Editor's evaluation: Canonical Wnt signaling and the regulation of divergent mesenchymal Fgf8 expression in axolotl limb development and regeneration

Field: Biochemistry, Genetics and Molecular Biology (Life Sciences)

Article Figures and data Abstract Editor's evaluation Introduction Results Discussion Materials and methods Data availability References Decision letter Author response Article and author information Metrics Abstract The expression of fibroblast growth factors (Fgf) ligands in a specialized epithelial compartment, the Apical Ectodermal Ridge (AER), is a conserved feature of limb development across vertebrate species. In vertebrates, Fgf 4, 8, 9, and 17 are all expressed in the AER. An exception to this paradigm is the salamander (axolotl) developing and regenerating limb, where key Fgf ligands are expressed in the mesenchyme. The mesenchymal expression of Amex.Fgf8 in axolotl has been suggested to be critical for regeneration. To date, there is little knowledge regarding what controls Amex.Fgf8 expression in the axolotl limb mesenchyme. A large body of mouse and chick studies have defined a set of transcription factors and canonical Wnt signaling as the main regulators of epidermal Fgf8 expression in these organisms. In this study, we address the hypothesis that alterations to one or more of these components during evolution has resulted in mesenchymal Amex.Fgf8 expression in the axolotl. To sensitively quantify gene expression with spatial precision, we combined optical clearing of whole-mount axolotl limb tissue with single molecule fluorescent in situ hybridization and a semiautomated quantification pipeline. Several candidate upstream components were found expressed in the axolotl ectoderm, indicating that they are not direct regulators of Amex.Fgf8 expression. We found that Amex.Wnt3a is expressed in axolotl limb epidermis, similar to chicken and mouse. However, unlike in amniotes, Wnt target genes are activated preferentially in limb mesenchyme rather than in epidermis. Inhibition and activation of Wnt signaling results in downregulation and upregulation of mesenchymal Amex.Fgf8 expression, respectively. These results implicate a shift in tissue responsiveness to canonical Wnt signaling from epidermis to mesenchyme as one step contributing to the unique mesenchymal Amex.Fgf8 expression seen in the axolotl. Editor's evaluation The authors investigate the upstream regulators of axolotl Fgf8 expression in limb development and regeneration with the aim of identifying the reasons for its unique mesenchymal localization. The results suggest some refractoriness of the axolotl epidermis to activate Wnt signaling although Fgf8 expression in the anterior mesenchyme is still under Wnt control. https://doi.org/10.7554/eLife.79762.sa0 Decision letter eLife's review process Introduction Signaling mediated by members of the fibroblast growth factor (Fgf) family is necessary for normal limb development and for limb regeneration in the salamander Ambystoma mexicanum (axolotl), a primary model for regeneration research (Nacu et al., 2016; Purushothaman et al., 2019). In less-regenerative tetrapods, like mouse (Mus musculus), Fgf signaling is similarly required for limb development (Lewandoski et al., 2000; Moon and Capecchi, 2000; Sun et al., 2002; Mariani et al., 2008). In mammals, Fgfs 4, 8, 9, and 17 are produced by cells in the Apical Ectodermal Ridge (AER), a thickened epidermal structure at the distal dorsoventral boundary of the limb bud. Fgf signals from the AER act on the underlying mesenchyme to sustain its growth and patterning. Of the AER Fgfs, Mmu.Fgf8 is the only one that is individually required for normal limb development in mice (Mariani et al., 2008). Limb regeneration in the axolotl proceeds through the formation of the blastema, a structure that resembles the limb bud, both at the morphological and transcriptional level (Gerber et al., 2018). In both the axolotl limb blastema and limb bud, Amex.Fgfs 8, 9, and 17 are expressed in the mesenchymal compartment, unlike in other vertebrates, while Amex.Fgf4 transcription is negligible or absent (Christensen et al., 2002; Nacu et al., 2016; Purushothaman et al., 2019). Restriction of Amex.Fgf8 expression to the mesenchymal compartment is to our knowledge unique to the salamander limb, where it serves a crucial role in connecting positional identity with growth and patterning during limb regeneration. Nacu et al. showed that Amex.Fgf8 expression is restricted to the anterior mesenchyme of the regenerating blastema, while cells of the posterior blastema mesenchyme do not express Amex.Fgf8 (Nacu et al., 2016). When a limb amputation is performed, anterior cells express Amex.Fgf8 and posterior cells express Amex.Shh, initiating a positive feedback loop in which Amex.Fgf8 and Amex.Shh reciprocally maintain each other’s expression: prolonged expression of both signaling molecules sustains blastema growth and limb regeneration. The anterior mesenchymal expression of Amex.Fgf8 therefore mediates the communication between anterior and posterior cells in the blastema. The incompetence of other blastema cells to transcribe Amex.Fgf8 acts as a safeguard to restrict the initiation of blastema formation to injuries involving both anterior and posterior limb cells. It is interesting to note that the positive feedback relationship between Fgfs and Shh was initially described in the mammalian limb, between Shh in the posterior mesenchyme and Fgf8 in the epidermis (Laufer et al., 1994; Niswander et al., 1994). Expression of Amex.Fgf8 in the mesenchyme could serve to couple limb outgrowth with anterior–posterior positional information, a capacity that is restricted to mesenchymal cells in the axolotl (Carlson, 1975). Given the interesting role mesenchymal Amex.Fgf8 expression plays in axolotl limb regeneration, we asked: how has the axolotl limb signaling network been rewired to elicit this adaptation? In the mouse limb bud AER, expression of Mmu.Fgf8 is controlled at the genetic level by a set of enhancers proximal to the gene locus (Marinić et al., 2013). Schlossnig et al. recently determined that conserved orthologs of the majority of these enhancers are present in the axolotl genome, and that at least one of these drives expression of a reporter construct in the mesenchyme of the axolotl limb (Schloissnig et al., 2021). This suggests that changes at the level of transacting factors may determine the divergent expression of Amex.Fgf8 in the axolotl limb. Transcription factors and intercellular signaling molecules that regulate AER-associated Fgf8 expression have been identified in chicken (Gallus gallus) and mouse. In mice, AER expression of Mmu.Fgf8 has been shown to be dependent on mesenchymal Mmu.Fgf10 acting through the epidermis-specific receptor isoform Mmu.Fgfr2b (Xu et al., 1998; De Moerlooze et al., 2000; Zhang et al., 2006; Sekine et al., 2019). Using the chick model, it was established that Gga.Fgf10 induces epidermal expression of the canonical Wnt ligand Gga.Wnt3a. Wnt3a (chick) and Wnt3 (mouse), together with BMPR1a-dependent signaling, activate canonical Wnt signaling within AER cells to induce Fgf8 (Kengaku et al., 1998; Kawakami et al., 2001; Barrow et al., 2003). In mice, genetic removal of epidermal Mmu.Wnt3 or the canonical Wnt signaling effector β-catenin is sufficient to abolish Mmu.Fgf8 expression in the limb, while expression of a constitutively active β-catenin markedly expands the Mmu.Fgf8 expression domain and induces ectopic Mmu.Fgf8 expression in the dorsal and ventral epidermis (Barrow et al., 2003; Soshnikova et al., 2003). Furthermore, mesenchymal expression of a constitutively active β-catenin construct appeared to potentially induce sporadic mesenchymal expression of Mmu.Fgf8 (Hill et al., 2006). Similarly in the chick developing limb, misexpression of β-catenin, Gga.Wnt3a or the Wnt transcription factor Gga.Lef1 is sufficient to induce Gga.Fgf8 expression ectopically in the dorsal and ventral epidermis (Kengaku et al., 1998). In addition to Wnt signaling components, Buttonhead transcription factors Sp8 and Sp6 have also been shown to be necessary for Fgf8 expression and act downstream of canonical Wnt signaling in the mammalian limb (Kawakami et al., 2004; Talamillo et al., 2010; Lin et al., 2013; Haro et al., 2014). Finally, Dlx factors Dlx5 and Dlx6 have been implicated in the maintenance of the AER and are required to sustain Fgf8 expression, but are dispensable for its induction (Robledo et al., 2002). Little is known about the Amex.Fgf8 regulatory machinery in the axolotl limb. Amex.Fgf10 is expressed in the mesenchyme, along with Fgfs 8, 9, and 17 (Christensen et al., 2002; Nacu et al., 2016; Purushothaman et al., 2019). This challenges the epithelial–mesenchymal interaction model of the mammalian and avian limb where mesenchymal Fgf10 and epidermal Fgf8 reciprocally sustain each other’s expression acting on different, tissue-specific, receptor isoforms. A recent publication reported that no Fgf receptor is expressed in the axolotl epidermis (Purushothaman et al., 2019), suggesting that the epidermal side of the interaction may be completely absent. This is at odds with previous studies that showed Fgfr2b expression in the basal epidermis of the newt blastema (Poulin and Chiu, 1995). The expression domain of Amex.Wnt3 ligands, the major Fgf8 AER regulators, has not been determined in the axolotl limb although Wnt3a expression is present in the salamander limb blastema as determined by qPCR (Singh et al., 2012). Another canonical Wnt ligand, Amex.Wnt10 was found in similar amounts in lateral injuries (surface wounds that do not trigger regeneration) and limb amputations, suggesting its role may be restricted to wound response (Knapp et al., 2013). Multiple studies reported that chemical activation of the canonical Wnt pathway increases cell proliferation in the salamander regenerating blastema (Singh et al., 2012; Wischin et al., 2017) but it has not been elucidated if the main site of canonical Wnt signaling activity is the blastema epidermis or mesenchyme. Although overexpression of the Wnt antagonist Axin1 is sufficient to block limb development and regeneration in the axolotl (Kawakami et al., 2006), it is unclear if this effect is mediated through downregulation of Amex.Fgf8. In the axolotl limb, it has also not been determined whether the transcription factors Amex.Sp6, Amex.Sp8, Amex.Dlx5, Amex.Dlx6, and Amex.Lef1 display mesenchymal or epidermal expression, nor if their function in regulating Amex.Fgf8 is conserved. Despite the expression of Fgf ligands in the mesenchymal compartment, classical experiments have shown that the epidermis of the axolotl blastema is necessary for both initial accumulation and proliferation of cells in the blastema (Thornton, 1957; Campbell and Crews, 2008). For example, Axolotl MARCKS like protein has been identified as an epidermal factor that induces the initial proliferation response in the blastema (Sugiura et al., 2016). Recent single-cell studies have highlighted that different cell populations are present in the regenerating axolotl epidermis (Leigh et al., 2018; Rodgers et al., 2020; Li et al., 2021), and previous studies have indicated that the basal layer of the axolotl epidermis expresses some markers of the mammalian AER, including Amex.Fibronectin1 and Amex.Dlx3 (Christensen and Tassava, 2000). In this study, we take advantage of published single-cell RNA sequencing (scRNA seq) datasets for the axolotl limb bud (Lin et al., 2021) and blastema (Li et al., 2021) to elucidate the tissue compartment specificity of candidate Amex.Fgf8 regulators. To precisely visualize tissue transcripts in the anatomical complexity of the developing and regenerating axolotl limb, we further develop a protocol that combines Hybridization Chain Reaction (HCR) in situ hybridization (Choi et al., 2018) with Ce3D tissue clearing (Li et al., 2017; Li et al., 2019) and light sheet imaging. We then implemented semiautomated image analysis pipelines to quantitatively compare transcript abundances in different tissue compartments and upon pharmacological perturbation. We identify conserved and divergent features of the axolotl limb signaling network and implicate enhanced canonical Wnt signaling in the axolotl limb mesenchyme as one of the regulators of Amex.Fgf8 mesenchymal expression. Results Expression of transcription factors known to regulate Fgf8 in the chick and mouse AER Several transcription factors have been functionally linked with the induction or maintenance of Fgf8 in the mammalian AER (Zuniga and Zeller, 2020). To determine whether these transcription factors are potentially upstream of axolotl Amex.Fgf8, we investigated if they are expressed in the epidermis and/or mesenchyme in the axolotl developing and regenerating limb. To this end, we first analyzed published scRNA seq datasets for the axolotl stage 52 axolotl hindlimb bud (Lin et al., 2021) and 7-day postamputation axolotl forelimb blastema (Li et al., 2021). Both single-cell datasets were reanalyzed to match the latest axolotl transcriptome annotation (Schloissnig et al., 2021) and reclustered, using known markers for cluster assignment (Figure 1—figure supplements 1 and 2). We then used HCR in situ hybridization in tissue sections and whole-mount cleared limb preparations of forelimb buds and blastemas of comparable stages (stage 45–46 forelimb bud, 7-day postamputation forelimb blastema) to validate the expression domains and acquire spatial information. To achieve precise visualization of transcripts in the anatomical complexity of the entire axolotl developing and regenerating limb, we combined HCR in situ hybridization with Ce3D tissue clearing and light sheet imaging (see Materials and methods). Buttonhead transcription factors Mmu.Sp6 and Mmu.Sp8 are expressed in the AER of the mouse developing limb, and their knockout abolishes Mmu.Fgf8 expression (Lin et al., 2013; Haro et al., 2014). Analysis of scRNA seq data from the axolotl limb bud and blastema (Li et al., 2021; Lin et al., 2021) revealed expression of Amex.Sp6 and Amex.Sp8 in the epidermis and not in mesenchyme (Figure 1A, A’, B, B’). We performed whole-mount HCR staining in regenerating axolotl limb blastemas and limb buds (Figure 2B, B’) and confirmed that Amex.Sp6 is expressed in the epidermis of the axolotl limb bud and regenerating blastema. Interestingly, we detected Amex.Sp6 also in the mesenchyme of the axolotl limb bud, in a domain proximal to Amex.Fgf8 expression (Figure 2—figure supplement 1). This proximal mesenchymal expression of Amex.Sp6 was absent in the blastemas that we analyzed and is unlikely to regulate the more distal Amex.Fgf8 expression in the axolotl limb bud. Using HCR in situ hybridization, we then found that Amex.Sp8 is exclusively expressed in the epidermis (Figure 2C, C’), with a strong enrichment in the basal layer (the inner layer facing the mesenchyme) in both the axolotl and limb bud and blastema. This indicates that Buttonhead factors are unlikely to directly regulate Amex.Fgf8 expression in the mesenchyme and that the lack of Amex.Fgf8 expression in the axolotl epidermis is not due to the absence of these factors. Figure 1 with 2 supplements see all Download asset Open asset Expression of AER transcription factors in the axolotl limb bud and limb blastema assessed by reanalysis of Axolotl scRNA seq datasets. Reanalysis of scRNA seq data from blastema (A–E, data from Li et al., 2021) or limb bud (A’–E’, data from Lin et al., 2021). (A, A’) UMAPs of Axolotl scRNA seq expression data for Sp6. Expression is detected in the epidermis of the axolotl blastema and limb bud. (B, B’) UMAPs of Axolotl scRNA seq expression data for Sp8. Expression is detected in the epidermis of the axolotl blastema and limb bud. (C, C’) UMAPs of Axolotl scRNA seq expression data for Dlx5. Expression is detected in the epidermis and mesenchyme of the axolotl blastema and limb bud. (D, D’) UMAPs of Axolotl scRNA seq expression data for Dlx6. Expression is detected in the epidermis and mesenchyme of the axolotl limb bud. Expression is detected only in few blastema cells. (E, E’) UMAPs of Axolotl scRNA seq expression data for Lef1. Expression is detected in the epidermis and mesenchyme of the axolotl blastema and limb bud. Figure 2 with 1 supplement see all Download asset Open asset Expression of AER transcription factors in the axolotl limb blastema and limb bud assessed by HCR in situ hybridization. (A, A’) Schematic outlining the mesenchymal and epidermal compartments in a longitudinal section of an axolotl blastema and limb bud. (B, B’) Expression of Sp6 in the epidermis and of Fgf8 in the mesenchyme of the axolotl blastema and limb bud revealed by HCR (single planes from whole-mount images, n = 4). (C, C’) Expression of Sp8 in the epidermis and of Fgf8 in the mesenchyme of the axolotl blastema and limb bud revealed by HCR (single planes from whole-mount images, n = 4). (D, D’) Expression of Fgf8 in the mesenchyme as well as of Dlx5 in the mesenchyme and basal epidermis of the axolotl limb bud and blastema (single planes from whole-mount images, n = 4). (E, E’) Expression of the mesenchymal marker Prrx1, and of Lef1 in the mesenchyme and basal epidermis of the axolotl blastema and limb bud (single planes from whole-mount images, n = 4). For microscopy images right panels represent magnifications of the outlined boxes, M = mesenchyme, E = epidermis. Dashed lines demarcate epidermal–mesenchymal boundaries. Dlx factors Mmu.Dlx5 and Mmu.Dlx6 are also expressed in the mouse AER, where they function to maintain Mmu.Fgf8 expression (Robledo et al., 2002). Single-cell analysis of the axolotl blastema and limb bud detected transcripts in both the epidermal and mesenchymal compartments (Figure 1C, C’, D, D’). Whole-mount HCR staining revealed that expression of Amex.Dlx5 is enriched in the distal mesenchyme and in the basal epidermis of the blastema (Figure 2D) and limb bud (Figure 2D’). Expression of Amex.Dlx5 in the limb epidermis suggests that in the axolotl, similar to the mouse, Amex.Dlx5 is not sufficient to induce Amex.Fgf8 expression. The strong mesenchymal Amex.Dlx5 expression distinguishes the axolotl limb from the mammalian one and could be consistent with a conserved role in maintaining Amex.Fgf8 expression. The canonical Wnt pathway is a main regulator of Fgf8 expression in the chicken and mouse developing limb. In the chick, the Wnt transcription factor Gga.Lef1 is required for AER Gga.Fgf8 expression. Furthermore, Gga.Lef1 overexpression is sufficient to induce ectopic Gga.Fgf8 expression in the non-AER epidermis (Kengaku et al., 1998). In mouse, the double knockout of the Wnt transcription factors Mmu.Lef1 and Mmu.Tcf1 abolishes AER Mmu.Fgf8 expression (Galceran et al., 1999). In our scRNA seq analysis, we could detect expression of Amex.Lef1 in both epidermal and mesenchymal cells (Figure 1E, E’). Using HCR, we found that Amex.Lef1 is expressed broadly in the limb bud and blastema mesenchyme (Figure 2E, E'). We also detected expression in the limb bud and blastema epidermis, strongest in the distal portion of the basal epidermis (Figure 2E, E’). Considering that overexpression of Gga.Lef1 in chick is sufficient to induce ectopic Gga.Fgf8 expression (Kengaku et al., 1998), we note here that, at least at the levels observed, it is not sufficient to allow Amex.Fgf8 expression in the axolotl limb epidermis. The broad expression pattern of Amex.Lef1 in the axolotl limb mesenchyme could be compatible with a role in inducing or maintaining mesenchymal Amex.Fgf8 expression in axolotl. Our analysis revealed that the transcription factors Amex.Sp8, Amex.Sp6, Amex.Dlx5, Amex.Dlx6, and Amex.Lef1 are expressed in the axolotl developing and regenerating limb epidermis, in particular all are present in the basal epidermis. This indicates that, in the axolotl limb epidermis, the combined endogenous expression of these factors is not sufficient to activate Amex.Fgf8 transcription. While expression of Sp factors appeared uniquely epidermal, Amex.Dlx5, Amex.Dlx6, and Amex.Lef1 are expressed in the limb bud and blastema mesenchyme, leaving open the possibility that they have a role in the regulation of Amex.Fgf8 expression in this tissue compartment. Conserved and divergent expression of canonical Wnt pathway components and upstream regulators Canonical Wnt signaling controls AER-associated Fgf8 expression and mediates the communication between the epidermal and mesenchymal limb compartments in chick and mouse. To verify if this interaction is conserved in axolotl, we decided to investigate the expression of the canonical Wnt3 ligands and of the components that are known to induce their expression in the mammalian limb bud AER, primarily Fgf10 and its receptor Fgfr2b. We furthermore profiled Rspo2, an extracellular potentiator of Wnt signaling that is necessary for normal limb development (Xu et al., 1998; De Moerlooze et al., 2000; Zhang et al., 2006; Sekine et al., 2019). Finally, to localize the site of canonical Wnt signaling, we examined the expression of Axin2, a target of the canonical Wnt pathway that is often used as a readout of signaling activation strength (Nam et al., 2007; Itou et al., 2011; Eckei et al., 2016; Nusse and Clevers, 2017). Conserved gene expression features In both blastema and limb bud scRNA seq datasets, we detected Amex.Fgf10 transcripts in the mesenchymal cluster (Figure 3A, A’), consistent with previous reports (Christensen et al., 2002; Nacu et al., 2016; Purushothaman et al., 2019). Using HCR in situ hybridization we confirmed strong and broad mesenchymal expression (Figure 4B, B’). Surprisingly, we additionally found Amex.Fgf10 transcripts in a small and distinct cell population located in the basal layer of the distal epidermis. To exclude that this population is of mesenchymal origin we performed HCR in situ hybridization and lineage tracing using Prrx1-Cre transgenic axolotls (Figure 4—figure supplement 1; Gerber et al., 2018). During mammalian limb development, mesenchymal Fgf10 signals to the epidermis via the specific receptor isoform Fgfr2b (Xu et al., 1998; De Moerlooze et al., 2000; Zhang et al., 2006; Sekine et al., 2019). In the axolotl, using HCR, we detected Amex.Fgfr2b transcripts through the entire basal epidermis of the blastema (Figure 4C) and limb bud (Figure 4C’), consistent with Amex.Fgf10 signaling to the epidermis as in other vertebrates. Figure 3 Download asset Open asset Expression of Fgf10 and of the canonical Wnt pathway components Wnt3a, Axin2, Rspo2 in the axolotl limb bud and limb blastema assessed by reanalysis of Axolotl scRNA seq datasets. Reanalysis of scRNA seq data from blastema (A–D, data from Li et al., 2021) or limb bud (A’–D’, data from Lin et al., 2021). (A, A’) UMAPs of Axolotl scRNA seq expression data for Fgf10. Expression is detected in the mesenchyme of the axolotl blastema and limb bud. (B, B’) UMAPs of Axolotl scRNA seq expression data for Wnt3a. Expression is detected in the epidermis of the axolotl blastema and limb bud. (C, C’) UMAPs of Axolotl scRNA seq expression data for Axin2. Expression is detected in the prevalently in the mesenchyme of the axolotl blastema and limb bud. (D, D’) UMAPs of Axolotl scRNA seq expression data for Rspo2. Expression is detected in the mesenchyme of the axolotl limb bud. Expression is detected only in few blastema cells. Figure 4 with 2 supplements see all Download asset Open asset Expression of Fgf10, Fgfr2b and of the canonical Wnt pathway components Wnt3a, Axin2, Rspo2 in the axolotl limb bud and limb blastema evaluated by HCR in situ hybridization. (A, A’) Schematic outlining the mesenchymal and epidermal compartments in a longitudinal section of an axolotl blastema and limb bud. (B, B’) Expression of Prrx1 and Fgf10 in the mesenchyme of the axolotl blastema and limb bud revealed by HCR (single planes from whole-mount images, n = 4). Arrows point to weak Fgf10 expression present in the distal basal epidermis of the blastema and limb bud. (C, C’) Expression of Fgf8 in the mesenchyme and expression of Fgfr2b in the basal epidermis of the axolotl blastema and limb bud revealed by HCR (single planes from whole-mount images, n = 4). (D, D’) Expression of Wnt3a in the basal epidermis and Axin2 in the mesenchyme and basal epidermis of the axolotl blastema and limb bud (single planes from whole-mount images, n = 4). Axin2 expression is stronger in the mesenchyme and weaker in the epidermis. (E, E’) Expression of Rspo2 in the mesenchyme and Axin2 in the mesenchyme and basal epidermis of the axolotl blastema and limb bud (single plane from whole-mount image, n = 4). Bright green structures (*) in the blastema outer epidermis are For microscopy images right panels represent magnifications of the outlined boxes, M = mesenchyme, E = epidermis. Dashed lines demarcate epidermal–mesenchymal boundaries. Fgf10 signaling is to be required for expression of Mmu.Wnt3 in mouse and Gga.Wnt3a in We therefore assessed expression of both Amex.Wnt3 and we found Amex.Wnt3a transcripts in the epidermal cluster (Figure while we could only detect negligible Amex.Wnt3 transcripts in cell HCR data confirmed that Wnt3a expression is present in the axolotl limb bud and blastema basal epidermis (Figure D’). These results suggest that the epidermal induction of Amex.Wnt3a is downstream of mesenchymal Amex.Fgf10 signaling in the axolotl limb as in mouse. Wnt signaling activity and the Rspo2 divergent gene expression features In mouse and chick limb development, epidermal signaling from Mmu.Wnt3 or Gga.Wnt3a is to induce canonical Wnt signaling in the AER (Xu et al., 1998; De Moerlooze et al., 2000; Zhang et al., 2006; Sekine et al., 2019). This activation results in strong AER expression of Axin2, a canonical Wnt target that is often used to determine pathway activity (Nam et al., 2007; Itou et al., 2011; Eckei et al., 2016; Nusse and Clevers, 2017). Wnt3 ligands act also on the underlying mesenchyme, where they induce expression of Axin2, at levels (Kengaku et al., 1998). To determine the site of canonical Wnt signaling in the axolotl limb, we evaluated the expression of Single-cell analysis indicated that expression in the axolotl limb is the mesenchymal compartment (Figure Using whole-mount HCR, we confirmed strong expression in the mesenchyme, with a distal and enrichment in both the axolotl blastema (Figure and limb bud (Figure E’). expression is also but The mesenchymal enrichment in expression distinguishes the axolotl limb from the mammalian a ligand that acts as an for Wnt signaling, is expressed in the mouse limb AER where it to canonical Wnt signaling in the Rspo2 gene limb in mice, and of in (Nam et al., 2007; et al., et al., et al., et al., 2018). Our single-cell analysis revealed that expression is absent from the axolotl developing and regenerating limb epidermis but present in the mesenchyme (Figure D’). We confirmed the mesenchymal expression of using HCR (Figure E’). In both the axolotl limb bud and blastema, mesenchymal expression is along the dorsoventral suggesting a role in dorsoventral limb patterning (Figure 4—figure supplement 2). Amex.Fgf8 expression is to pharmacological of Wnt signaling The mesenchymal enrichment of transcripts to if canonical Wnt signaling Amex.Fgf8 expression in the mesenchyme of the axolotl regenerating limb, and whether Wnt signaling in the epidermis be sufficient to induce ectopic Amex.Fgf8 expression. To this end, we regenerating axolotls with a chemical Wnt or antagonist and used HCR in whole-mount blastema to visualize the effect on transcription of Amex.Fgf8, using as a readout for pathway perturbation. To the effect of the we a semiautomated image analysis that the of gene expression the removal of the from cells in the blastema, and the of gene expression levels (Figure supplement Materials and methods). to the role of Wnt signaling in maintaining Amex.Fgf8 expression, we performed in regenerating were at to a blastema and then were for in a of an of the canonical Wnt pathway that acts by Axin2 and the of a similar in and Amex.Fgf8 transcripts to controls (Figure suggesting that canonical Wnt signaling is required to sustain expression of Amex.Fgf8 in the axolotl blastema mesenchyme. Figure with 3 supplements see all Download asset Open asset of pharmacological of Wnt signaling on Fgf8 expression in the axolotl blastema. expression levels of Fgf8 or Axin2 as assessed by fluorescent of HCR the gene expression the from one blastema, each for each the of the