confined to 1 to 2 layers of cells, Wnt8 exhibited a broader expression pattern (Fig. 2a). Quantitative PCR (qPCR) further confirmed the higher expression level of Wnt8 (Fig. 2b) and the peripheral localization of three Wnt genes (Fig. 2c and d). Interestingly, WntA and WntB were localized to the lower cell layer, as indicated by the absence of auto-fluorescent particles, which mark upper-layer cells (Varoqueaux et al. 2018) (Fig. 2e). In contrast, Wnt8 was expressed in cells from both upper and lower layers (Fig. 2e). To determine the specific cell types expressing these Wnt genes, we further utilized single-cell transcriptome analysis (Najle et al. 2023). This analysis revealed that WntA and WntB were primarily expressed in a subset of peptidergic cells, while Wnt8 was expressed in both peptidergic cells and dorsal epithelial cells (Fig. 2f and g).

Figure 2.

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Expression of Wnt signaling ligands and receptors in T. adhaerens. a) FISH results showing the expression of three Wnt genes at the periphery of T. adhaerens. b) Comparison of gene expression levels of three Wnt genes. c) Live image of a T. adhaerens individual. The yellow line indicates a manually defined boundary at approximately one-third of the radius from the edge, used to separate the central and peripheral regions for analysis. d) Comparison of Wnt expression levels between the central and peripheral zones. e) Confocal images showing the FISH staining results of Wnt genes (red). The green channel indicates the auto-fluorescent signals from the dorsal layer. D: dorsal, V: ventral, AF: autofluorescence. f) Unsupervised UMAP of scRNA-seq dataset from T. adhaerens showing the cluster of different cell types as indicated. g) Gene expression patterns of Wnt genes on different cell types from scRNA-seq data. h) FISH results showing the expression of two Frizzled receptors and a Lrp5/6 co-receptor. i) Comparison of Frizzled and Lrp5/6 expression levels between the central and peripheral regions. j) Relative expression levels of Frizzled and Lrp5/6 genes in different cell clusters. In panels a, e, and h, nuclei were counterstained with DAPI (blue). In panels b, d, and i, each dot corresponds to an independent biological replicate. The number of animals examined is shown in the bottom left of each panel. Scale bars: 100 μm (a, h), 20 μm (e), 250 μm (c), and 50 μm in enlarged views in (a, h).

We also examined the expression of several Wnt receptor genes, including Frizzled and Lrp5/6, to identify the cells capable of receiving Wnt signals. T. adhaerens contains two Frizzled genes, FzdA and FzdB (Schenkelaars et al. 2015) (Fig. 1d). FzdA was primarily expressed in the peripheral region, whereas FzdB was localized to central regions (Fig. 2h and i). The Lrp5/6 co-receptor gene showed a broader expression pattern, encompassing both peripheral and central regions (Fig. 2h and i). Transcriptome analysis further indicated that these receptor genes are widely expressed in multiple cell types, including epithelial cells, peptidergic cells, and fiber cells (Fig. 2j). Collectively, these findings suggest that Wnt genes are predominantly expressed in peripheral peptidergic cells and dorsal epithelial cells, while Wnt signal-receiving cells are distributed across both outer and inner regions of the animal.

In bilaterians, Wnt signaling is crucial for establishing the AP axis during embryonic development (Petersen and Reddien 2009). In contrast, T. adhaerens rarely exhibits embryonic development in laboratory cultures, as it predominantly reproduces asexually through binary fission. To explore the dynamics of Wnt signaling, we monitored the growth of individuals immediately following division. These newly divided animals gradually increased in size and typically underwent another division within 10 d (Fig. 3a). Remarkably, Wnt expression was minimal during the early stages of these small individuals (Fig. 3b). As the animals grew, the number of cells expressing Wnt8 increased gradually, revealing a dynamic pattern of Wnt gene expression associated with growth (Fig. 3c and d).

Figure 3.

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Spatiotemporal expression of Wnt8 in T. adhaerens. a) Growth of newly divided animals from days 1 to 10. b) Comparison of Wnt8 expression levels among different stages during animal growth. c) FISH results showing the expression of Wnt8 at different times during animal growth. d) Quantification of Wnt8 signal density at different time points. In panel b, each dot corresponds to an independent biological replicate. In panel d, each data point represents the density of an individual animal. A.U., arbitrary unit. In panel c, nuclei were counterstained with DAPI (blue). Scale bars: 250 μm (a), 100 μm (c), and 20 μm in enlarged views in (c).

Wnt signaling regulates growth in T. adhaerens

To investigate the role of Wnt signaling in T. adhaerens, we overexpressed the Wnt genes via electroporation under the control of a ubiquitous T. adhaerens β-actin promoter (Jin et al. 2026). qPCR confirmed significant upregulation in the expression levels of each Wnt gene (Figure S2a). The expression of Axin, a well-established target of Wnt/β-catenin signaling, was also markedly elevated in transfected animals (Fig. 4a) (Jho et al. 2002; Kozmikova and Kozmik 2020). Notably, Tcf transcript levels were also increased (Figure S2b), suggesting the presence of a potential feedback regulatory mechanism within the Wnt signaling network (Broun et al. 2005; Holstein 2022; Ross et al. 2025). Phenotypically, animals overexpressing Wnt genes exhibited a significant increase in body size compared to control animals (Fig. 4b and c, Figure S2c). Growth curve analysis further revealed that Wnt overexpression stimulated animal growth beginning at 3 d post-transfection (Fig. 4c).

Figure 4.

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Wnt signaling regulates growth in T. adhaerens. a) qPCR analysis showing the expression of Axin following overexpression of different Wnt genes. b) Representative images showing animals transfected with WntB genes at different time points following electroporation. c) Statistical analysis showing relative animal size following electroporation with different Wnt genes. n = 26 for Control, n = 24 for WntA, n = 27 for WntB and n = 22 for Wnt8. d) qPCR analysis showing the expression of WntA, WntB, and Wnt8 in different knockdown groups as indicated. e) Axin expression levels in animals after knocking down of different Wnt genes. f) Statistical analysis showing relative animal size following knocking down of different Wnt genes. n = 38 for NC, n = 32 for WntA, n = 35 for WntB, n = 39 for Wnt8, n = 39 for WntB  +  Wnt8 and n = 36 for triple knockdown group. In panels a, d, and e, each dot corresponds to an independent biological replicate. Scale bar = 250 μm. See also Figure S2.

Next, we performed knockdown experiments to further investigate the role of Wnt signaling. Using shRNA-mediated RNA interference (RNAi), we successfully silenced each Wnt gene individually in T. adhaerens (Fig. 4d). Notably, knocking down a single Wnt gene often resulted in the upregulation of other Wnt genes, suggesting functional redundancy among them (Fig. 4d). To further suppress Wnt signaling, we conducted double and triple knockdown experiments (Fig. 4d). Silencing these Wnt genes not only reduced Axin and Tcf expression but also significantly impaired animal growth (Fig. 4e and f, Figure S2d and e).

Wnt signaling regulates body plan formation by promoting peripheral cell proliferation

The rapid growth observed in animals overexpressing Wnt genes is likely driven by increased cell proliferation. We quantified proliferating cells using pH3 antibody labeling and further enhanced its detection by treating the animals with colchicine, which arrests cells in metaphase by inhibiting tubulin polymerization. Compared to the control group transfected with β-actin::GFP plasmid, Wnt overexpression significantly increased the number of proliferating cells, with this effect predominantly restricted to the peripheral regions (Fig. 5a and b, Figure S3a). Treatment with 6-bromoindirubin-3′-oxime (BIO), an effective GSK3β inhibitor and activator of canonical Wnt signaling, similarly elevated Axin and Tcf expression and the number of pH3-positive cells (Figure S3b—to d). Conversely, knocking down Wnt genes reduced the number of proliferating cells (Fig. 5c, d, Figure S3e).

Figure 5.

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Wnt signaling regulates body plan formation by promoting peripheral cell proliferation. a) Representative images showing pH3 staining results in control and WntB-overexpressing animals. b) Quantification of pH3 signal density in T. adhaerens. A.U., arbitrary unit. c) Representative images of pH3 antibody labeling in control and Wnt gene triple RNAi animals. d) Quantification of pH3 signal density in different RNAi groups. e) Gene expression pattern of PWN on different cell types from scRNA-seq data. Relative expression level in different clusters is shown on the right. f) Representative images of FISH staining results with probes against PWN in control and WntB-overexpressing animals. g) Quantification of PWN+ cell densities in control and Wnt gene overexpression groups. h) Representative images showing dual staining with PWN probes and pH3 antibody labeling. Magnified insets highlight proliferating PWN+ cells. i) Proliferation rate of PWN+ cells, calculated as the percentage of PWN+/pH3+ double-positive cells among total PWN positive cells. j) Representative images illustrating Trypsin gene expression in control and WntB-overexpressing animals, with a yellow line marking the central expression region. k) Quantification of Trypsin-expressing regions, presented as the percentage of the total body area. In panels a, c, f, and j, nuclei were counterstained with DAPI (blue). In panels b, d, g, i, and k, each data point represents an individual animal. Scale bars: 200 μm (a), 100 μm (c, j), 50 μm (f, h), and 10 μm in enlarged views in (h). See also Figures S3 and S4.

To better understand the role of Wnt signaling in cell proliferation, we focused on peptidergic cells, which showed high expression levels of Wnt receptors (Fig. 2j). PWN is a neuropeptide expressed specifically in a distinct type of peptidergic cell (Najle et al. 2023) (Fig. 5e). Further FISH analysis revealed that PWN-positive cells were primarily localized to the animal's margins (Fig. 5f). Strikingly, overexpression of Wnt genes significantly increased the number of PWN-positive cells (Fig. 5f and g, Figure S4a). pH3 labeling also further confirmed the enhanced cell proliferation in these regions (Fig. 5h and i, Figure S4b). In contrast, Wnt gene knockdown reduced the number of PWN-positive cells and overall cell proliferation (Figure S4c to e).

Finally, considering the different spatial distribution of Wnt genes and their receptors, we asked whether Wnt signaling can balance the growth of central and peripheral regions. In T. adhaerens, peripheral cells exhibit greater diversity and are crucial for environmental interaction and movement, while central cells primarily contribute to structural stability and digestion (Varoqueaux et al. 2018; Mayorova et al. 2019; Li et al. 2025). For example, Trypsin expression, a marker for gland cells, is confined to the central region (Mayorova et al. 2019) (Fig. 5j). In control animals, the Trypsin expression region accounted for approximately 45% to 55% of the total body size (Fig. 5k). Strikingly, in Wnt-overexpressing animals, the relative area of the Trypsin expressing region was significantly reduced (Fig. 5j and k, Figure S4f and g). Triple knockdown of Wnt genes, however, increased the ratio of the Trypsin expressing region (Figure S4h and i).

In summary, these findings demonstrate that Wnt signaling is crucial for the growth and body plan formation of T. adhaerens. Specifically, Wnt signaling promotes peripheral cell proliferation and relatively inhibits the development of the central region, thereby coordinating the development between the inner and outer cells.

A central–peripheral axis in T. adhaerens

Given the role of Wnt signaling in body axis determination (Petersen and Reddien 2009), the distinct expression patterns of Wnt genes in the peripheral and central regions prompted us to investigate whether a primary body axis is present in this organism. To address this, we performed transcriptome analysis comparing gene expression patterns between the central and peripheral regions. Our results revealed distinct gene expression profiles between these two regions (Fig. 6a). Hierarchical clustering of differentially expressed genes (DEGs) showed that approximately 80% of these genes were upregulated in the peripheral region. To further characterize these region-specific gene expression patterns, we conducted Gene Ontology (GO) enrichment analysis on the top 100 genes specific to the peripheral region. These genes were predominantly associated with biological processes such as G protein-coupled receptor signaling, regionalization, and locomotion regulation (Fig. 6b). In terms of molecular function, many of these genes encode proteins involved in DNA binding transcription factor activity, and signaling receptor activity (Fig. 6b).

Figure 6.

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Transcriptional differences between the central and peripheral regions of T. adhaerens. a) Hierarchical clustering analysis of DEGs between the central and peripheral regions of T. adhaerens. Three replicate samples for each region are shown, with red indicating upregulated genes and blue indicating downregulated genes. b) GO enrichment analysis of DEGs upregulated in the peripheral region, categorized into Biological Process (BP) and Molecular Function (MF). c) Comparative transcriptomic analysis reveals conserved axis-patterning genes between T. adhaerens and H. vulgsaris. Dashed boxes outline region-specific gene clusters in the heatmap, with color-coded gene categories representing different transcription factor families and Wnt signaling components. Putative orthologs between the two species are marked in red and connected by lines, showing that most peripheral-enriched genes in T. adhaerens are highly expressed in the hypostome of H. vulgaris. d) Diagram illustrating the relative expression positions of key polarity-related genes in the central and peripheral regions of T. adhaerens. e) Phylogenetic diagram depicting the presence of the primary body axis across metazoans. Red color highlights the expression regions of Wnt genes along the body axis. The role of Wnt genes in body axis determination remains unresolved in ctenophores. See also Figure S5.

Similar to placozoans, cnidarians are radially symmetric animals characterized by a primary OA axis, which has been proposed to share similarities with the AP axis in bilaterians (Schaffer et al. 2016; Lebedeva et al. 2021). Next, we compared the transcriptomic profiles between the central and peripheral regions in T. adhaerens with those from distinct positions along the OA axis of Hydra vulgaris (Ferenc et al. 2021) (Figure S5a). Considering that transcription factors, such as homeobox, forkhead, zinc finger, and T-box gene families, constituted the majority of the identified body axis-associated genes, we compared the transcription profiles between T. adhaerens and H. vulgaris by focusing on these transcription factors, as well as the Wnt signaling-related genes. The comparative transcriptomic analysis revealed a pronounced similarity between the peripheral region of T. adhaerens and the hypostome of H. vulgaris—a domain marked by strong expression of Wnt genes (Lengfeld et al. 2009) (Fig. 6c, Figure S5a). In contrast, the transcriptional profile of the central region of T. adhaerens closely mirrored that of the Hydra foot (Fig. 6c, Figure S5a). Further qPCR analysis also confirmed the enrichment of these transcription factors in the peripheral region of T. adhaerens, including Otp and Trox-2 (Jakob et al. 2004) (homeobox), FoxB and FoxD (forkhead), and zinc finger transcription factors Sp9 and Zic4 (Figure S5b). In contrast, the transcription factor FoxQ2 was predominantly expressed in the central region (Figure S5b). Moreover, we found that not only were multiple Wnt genes enriched in the peripheral region (Fig. 2d), but also the conserved Wnt secretion regulator Wntless (Figure S5b). Conversely, the Wnt inhibitor secreted frizzled-related protein (Sfrp) was predominantly localized to the central region (Figure S5b). Taken together, these results reveal a clear regionalized expression of both transcription factors and Wnt signaling components, providing strong molecular evidence for the existence of a central–peripheral body axis in T. adhaerens, which may be analogous to the aboral–oral axis in cnidarians and the AP axis in bilaterians (Fig. 6d and e).

Discussion

Wnt signaling is an ancient and highly conserved pathway unique to metazoans. While Wnt ligands are found in all metazoans, most bilaterian Wnt proteins first appeared in cnidarians, with only a few members identified in Ctenophora, Porifera, and Placozoa (Richards and Degnan 2009) (Fig. 1f). In bilaterians and cnidarians, Wnt signaling is essential for AP or OA axis determination during embryonic development. However, the mechanisms underlying body plan establishment in basal metazoans remain unclear. Using T. adhaerens as a model for placozoans, we demonstrate that Wnt signaling also regulate body plan formation in this phylum. Wnt genes are predominantly expressed in peripheral cells, where they promote cell proliferation and influence the spatial balance between central and peripheral regions. Overexpression of Wnt genes accelerates growth, increases peripheral cell proliferation, and reduces the relative size of the central region. Conversely, Wnt knockdown impairs growth and proliferation, highlighting the essential role of Wnt signaling in shaping the body plan of T. adhaerens. Interestingly, while several well-known Wnt antagonists, including Dickkopf (Dkk), Wnt-inhibitory factor 1 (WIF-1), and Cerberus, are absent in the T. adhaerens genome, secreted frizzled-related protein (SFRP), a classical Wnt antagonist, is strongly expressed in the central region (Fig. 6c and d, Figure S5b). This suggests that Wnt signaling may also play a negative regulatory role in central region growth.

In bilaterians, Wnt signaling is primarily involved in AP axis formation during embryonic development, where Wnt gene expression is typically restricted to the posterior region (Fig. 6e). Notably, sexual reproduction is rarely observed in placozoans under laboratory conditions, and it remains controversial whether embryonic development occurs in these animals. Although we did not detect Wnt gene expression during embryogenesis, we observed dynamic Wnt gene expression during individual growth following asexual division (Fig. 3). Interestingly, proliferating cells were also preferentially localized to the peripheral region during growth, consistent with the spatial distribution of Wnt expression (Figure S6). These findings suggest that Wnt-mediated axial patterning is established during organismal growth and maintained into adulthood. A comparable polarized distribution of Wnt signaling in adult stages has also been reported in other early-branching metazoans. For instance, in the Homoscleromorph sponge Oscarella, Wnt genes are specifically expressed at the adult ostia (canal openings), and activation of the Wnt/β-catenin pathway provokes the formation of ectopic ostia and increases ostia density (Lapebie et al. 2009). Similarly, Wnt genes are also expressed at the posterior pole of the larva in the demosponge Amphimedon queenslandica, a region considered to be the precursor of the ostia (Adamska et al. 2010). These observations further support the view that Wnt-mediated patterning of the primary body axis represents an ancient and conserved mechanism in metazoan evolution (Fig. 6e).

The transcriptional differences between the central and peripheral regions, along with the striking transcriptional similarity between the OA axis of cnidarians and the central–peripheral regions of placozoans, strongly support the existence of a central-peripheral body axis in T. adhaerens. Recent studies have highlighted molecular similarities between the OA axis of cnidarians and the AP axis of bilaterians, with the aboral side of cnidarians resembling the anterior region of bilaterians, while the oral side corresponds to the posterior region (Schaffer et al. 2016; Rentzsch and Holstein 2018). We hypothesize that the central–peripheral axis of Trichoplax may resemble the OA region of cnidarians or the AP axis of bilaterians. Interestingly, in the sea star Patiria miniata, the proposed “ambulacral–anterior” model also suggests that head-like characteristics are centrally positioned (Formery et al. 2023). In addition to this putative central–peripheral axis, a DV-like polarity has been suggested in placozoans (DuBuc et al. 2019). Expression analyses revealed that canonical DV regulators, including Bmp2/4 and Chordin-like genes, are co-localized on the same side of the animal (DuBuc et al. 2019). In cnidarians, Bmp and Chordin orthologs are expressed at the oral pole in an asymmetric manner, contributing to the establishment of the directive axis, a secondary body axis oriented perpendicular to the OA axis (Finnerty et al. 2004; Matus et al. 2006; Rentzsch et al. 2006; Saina et al. 2009; Kraus et al. 2016). Whether placozoan DV-like patterning reflects an ancestral condition related to bilaterian DV polarity, or instead represents a lineage-specific deployment of conserved signaling modules, remains unresolved.

Hox genes play central roles in bilaterian body plan regionalization. In placozoans, only a single Hox/ParaHox-related gene, Trox-2, has been identified. Trox-2 is predominantly expressed in the peripheral region of Trichoplax (Figure S5b), consistent with a role in axial patterning. However, knockdown of Trox-2 disrupts growth and fission (Jakob et al. 2004); it remains unclear whether this ancestral Hox-related gene contributes directly to body plan regionalization in placozoans. Given that neither a canonical DV axis nor Hox genes have been identified in sponges and ctenophores, the AP axis may represent one of the earliest axial systems in metazoan evolution (Martindale 2005; Genikhovich and Technau 2017). Clarifying the origin and subsequent diversification of body axes will therefore be essential for a comprehensive understanding of metazoan body plan evolution.

In summary, our results suggest that Wnt signaling may represents an evolutionarily conserved mechanism guiding body plan organization across metazoans, from basal lineages to vertebrates. The polarized expression of Wnt genes and their involvement in defining the central–peripheral axis in placozoans highlight the evolutionary flexibility of this pathway in patterning diverse animal body plans.

Materials and methods

Animal culture

Trichoplax adhaerens were generously provided by Dr. Carolyn L. Smith (NIH). Animals were maintained in Petri dishes containing filtered (0.22 μm) and autoclaved natural seawater at an environmental chamber and fed with Rhodomonas salina, as described previously (Jin et al. 2024).

Identification of Wnt signaling genes and phylogenetic analysis

Homologous sequences of core components of the T. adhaerens Wnt signaling pathway were identified using the NCBI protein BLAST program, and IDs of these homologs are provided in Table S1. To validate these homologs, a reverse BLAST analysis was performed, where candidate sequences identified in different phyla or species were used to confirm their evolutionary conservation. Once homologous sequences were validated, their domain organization was analyzed using SMART (Letunic et al. 2021).

To investigate the evolutionary relationships of T. adhaerens Wnt proteins, phylogenetic trees were constructed alongside Wnt proteins from various metazoans. The sequences used for phylogenetic analysis are provided in Table S2. Sequence alignment was performed using MUSCLE, and phylogenetic trees were inferred using IQ-TREE2 to generate an approximate ML tree (Minh et al. 2020). Tree robustness was assessed with 1,000 ultrafast bootstrap replicates, and the resulting tree was visualized using the iTOL web server. Additionally, Bayesian phylogenetic trees were reconstructed using MrBayes v3.2.7. The analysis was conducted under the invgamma rate model, with the amino acid substitution model set to prset aamodelpr = fixed (LG). A minimum of 1 × 106 generations were run across four chains, with sampling every 1,000 generations. The first 25% of samples were discarded as burn-in, and the remaining samples were used to construct the consensus tree. Convergence of the chains was evaluated by running two independent analyses simultaneously. The Bayesian phylogenetic tree was visualized and annotated using FigTree v1.4.4.

Plasmid construction and electroporation

Overexpression of Wnt genes was carried out following our recently published protocol (Jin et al. 2026). Briefly, a 2.18 kb β-actin promoter was amplified from the genomic DNA of T. adhaerens and ligated into a linearized CMV::GFP plasmid to generate a β-actin::GFP fusion construct. The full open reading frames of various Wnt genes were then amplified from cDNA and inserted into the linearized β-actin::GFP plasmid to replace the GFP sequence, resulting in Wnt expression constructs. All primers used are listed in Table S3. Electroporation was performed following a previously established protocol (Jin et al. 2026), and the parameters were set as follows: 65 V, 2,000 μF, 1 pulse.

RNA interference

The RNAi experiments were similar to those previously described (Jin et al. 2024; Li et al. 2025). Briefly, 4 μg of shRNA (mixture of three targeting shRNA) were incubated with 10 μL of 10 μg/μL homemade spike silica nanoparticles for 15 min at room temperature. Then the mixture was added to the wells to a final volume of 400 μL and incubated for 48 h in the darkness. For double and triple gene knockdown experiments, the volume of the mixture was doubled. The sequences of shRNA are listed in Table S3.

Whole-mount immunofluorescence

To assess cell proliferation during the growth of T. adhaerens, animals were placed at the bottom of a 35 mm plastic Petri dish containing 1 mL of seawater. The seawater was rapidly replaced with prechilled methanol (−80 °C), and the dish was transferred to a −80 °C freezer for overnight fixation. Following fixation, samples were rehydrated through sequential incubation to PBS. Blocking was performed at room temperature for 1 h using a solution of 3% goat serum, 2% horse serum, and 1% bovine serum albumin in PBS. Samples were then incubated overnight at 4 °C with the primary antibody against phospho-histone H3 (pH3) (Cell Signaling Technology, #3377), diluted 1:500 in blocking solution. Afterward, secondary antibodies were applied and incubated overnight at 4 °C. Nuclei were counterstained with DAPI for 30 min at room temperature. Images were captured using a Zeiss LSM900 confocal microscope. For proliferation analysis following functional perturbation, animals were treated with 0.1% colchicine for 6 h prior to fixation to arrest cells in mitosis.

Whole-mount FISH

Samples were fixed in methanol and rehydrated following the same protocol as immunofluorescence. After rehydration, specimens were permeabilized with 10 μg/mL Proteinase K for 1 to 2 min, followed by an additional fixation step in 4% paraformaldehyde (PFA) prior to hybridization. DIG-labeled probes were hybridized to T. adhaerens for 16 h at 58 °C in hybridization buffer containing 50% formamide, 5× SSC, 50 μg/mL heparin salts, 100 μg/mL sheared salmon sperm DNA, 1% SDS, and 0.5% Tween-20. Post-hybridization, samples underwent sequential washes in 50% formamide/2× SSCT, 2× SSCT, 0.2× SSCT, and PBS. Following hybridization, samples were blocked and incubated overnight at 4 °C in the darkness with a peroxidase-conjugated anti-DIG antibody (1:2,000; Roche). The signal was developed using the TSA kit (Akoya Biosciences) in the darkness. Nuclei were counterstained with DAPI, and slides were sealed before imaging with a Zeiss LSM900 confocal microscope. All primers used for probe synthesis are listed in Table S3.

Growth and area measurement of T. adhaerens

To monitor the growth of T. adhaerens, small individuals of similar body size were transferred into 35 mm Petri dishes for individual culture. Animals were imaged using a ZEISS Stemi508 stereomicroscope equipped with a WEITU camera. Images were captured with WT-1000GM software, converted to 8-bit format in Fiji, and the area of each animal was measured by manually outlining its shape. At selected time points during growth, animals were either harvested for total RNA extraction and subsequent qPCR analysis or fixed for whole-mount FISH to examine Wnt gene expression. To construct the growth curve, the area measured for each animal was normalized to the mean initial area of its group.

Gene expression analysis

Total RNA was extracted from approximately 20 animals using the RNA Isolation Kit-BOX2 (Vazyme). Reverse transcription was carried out with the HiScript III 1st Strand cDNA Synthesis Kit (Vazyme), and gene expression levels were quantified using ChamQ Universal SYBR qPCR Master Mix (Vazyme). The Ef1α gene of T. adhaerens served as the internal control for expression analysis. Each experiment was repeated at least three times. All the primers used are listed in Table S3.

Pharmaceutical treatments

To activate the Wnt/β-catenin signaling pathway, T. adhaerens was treated with BIO for 2 d. BIO was first dissolved in dimethyl sulfoxide (DMSO) to prepare a 50 mM stock solution. The optimal working concentration of BIO (0.25 μM) was determined based on a preliminary dose-response experiment. The working solution was prepared by diluting the stock solution in 3 mL of seawater, while the control group received an equivalent volume of DMSO.

RNA-seq transcriptome analysis

To examine gene expression differences between the central and peripheral regions, T. adhaerens were dissected along a radial line approximately one-third of the radius from the edge, using a fine hair as a cutting tool. This procedure separated each specimen into an inner central region and an outer, ring-like peripheral region. Total RNA was extracted separately from each region and used for qPCR analysis or transcriptome sequencing.

For the RNA-seq data, raw read quality was assessed with FastQC. Cleaned reads were aligned to the T. adhaerens reference genome, and transcript quantification was performed using raw counts and normalized with the DESeq2 package in R. Differential expression analysis between the central and peripheral regions was also carried out using DESeq2. Prior to analysis, genes with low expression across all samples were filtered out. DEGs between experimental groups were identified based on a threshold of |log2FoldChange| ≥ 1 and adjusted P-value < 0.05. Principal component analysis (PCA) was conducted using the prcomp function in R to visualize sample clustering based on gene expression profiles. Heatmaps of DEGs were generated using the pheatmap package. GO enrichment analysis was performed on the DEGs using the ClusterProfiler package in R to identify significantly enriched biological processes and molecular functions.

Positional RNA-seq data of Hydra vulgaris were obtained from ArrayExpress database (accession numbers E-MTAB-9676), and the reference genome of H. vulgaris was obtained from the accession GCA_038396675.1. The data were processed using a consistent pipeline: quality checking with Fqtrim v0.9.7, alignment with STAR v2.7.6a, and quantification with FeatureCounts. Transcripts per million (TPM) were calculated in R for expression analysis. Orthologous genes between T. adhaerens and H. vulgaris were identified using OrthoFinder, and the resulting ortholog pairs were filtered and manually curated for downstream comparative analysis.

Single-cell RNA-seq data of T. adhaerens were downloaded from Figshare (https://doi.org/10.6084/m9.figshare.26798683), and the analysis was similar to that previously described (Jin et al. 2024). After quality control and filtering in R, cells were clustered using Seurat v5.0.1 with parameters dims = 1:10 and resolution = 0.5. Clustering was visualized via Uniform Manifold Approximation and Projection (UMAP), and marker genes were identified using the FindAllMarkers function for cluster annotation.

Statistical analysis

For quantitative statistical analysis, the signal densities of Wnt8 and pH3 staining were quantified as ×103 signals per square millimeter, while the density of PWN-positive cells was determined by counting the number of positive cells per millimeter along the body margin. These data were further normalized to arbitrary units. All data distributions were tested for normality using Prism 9 (GraphPad). For datasets following a normal distribution, statistical comparisons were performed using an unpaired two-tailed t-test. Statistical significance is indicated by the corresponding P-values: P < 0.05 (*), P < 0.01 (**), P < 0.001 (***), and P < 0.0001 (****); “ns” denotes no significant difference. Graphs were generated using Prism 9, with data presented as mean ± standard deviation (SD). Each experimental condition was tested in at least three independent biological replicates to ensure reproducibility.

Supplementary Material

msag089_Supplementary_Data
msag089_supplementary_data.docx (27.5MB, docx)

Acknowledgments

We thank members of the Zhao Lab and FANG Center for their kind help during the preparation of this manuscript. We also gratefully acknowledge the outstanding support from the core facilities of the IEMB and FANG Center at OUC.

Contributor Information

Guotao Di, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.

Minjun Jin, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.

Wanqing Li, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.

Meng Yuan, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.

Yifan Zhang, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China.

Chengtian Zhao, Fang Zongxi Center for Marine EvoDevo, MoE Key Laboratory of Marine Genetics and Breeding, College of Marine Life Sciences, Ocean University of China, Qingdao 266003, China; Institute of Evolution & Marine Biodiversity, Ocean University of China, Qingdao 266003, China; Laboratory for Marine Biology and Biotechnology, Qingdao National Laboratory for Marine Science and Technology, Qingdao 266003, China.

Author contributions

C.Z. and G.D. conceived the idea for the project. C.Z., G.D., and M.J. designed the experiments. G.D., M.J., and W.L. conducted the experiments. G.D., M.J., Y.Z., and M.Y. analyzed the results. C.Z. and G.D. wrote the paper. All authors reviewed the results and approved the final version of the manuscript.

Supplementary material

Supplementary material is available at Molecular Biology and Evolution online.

Funding

This work was supported by the National Natural Science Foundation of China (Nos. 32125015, W2411026 to C.Z., No. 324B2023 to W.L., No. 32500727 to M.J.), the Postdoctoral Fellowship Program of CPSF (GZC20251747) to M.J., and funds from Laoshan Laboratory (LSKJ202203204) to C.Z.

Data availability

All data supporting the findings of this study are available within the paper and its Supplementary Information. The transcriptome datasets for the Trichoplax adhaerens central and peripheral regions have been deposited in Figshare and can be accessed via DOI: 10.6084/m9.figshare.31808356. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

msag089_Supplementary_Data
msag089_supplementary_data.docx (27.5MB, docx)

Data Availability Statement

All data supporting the findings of this study are available within the paper and its Supplementary Information. The transcriptome datasets for the Trichoplax adhaerens central and peripheral regions have been deposited in Figshare and can be accessed via DOI: 10.6084/m9.figshare.31808356. Additional data supporting the findings of this study are available from the corresponding author upon reasonable request.

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