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Wnt proteins can direct planar cell polarity in vertebrate ectoderm

  1. Chih-Wen Chu
  2. Sergei Y Sokol  Is a corresponding author
  1. Icahn School of Medicine at Mount Sinai, United States
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Cite as: eLife 2016;5:e16463 doi: 10.7554/eLife.16463

Abstract

The coordinated orientation of cells across the tissue plane, known as planar cell polarity (PCP), is manifested by the segregation of core PCP proteins to different sides of the cell. Secreted Wnt ligands are involved in many PCP-dependent processes, yet whether they act as polarity cues has been controversial. We show that in Xenopus early ectoderm, the Prickle3/Vangl2 complex was polarized to anterior cell edges and this polarity was disrupted by several Wnt antagonists. In midgastrula embryos, Wnt5a, Wnt11, and Wnt11b, but not Wnt3a, acted across many cell diameters to orient Prickle3/Vangl2 complexes away from their sources regardless of their positions relative to the body axis. The planar polarity of endogenous Vangl2 in the neuroectoderm was similarly redirected by an ectopic Wnt source and disrupted after depletion of Wnt11b in the presumptive posterior region of the embryo. These observations provide evidence for the instructive role of Wnt ligands in vertebrate PCP.

https://doi.org/10.7554/eLife.16463.001

Introduction

Studies in Drosophila revealed the segregation of core PCP proteins to opposite sides of epithelial cells (Goodrich and Strutt, 2011; Peng and Axelrod, 2012). This mutually exclusive localization has been preserved in vertebrate tissues and is thought to be essential for multiple morphogenetic processes, including gastrulation and neurulation (Gray et al., 2011; Sokol, 2015; Tada and Heisenberg, 2012; Wallingford, 2012). Polarity cues causing the segregation of PCP complexes remain largely unknown (McNeill, 2010; Wang and Nathans, 2007). Wnt proteins have been proposed as candidates for these cues due to their involvement in many PCP-dependent processes (Gao et al., 2011; Mahaffey et al., 2013; Ossipova et al., 2015b; Qian et al., 2007; Wu et al., 2013; Yang and Mlodzik, 2015). However, whether vertebrate Wnt ligands play a permissive or instructive role in PCP remains controversial. While Wnt proteins can instruct PCP in the Drosophila wing and orient myocytes in chick somites (Gros et al., 2009; Matis et al., 2014; Wu et al., 2013), Wnt11 has been argued to act permissively in convergent extension during zebrafish gastrulation (Heisenberg et al., 2000).

The Xenopus larval epidermis contains multiciliated cells (MCCs) that are coordinately aligned to generate a unidirectional fluid flow (Konig and Hausen, 1993; Werner and Mitchell, 2012). This alignment is controlled by PCP proteins during gastrulation and neurulation (Butler and Wallingford, 2015; Mitchell et al., 2009; Yasunaga et al., 2011). Nevertheless, it has been challenging to document core PCP protein polarization in the ectoderm before late neurula stages (Butler and Wallingford, 2015; Chien et al., 2015; Ciruna et al., 2006; Ossipova et al., 2015b). In this study, we demonstrate that ectodermal PCP visualized by exogenous Prickle3 (Pk3)/Vangl2 complex in the epidermis and endogenous Vangl2 in the neuroectoderm can be instructed by Wnt ligands during gastrulation.

Results and discussion

To establish early PCP markers, we examined the subcellular localization of GFP-tagged Pk3, one of the core PCP proteins predominantly expressed in the epidermal ectoderm (Ossipova et al., 2015a). When supplied to the ectodermal tissue by RNA microinjection, GFP-Pk3 was homogeneously distributed in the cytoplasm and at the cell junctions (Figure 1A,B). We hypothesized that Pk3 is not polarized because another PCP component is limiting. Given that Drosophila Prickle physically associates with Van Gogh (Bastock et al., 2003; Jenny et al., 2003), we suspected that the limiting factor is a Van Gogh homologue. Indeed, when GFP-Pk3 was coexpressed with Vangl2, its binding partner (Chu et al., 2016), epidermal PCP became evident by the beginning of neurulation with GFP fluorescence enriched at the anterior cell boundary (Figure 1C). In early gastrula ectoderm, GFP-Pk3 was visible as multiple membrane patches (Figure 1D–D") but, at later stages, formed a single Vangl2-positive crescent- or chevron-shaped domain near the anterior cell vertex, i. e. the junction of more than two cells, with a ventral bias (Figure 1E–H). The anterior localization of GFP-Pk3 was confirmed by the analysis of mosaically-expressing cell clones (Figure 1E,F). This distribution might reflect biased stabilization of PCP proteins noted in a recent study (Chien et al., 2015). At the doses used, the exogenous PCP complexes did not cause any visible morphological defects. These findings establish the Pk3/Vangl2 complex as a sensor that allows direct visualization of PCP in Xenopus epidermal ectoderm by the end of gastrulation. This anteroposterior PCP is similar to the one observed in the Xenopus neural plate (Ossipova et al., 2015b) and other vertebrate embryonic tissues (Antic et al., 2010; Borovina et al., 2010; Ciruna et al., 2006; Davey et al., 2016; Devenport and Fuchs, 2008; Hashimoto et al., 2010; Nishimura et al., 2012; Roszko et al., 2015; Yin et al., 2008).

Figure 1 with 1 supplement see all
Polarization of Pk3/Vangl2 complexes at the anterior cell cortex.

(A) Experimental scheme. RNAs encoding GFP-Pk3 and mouse HA-Vangl2 (150 pg each) were injected into the animal-ventral region of four-cell embryos. Ectoderm was dissected from the ventral midline area of fixed embryos for imaging (red box). The anteroposterior (AP) and dorsoventral (DV) axes are indicated. (B, C) GFP fluorescence of stage 15 ectoderm expressing GFP-Pk3 alone (B) or with HA-Vangl2 (C). Arrows in C point to GFP-Pk3 at the anterior cortex. (D, E) Embryos mosaically-expressing GFP-Pk3 and HA-Vangl2 were fixed at stage 11 (DD’’) or stage 15 (EE’’). Staining of GFP and HA is shown as indicated. HA-Vangl2 polarization is poorly detected in some cells due to variation in protein levels. Scale bar, 20 µm. (F) A single cell expressing GFP-Pk3 and HA-Vangl2. Dashed line depicts the cell boundary. Pk3 orientation (white arrow) is defined as perpendicular to the line connecting the ends of the crescent (yellow bar). (G) Rose diagrams show the orientation of GFP-Pk3 crescents in the ventral ectoderm at the indicated stage. n, number of scored cells. (H) Summary of GFP-Pk3 polarization derived from data in (G). Each arrow in the polar plot displays the mean orientation of Pk3 crescents in a single embryo at stage 12.5 (blue) or stage 15 (red). Arrow length is 1 minus the circular variance around the mean. Data are representative of two independent experiments.

https://doi.org/10.7554/eLife.16463.002

To further analyze the interaction between Pk3 and Vangl2 that is essential for their polarization, we assessed which domain is responsible for Pk3 polarity. We generated a series of Pk3 deletion mutants and examined their subcellular localization in the presence of Vangl2 (Figure 1—figure supplement 1). Similar to full-length Pk3, the mutated proteins did not polarize in the absence of Vangl2 (data not shown). While the N terminus of Pk3 was dispensable for its polarization, the C-terminal domain was required for Pk3 membrane recruitment, in agreement with its ability to bind Vangl2 (Chu et al., 2016). By contrast, Pk3 C-terminus (Pk3-C) was recruited to the plasma membrane but failed to polarize, consistent with the previous study of Drosophila Prickle (Jenny et al., 2003). A Pk3 mutant lacking the LIM domains (Pk3ΔLIM) was also unable to polarize despite being associated with the cell membrane. Of note, deletion of the CAAX motif, previously implicated in Drosophila Prickle polarization and stability (Lin and Gubb, 2009; Strutt et al., 2013), did not interfere with Pk3 polarization. Removal of the PET domain had a partial effect (Figure 1—figure supplement 1A,B), contrary to the data obtained for Prickle2 (Butler and Wallingford, 2015). These data show that the C-terminus is both necessary and sufficient for Vangl2-dependent membrane recruitment of Pk3, which is a prerequisite for its polarization. Notably, Pk3-C overexpression inhibited the incorporation of MCCs into the superficial epidermal cell layer at tailbud stages (data not shown), confirming the involvement of Pk3 in radial cell intercalation (Ossipova et al., 2015a).

Having established the utility of Pk3/Vangl2 complex as a polarity sensor, we next wanted to determine a role of Wnt signaling in ectodermal PCP. Since several Wnt ligands, including Wnt3a, Wnt5a and Wnt11b, are expressed in Xenopus early embryos (Hikasa and Sokol, 2013; Kiecker and Niehrs, 2001; Ku and Melton, 1993; Moon et al., 1993), we monitored GFP-Pk3/Vangl2 complex polarization in embryos, in which Wnt signaling was downregulated. Expression of the extracellular domain of Fz8 (ECD8), a potent Wnt inhibitor (Itoh and Sokol, 1999), disrupted Pk3/Vangl2 complex polarization in 85% of injected embryos (n = 41), whereas only 40% of control embryos lacked Pk3/Vangl2 polarity (n = 29) (Figure 2—figure supplement 1). Since ECD8 inhibits the majority of Wnt proteins (Itoh and Sokol, 1999), we utilized more selective Wnt antagonists, DN-Wnt11 and the dominant negative ROR2 receptor Ror2-TM, both of which specifically interfere with Wnt5- and Wnt11-like signals (Bai et al., 2014; Hikasa et al., 2002; Oishi et al., 2003; Tada and Smith, 2000). The majority of cells expressing DN-Wnt11 and Ror2-TM lacked GFP-Pk3 polarity in 89% (n= 28) and 88% (n= 25) of injected embryos, respectively (Figure 2A–C). This loss of polarity was unlikely caused by Pk3 and Vangl2 degradation, judged by immunoblotting (Figure 2D). Together, these experiments suggest that Wnt5- and/or Wnt11-like proteins function to establish PCP in early ectoderm.

Figure 2 with 1 supplement see all
Effects of Wnt antagonists on Pk3 polarization in the epidermal ectoderm.

Embryos were injected with RNAs encoding GFP-Pk3 (150 pg), Xenopus HA-Vangl2 (120 pg), and LacZ (1 ng, A) or DN-Wnt11 (2 ng, B) or Ror2-TM (1 ng, C). (AC) GFP fluorescence is shown in the epidermal ectoderm of embryos fixed at stage 15. Anterior is to the top. Scale bar, 20 µm. (D) Protein levels of GFP-Pk3 and HA-Vangl2 in the ectoderm analyzed by immunoblotting. A non-specific band detected by anti-HA antibody reflects protein loading.

https://doi.org/10.7554/eLife.16463.004

We next studied whether Wnt5a can induce ectopic Pk3 polarization in gastrula ectoderm. RNAs encoding GFP-Pk3 and Vangl2 were injected into one ventral animal blastomere of the 32-cell embryo, whereas Wnt5a RNA was coinjected with TurboFP635 (TFP) RNA as a tracer into the adjacent blastomere across the midline (Figure 3A,B,“L-R”). At stage 11.5, GFP-Pk3 patches formed at the cell membrane without apparent planar polarity in control embryos (Figure 3C). Remarkably, Wnt5a promoted early formation of polarized GFP-Pk3/Vangl2 crescents that were oriented away from the Wnt-expressing clone in 73% of injected embryos (Figure 3D,I,J, n = 40). These data demonstrate that Wnt5a can induce an exogenous PCP axis in non-polarized ectoderm.

Figure 3 with 2 supplements see all
The instructive role of Wnt5a in the establishment of Pk3 polarity.

(A) Experimental scheme. RNAs encoding GFP-Pk3 (150 pg) and Xenopus HA-Vangl2 (60 pg) were injected animally into a ventral blastomere of 32-cell embryos, followed by injection of TurboFP635 (TFP) RNA (150 pg, lineage tracer) with or without Wnt5a RNA (500 pg) into a blastomere either to the right (L–R) or anterior (A–P) of the Pk3-injected blastomere. The injected embryos were fixed at indicated stages, ectodermal explants were dissected, and the orientation of Pk3 crescents was evaluated by GFP fluorescence. (BD) Cell orientation in L-R-positioned clones. (B) Low magnification view of a stage 11.5 explant. Orientation of individual cells was quantified relative to the dashed line approximating TFP clone border (boxed area). The antero-posterior and left-right axes are indicated. (C) Control embryo, (D) Wnt5a-expressing embryo. (EH) Cell orientation in A–P-positioned clones at indicated stages. (E) Low magnification view. (F) Control embryo, (G, H) Wnt5a RNA-injected embryos. Arrows indicate cell orientation relative to the TFP clone (D, G, H). Scale bar, 50 µm. (I, K) Rose diagrams show Pk3 patch orientation in L–R (I) or A–P (K) experimental groups. Cell orientation was defined by an angle between the line joining the two ends of each Pk3 patch and the line approximating TFP clone border. (M) Orientation of Pk3 crescents in the A–P group at stage 15. See Figure 1F–H legend for quantification details. n, number of scored cells. p values were obtained by comparing the Wnt group to the control group using Chi-squared test. (J, L, N) Polar plots derived from (I, K, M), respectively, depict the mean Pk3 orientation in individual embryos. Arrow length is 1 minus the circular variance around the mean. Data were collected from two independent experiments.

https://doi.org/10.7554/eLife.16463.006

To further assess whether Pk3 polarization is defined by the location of the Wnt source, we generated Wnt5a-expressing clones to the anterior of the Pk3/Vangl2-expressing clone (Figure 3A,E, “A-P”). By comparing the effects of Wnt5a at the lateral and anterior locations, we found that the majority of GFP-Pk3 crescents were oriented away from the Wnt5a-expressing clone regardless of its position in the ectoderm (Figure 3B-G,I-L). Moreover, this effect of Wnt5a persisted until neurula stages, leading to reversal of Pk3 orientation in 77% of embryos expressing Wnt5a (Figure 3H,M,N, n = 30). Together, these findings support the instructive role of Wnt5a in ectodermal PCP.

To find out whether the observed effect on PCP is specific to Wnt5a or can be mediated by other Wnt ligands, we evaluated the ability of Wnt3a, Wnt11 and Wnt11b, known to be expressed in the early embryo, to modulate PCP in a similar assay (Figure 4A). Wnt3a had little effect on GFP-Pk3 polarity (Figure 4B,E,F). By contrast, Wnt11 and Wnt11b behaved similarly to Wnt5a by orienting the Pk3/Vangl2 crescents away from the Wnt-expressing clone (Figure 4C–F). GFP-Pk3 was reoriented in 57%, 36% and 36% of the examined embryos expressing Wnt5a, Wnt11, or Wnt11b RNA, respectively (n >10). These results suggest that PCP can be instructed by these Wnt ligands, but less so by Wnt3a that acts preferentially through the β-catenin-dependent pathway (Kikuchi et al., 2009; Semenov et al., 2007).

Establishment of Pk3 polarity in response to different Wnt ligands.

(A) Experimental scheme. RNAs encoding GFP-Pk3 and mouse HA-Vangl2 (150 pg each) were injected into the left animal-ventral blastomere of eight-cell embryos, followed by coinjection of a Wnt RNA (500 pg) and TFP RNA into the right animal-ventral blastomere. (BD) Cell orientation in stage 11.5 ectoderm of embryos injected with Wnt3a (B), Wnt11 (C) and Wnt11b (D) RNAs. The antero-posterior and left-right axes are indicated. Scale bar, 50 µm. (E) Rose diagrams show Pk3 patch orientation in clones adjacent to control (H2O) or Wnt-expressing clones. See Figure 3I for details. n, number of scored cells. p values were obtained using Chi-squared test. (F) Polar plots derived from (E) depict the average Pk3 orientation in individual embryos. Arrow length is 1 minus the circular variance around the mean. Data were collected from two independent experiments.

https://doi.org/10.7554/eLife.16463.009

We next attempted to find an endogenous marker or morphological structure that would provide additional evidence of early ectodermal PCP manifested by the exogenous GFP-Pk3/Vangl2 complex. Since microtubules play a critical role in PCP (Chien et al., 2015; Matis et al., 2014; Vladar et al., 2012), we examined the microtubule orientation at midgastrula stages by monitoring the movement of CLIP-170-GFP and EB1-GFP, two microtubule plus-end-binding proteins (Akhmanova and Steinmetz, 2008). In a similar experimental setting (Figure 4A), control embryos showed a weak alignment of CLIP170-GFP traces along the TFP clone border (Figure 3—figure supplement 1, Video 1). A slight reorientation of CLIP170-GFP traces was detected towards the border of the Wnt5a clone, yet the difference was insignificant (Figure 3—figure supplement 1). In addition, neither live imaging of EB1-GFP nor analysis of stable microtubules visualized by the microtubule-binding protein Ensconsin-GFP revealed a significant effect of Wnt5a on microtubule alignment (data not shown). Thus, Wnt signaling might regulate core PCP proteins without reorganizing microtubules in this system, as opposed to the Drosophila wing (Matis et al., 2014). Similarly, there was no detectable bias in the position of centrosomes marked by γ-tubulin staining (data not shown). Since core PCP proteins likely represent an early response to Wnt signaling, morphological manifestations of PCP may not be fully apparent until later developmental stages.

Video 1
Microtubule orientation visualized by the movement of Clip170-GFP foci.

Time-lapse imaging of Clip170-GFP comets in an ectodermal cell of a stage 11 embryo. See Figure 3—figure supplement 1 for details.

https://doi.org/10.7554/eLife.16463.010

To demonstrate the effect of Wnts on an endogenous PCP marker, we evaluated Vangl2 that is polarized in neuroectoderm but is poorly detectable in the epidermis (Ossipova et al., 2015b). Compared to its anterior polarization in control neuroectodermal cells, Vangl2 was reoriented away from a source of Wnt5a (Figure 5A–D). Such effect was observed in 90% of injected embryos (n = 46), but it was only visible in cells located one to four cell diameters away from the border of the Wnt5a clone. This finding supports our conclusions obtained for ectopic Pk3/Vangl2 complexes and suggests that the anterior polarization of Vangl2 results from endogenous Wnt proteins secreted from the posterior end of the embryo. To elucidate which Wnt ligands might be responsible, we knocked down Wnt5a and Wnt11b, two non-canonical Wnt ligands expressed at the posterior region of gastrula embryos (Ku and Melton, 1993; Moon et al., 1993), using previously characterized morpholino oligonucleotides (Pandur et al., 2002; Schambony and Wedlich, 2007). Whereas Vangl2 was accumulated at the anterior borders of cells in control embryos (87%, n = 24) and Wnt5a-depleted embryos (90%, n = 28), this polarity was retained only in 59% of embryos depleted of Wnt11b (n = 32) (Figure 5E,F and data not shown). These observations suggest the involvement of Wnt11b in anteroposterior PCP, consistent with its proposed activity in the gastrocoel roof plate (Walentek et al., 2013). Taken together, our gain- and loss-of-function assays support the idea that Wnt11b acts from the posterior region to establish an anteroposterior PCP across many cell diameters. Nevertheless, since the morpholino injection into vegetal blastomeres might partially interfere with the local production of Wnt11b in the neural plate, currently we cannot discriminate between long-range diffusion and local effects of Wnt proteins propagated by a signal relay system or cell division (Alexandre et al., 2014; Farin et al., 2016; Zecca et al., 1996).

Wnt signaling instructs Vangl2 polarization in the neural plate.

(A) Experimental scheme. Histone-GFP RNA (100 pg, nuclear lineage tracer, green) alone or with Wnt5a-Myc DNA (100 pg) was targeted to the border of the neural plate (pink), followed by immunostaining of Vangl2. (B) The neural plate of a stage 15 embryo with a clone of Histone-GFP-expressing cells (asterisk). Dotted line depicts the midline, and the antero-posterior (A–P) and medial-lateral (M–L) axes are indicated. (CD') Neural plates of embryos injected with Histone-GFP RNA alone (CC') or with Wnt5a-Myc DNA (DD') were immunostained for GFP and Vangl2. Vangl2 polarization is evident at the anterior cell borders (arrowheads). In cells adjacent to the Wnt5a-Myc clone, Vangl2 is oriented away from the clone (arrows). Images are representative of three independent experiments. (EF) Wnt11b is required for Vangl2 polarization. Each vegetal blastomere of eight-cell embryos was injected with 20 ng of either control MO (CoMO) (E) or Wnt11b MO (F). Neural plate explants at stage 15 were stained to visualize Vangl2. Scale bar, 20 µm. Images are representative of three independent experiments.

https://doi.org/10.7554/eLife.16463.011

Our findings support a function of Wnt5- and/or Wnt11-like proteins as biochemical polarity cues. With the demonstration that Frizzled proteins function as Wnt receptors (Bhanot et al., 1996), Wnt ligands were proposed to control PCP (Adler et al., 1997), yet no conclusion has been reached regarding the underlying mechanism (Gros et al., 2009; Lawrence et al., 2002; Wu et al., 2013). Wnt signaling may directly affect core PCP proteins by regulating PCP protein post-translational modifications (Gao et al., 2011) or stability (Chien et al., 2015; Strutt et al., 2011). Wnt ligands were also proposed to function in PCP by blocking the Frizzled-Van Gogh interaction (Wu and Mlodzik, 2008). The latter explanation is less likely, because ECD8, expected to compete with Frizzled for Vangl2 binding, interfered with Pk3 polarization, instead of instructing it similar to Wnt5a (Figure 2—figure supplement 1, Figure 3—figure supplement 2). Alternatively, given the role of Wnt signaling in gastrulation (Habas et al., 2001), Wnt proteins might generate mechanical strains to modulate PCP (Aigouy et al., 2010; Chien et al., 2015; Heisenberg and Bellaiche, 2013). While the effect of mechanical forces on PCP is thought to require microtubule reorganization (Chien et al., 2015), we did not detect a significant change of microtubule orientation in response to Wnt5a. Although our results demonstrate that Wnt proteins can instruct Pk3 polarization in our specific experimental conditions, the immediate morphological manifestations of this activity remain obscure and whether such function involves mechanical or chemical signaling should be established by future studies.

Our observations provide support to the instructive role of Wnt proteins in PCP. By contrast, ubiquitously expressed Wnt11 can partially rescue zebrafish embryos with a mutation in the wnt11 gene (Heisenberg et al., 2000). Whereas this finding suggests a permissive effect, lack of complete rescue may be also explained by the absence of proper instructions. At present, it is still unclear whether the proposed instructive mechanism operates to direct PCP during normal embryonic development.

Materials and methods

Plasmids, mRNA synthesis and morpholinos

GFP-Pk3, GFP-Pk3-C and GFP-Pk3∆PET in pXT7 have been described (Chu et al., 2016; Ossipova et al., 2015a). All Pk3 deletion mutants were obtained by PCR and subcloned into pXT7-GFP. The following Pk3 constructs were made: ∆N (69–538), ∆C (1–372), C (373–538), ∆PET (deletion of amino acids 69–170), ∆LIM (deletion of 179–372), ∆CAAX is missing the last 4 amino acids. Numbers in parentheses refer to amino acid position deduced from the cDNA clone (GenBank accession number BC154995). HA-tagged Xenopus Vangl2 in pCS2 was generated by PCR. Details of cloning are available upon request. Wnt5a-myc was subcloned into pCS2 from a plasmid obtained from R. Moon (unpublished).

Capped mRNAs were synthesized using mMessage mMachine kit (Ambion, Austin, TX) from the linearized DNA templates encoding Pk3 derivatives and the following previously described plasmids: mouse HA-Vangl2 (Gao et al., 2011), Wnt3a (Wolda et al., 1993), Wnt5a (Moon et al., 1993), Wnt11/Wnt11R (Garriock et al., 2005)(a gift of P. Krieg), Wnt11b (Tada and Smith, 2000), extracellular domain of Frizzled8 (ECD8) (Itoh and Sokol, 1999), Ror2-TM (Hikasa et al., 2002), DN-Wnt11 (Tada and Smith, 2000). Human histone H2B-GFP-pCS2 was a gift of P. Skourides and Chenbei Chang. TurboFP635-pCS2 was made from the TurboFP635 (Katushka) plasmid obtained from A. Zaraisky.

The following morpholinos (MOs) were used: standard control oligo (CoMO) (Gene Tools), Wnt5a MO (Schambony and Wedlich, 2007) and Wnt11b MO (Pandur et al., 2002).

Xenopus embryo culture and microinjections

In vitro fertilization and culture of Xenopus laevis embryos were carried out as previously described (Dollar et al., 2005). Staging was according to (Nieuwkoop and Faber, 1994). For microinjections, embryos were transferred into 3% Ficoll in 0.5 × MMR buffer and 5–10 nl of mRNA mixture or morpholinos was injected into one or more blastomeres. Amounts of injected mRNA per embryo have been optimized in preliminary dose-response experiments (data not shown) and are indicated in Figure legends.

Immunoblot analysis

For protein analysis, five stage 15 embryos expressing Pk3 deletion mutants were lysed in the buffer containing 50 mM Tris-HCl pH 7.6, 50 mM NaCl, 1 mM EDTA, 1% Triton X-100, 10 mM NaF, 1 mM Na3VO4, 25 mM β-glycerol phosphate, 1 mM PMSF. For analysis of Pk3 and Vangl2, animal caps were dissected at stage 10 and incubated in 0.6 x MMR until the equivalent of stage 15 when they were lysed. After centrifugation at 15,000 g, the supernatant was subjected to SDS-PAGE and Western blot analysis using standard techniques as described (Itoh et al., 1998). Sample loading was controlled by staining with Ponceau S (Sigma, St. Louis, MO). Chemiluminescence was captured by the ChemiDoc MP imager (BioRad, Hercules, CA).

Immunofluorescence, image analysis and quantification

For GFP and TFP fluorescence and immunofluorescence staining, embryos were manually devitellinized, ectoderm was dissected and fixed with MEMFA (0.1 M MOPS, pH 7.4, 2 mM EGTA, 1 mM MgSO4 and 3.7% formaldehyde) for 30 min at room temperature. Indirect immunofluorescence staining was performed as described previously (Ossipova et al., 2014). The following primary antibodies were used: rabbit anti-Vangl2 (1:100, (Ossipova et al., 2015b)), mouse anti-GFP (B-2, 1:200, Santa Cruz Biotechnology, Dallas, TX) and rabbit anti-HA (1:3000, Bethyl Labs. Montgomery, TX). Secondary antibodies were Alexa Fluor 488-conjugated (1:400, Invitrogen, Waltham, MA) or Cy3-conjugated (1:400, Jackson ImmunoResearch). Stained explants were mounted for observation in the Vectashield mounting medium (Vector Labs, Burlingame, CA). Images were captured using a Zeiss AxioImager microscope with the Apotome attachment (Zeiss, Germany). Images shown are representative of 2–4 independent experiments with 6–8 embryos per group.

To quantify cell orientation, we selected embryos with clearly separable Wnt- and Pk3-expressing clones with the expected position relative to the body axis. At stage 15, scoring was done only for the cells displaying unambiguous GFP-Pk3 signal as a single crescent. Cell orientation was defined by an arrow perpendicular to the line connecting the ends of each Pk3 crescent and quantified by ImageJ (NIH). Since Pk3 forms patches rather than crescents in stage 11.5 embryos, cell polarity was quantified differently. In this case, cell orientation was defined as an angle between the line approximating each Pk3 patch and the line tangential to TFP clone border and was measured by ImageJ. Data were collected from GFP-Pk3-expressing cells within 10 cell diameters from the TFP clone border. Rose diagrams were drawn using Oriana 3 (Kovach Computing Services, UK), and two-sample Chi-squared test was used for statistical analysis. The mean vector of Pk3 orientation per embryo was presented by polar plots.

Microtubule end-tracking

Microtubule polarity was visualized in embryos injected with Clip170-GFP RNA, synthesized from the pCS2-CLIP170-GFP plasmid (Werner et al., 2011). The movement of Clip170-GFP comets was assessed under a Zeiss LSM 880 confocal microscope with a 63X oil objective. Videos of individual cells were taken at a rate of 1 frame/2.5 s and contained 12 frames. The data were processed using ImageJ. Each video was temporally color-coded to define microtubule polarity, and the angle of Clip170-GFP traces relative to the TFP clone border was measured. Oriana 3 was used to plot rose diagrams and calculate mean axial vectors of individual embryos from mean axial vectors of individual cells.

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Decision letter

  1. Jeremy Nathans
    Reviewing Editor; Johns Hopkins University School of Medicine, United States

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

Thank you for submitting your article "Wnt proteins direct planar cell polarity axis in the vertebrate epidermis" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Reviewing Editor and Janet Rossant as the Senior Editor. One of the three reviewers has agreed to reveal her identity: Cecilia Moens (Reviewer #1).

The reviewers have discussed the reviews with one another and the Reviewing Editor has drafted this decision to help you prepare a revised submission.

As stated above, the manuscript has been reviewed by three expert reviewers, and their assessments, together with my own (Jeremy Nathans, Reviewing Editor), forms the basis of this letter. I am including the three reviews at the end of this letter, as there are various specific comments in them that will not be repeated in the summary here.

All of the reviewers were impressed with the importance of your work. Overall, the experiments appear to be carefully executed. However, there is a general consensus that the data should be more cautiously interpreted and that various experiments that could strengthen (or weaken) the conclusions are feasible.

In particular, the experiments are really a demonstration of what is possible with an experimental perturbation, but not necessarily what actually happens in vivo, which would require a true loss-of-function experiment. Describing it as such is important. Also, the connection between polarized PCP protein distribution and some biological structure (e.g. cytoskeleton or cilia) is not demonstrated here. Finally, there are technical caveats related to over-expressing a Frizzled CRD or dominant Wnt that should be critically addressed.

We would like to encourage you to resubmit a revised manuscript that addresses the specific issues raised in the reviews and summarized above. We realize that several of the suggestions may be beyond the scope of the present work (e.g. doing a true loss of function analysis), but we have not removed them from the reviews since they convey the reaction to the work over-all.

Reviewer #1:

The planar cell polarity pathway (PCP) polarizes cells in the plane of an epithelium. Core components of the pathway include Fzd and Dsh family members, however the requirement for a Wnt ligand in planar polarization is variable: essential for some PCP processes and apparently dispensable for others. Even in contexts where a Wnt is essential, the loss of function experiments that demonstrate this do not distinguish between a permissive versus an instructive role in specifying the direction of polarization. This brief manuscript demonstrates that the polarization of PCP components occurs in response to a Wnt ligand. Using the robust Vangl2-dependent localization of Pk3 to anterior cell boundaries in the Xenopus non-neural ectoderm as a direct readout of PCP signaling in dominant-negative and gain-of-function experiments, they show a) that planar polarization depends on Wnt signaling and b) that localized expression of a Wnt ligand can repolarize epithelial cells several cell diameters away from the source. This is not the only mechanism by which PCP can be established; in other contexts, PCP can arise in response to tension, flow, or by unknown but Wnt-independent means. Nevertheless, the paper shows clearly that a directional Wnt signal can establish PCP and as such is an important addition to the literature. With the relatively minor revisions suggested below I feel that the paper is appropriate for publication in eLife.

Criticisms:

1) The quantification and statistics were done on a per-cell basis, with each N being the orientation of Pk3 in a single cell. However, it is not reasonable to expect that the behavior of one cell in an embryo is independent of the behavior of a neighboring cell in the same embryo. This is particularly true when we are dealing with planar polarity. Therefore, the data should be re-analyzed on a per-embryo basis, with each N being the average Pk3 polarization angle in a single embryo.

2) In the Results and Discussion the paper says: "Moreover, this effect of Wnt5a persisted to the neurula stage, resulting in PCP reversal in 77% of embryos”. How was this eventual polarity reversal demonstrated? Was it based on something other than the orientation of Pk crescents? This claim of tissue polarity reversal should be substantiated using a separate readout such as primary cilium orientation.

Reviewer #1 (Additional data files and statistical comments):

No additional data required. Statistics should be re-done on a per-embryo (rather than per-cell) basis.

Reviewer #2:

The central contention of this manuscript is that Wnt's provide a long range directional signal for PCP in the gastrula stage Xenopus ectoderm. The authors use coexpression of Pk3-GFP and Vangl2 as markers of GFP, and dominant negative Wnt and FzECD constructs to indicate a requirement for Wnt's, and Wnt ectopic expression to demonstrate the ability to determine directionality. The notion that Wnt's act as long range directional signals for PCP has been unsettled in all systems in which it has been addressed for as long as the idea has been floated. The evidence presented here that this is the case in the gastrula stage ectoderm is as good as any in any system so far, which is to say intriguing, but not entirely compelling. It has been clear for some time that Wnts can direct polarity. The key question is whether they do direct polarity, and it is here that this study, like several previous ones, falls just short of being entirely convincing. I'm on the fence about whether the arguments presented here warrant publication in a leading journal, but at a minimum there are several things that should be done to shore up the argument.

The interaction between Pk3 and Vangl2 is consistent with our understanding of PCP signaling and is not particularly novel, and the deletion analysis to identify required Pk3 domains is a bit of a distraction. Why Vangl2 should be limiting for Pk3 localization at these stages is an interesting and more puzzling question in light of recently published results. The failure to observe asymmetry of exogenous Pk3-GFP if endogenous Pk3 is asymmetrically localized is best explained if the Pk3-GFP is present in large excess, so that asymmetry that would otherwise be evident is swamped out by randomly localized and unassociated Pk3. The less interesting possibility is that endogenous Pk3 is not localized asymmetrically, but can be induced to do so in the presence of the necessary other players.

This contrasts with results from Butler and Wallingford, who showed that Pk2, Vangl1 and Dvl1 all localize asymmetrically when independently expressed at later stages. The puzzle here is the contrasting dynamics. Butler's results suggest a much later acquisition of polarity, as their probes were symmetric as late as stage 19, and only subsequently showed increasing asymmetry by the time of MCC differentiation. From these results, we would infer that there is little or no activity of the PCP components at the gastrula stage.

If that is the case, this raises the question of whether the authors are examining what is essentially the potential to polarize, or in other words, a synthetic polarization that can be created by expressing needed components (and that can then perhaps respond to endogenous or exogenous Wnt's), but that doesn't reflect an endogenous polarization that normally occurs. For example, we have no evidence that Pk3 is needed for this polarity, and there is no independent readout of polarization. In the GRP, Pk3 is normally at the basal bodies, though it can be induced to apicolateral asymmetry, and its role and localization in radial intercalation of MCCs (Sokol lab) are both potentially consistent with different mechanisms. Given these observations, it is plausible that the authors have created a synthetic PCP system that can respond to Wnt's, but that doesn't normally exist. The authors offer no other readout for PCP at this time.

On the other hand, asymmetry, or at least asymmetric activity, of Vangl2, Celsr1, Fzd3 was inferred by FRAP at this time by Chien et al. (although these assays also required the co-expression of exogenous components), and this was associated with polarized apical microtubules. However, these authors concluded that tissue strain provided the directionality to polarization, in contrast to the current contention that Wnt signaling directs polarization. If Chien et al. are correct, it may be that microtubules determine the direction of the PCP orientation, as opposed to being responsive to PCP protein polarization. Furthermore, these authors showed that microtubule orientation is responsive to strain, which might be providing the directional signal. If the authors suggest that microtubules are reading Wnt dependent polarity, it would stretch the imagination to conceive that strain reorganizes the pattern of Wnt expression. A more harmonious conclusion would be that if Wnt's are involved, they are involved in local cell to cell signaling rather than long range signaling.

Therefore, whether PCP signaling normally occurs, what if anything it is doing, and the upstream directional signal are not entirely established. Furthermore, Pk3 may or may not be involved, though even if not, it could be a legitimate marker of PCP.

The relevance of the Pk3/Vangl2 complex as a PCP marker in reading a Wnt signal would be bolstered if the authors could show that the polarized microtubules observed at this time depend on Pk3 or Vangl2. Even better, the authors should look at microtubules in their loss and gain of function experiments to determine their potential responsiveness to these inputs. If microtubules reorganize in response to Wnt's, they should then demonstrate that the requirement for normal microtubule orientation, proposed to be Wnt dependent, is lost upon knockdown of PCP components. If microtubules do not depend on Wnt endogenous or exogenous Wnt signals, then I would not feel confident that endogenous PCP signaling is occurring at gastrula stages.

Based on the currently provided results, the authors' statement "In this study, we use the complex of Pk3 and Vangl2 as a sensor of epidermal PCP to demonstrate that the polarity of this complex is directly defined by Wnt ligands during gastrulation" should instead say: "the polarity of this complex CAN BE directly defined by…".

Another important concern is the conclusion that can or cannot be drawn from the methods used to interfere with endogenous Wnt signaling. As discussed above, these are critical to the distinction between CAN and DO. The mechanisms by which the Fz8ECD and the dominant negative Wnt's act is not certain. While they may bind and block Wnt activity, it is also hypothesized that PCP involves direct interaction between Fz and Vangl proteins, and these reagents could potentially interfere with such an interaction to disrupt PCP. The authors could potentially address this concern if they could show that RNAi of the suspect Wnt's, either alone or simultaneously, would disrupt asymmetry of their PCP markers.

Last, the authors acknowledge that they don't truly distinguish between a long range Wnt signal providing a directional signal, and a paracrine signal involved in cell to cell signaling, as proposed by Adler many years ago, that would not provide a directional signal. The localized Wnt expression patterns (particularly since they are very low resolution old in situ studies) are not enough to force the former conclusion. Does this not undermine their thesis?

As is, I'm left to conclude that PCP signaling might be occurring at gastrula stage, this polarization might do something that matters, and Wnt's might provide a long range signal for this polarization. It would be good to feel a little more confident.

Reviewer #2 (Additional data files and statistical comments):

No additional data files needed, and assuming representative images are shown, the results as shown are compelling.

Reviewer #3:

The results presented support a role for Wnts in early PCP establishment in the Xenopus epidermis. The authors contend that this is an instructive role, and such a finding would stand in contrast to the recent report by Chien et al. (2015 Curr Biol 25, 2774-2784) that suggests mechanical cues are responsible for early PCP orientation in the Xenopus epidermis (although the authors don't make this point). However, as it stands, I think that while the authors make a case that Wnts can re-orient polarity, they do not provide good evidence that this normally happens and/or whether it happens when Wnts are expressed at physiological levels. It is equally likely that the observed results could be a dominant negative effect caused by Wnt binding to a Fzd CRD domain and blocking interactions with Vangl, similar to what Wu et al. 2013 see in Drosophila upon Wnt over expression.

The authors need better evidence to convince me that they have made any advance upon the previous view that Wnts might only play a permissive role in this context.

Other major comments:

1) This is not the first evidence for instructive role of Wnts in vertebrates, as Wnt5a has previously been shown to have an instructive role in the mouse limb bud, in a study that also impressively showed direct evidence of a Wnt-induced activity gradient (Gao et al. (2011) Dev Cell 20, 163-176).

2) Although the "fluorescent sensor for planar polarity in Xenopus" may be new (as indicated in the "Impact statement"), it is not really novel for a GFP fusion to be used to reveal polarity, as even in Xenopus this has been previously demonstrated (Butler and Wallingford (2015) Development 142, 3429-3439).

3) A structure-function dissection of Pk3 domains required for polarization is described, without any reference to the similar results for Pk2 described in Butler et al. 2015. I'm also concerned about how much can be concluded if endogenous protein is not first removed in a structure-function experiment like this.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

Thank you for submitting your revised manuscript "Wnt proteins can direct planar cell polarity in vertebrate ectoderm" to eLife. The revised manuscript has been reviewed by two expert reviewers, and their assessments, together with my own (Jeremy Nathans, Reviewing Editor), forms the basis of this letter. I am including the two reviews at the end of this letter, as the specific suggestions in them will not be repeated in the summary here.

We would like to encourage you to make some additional text modifications to the manuscript that addresses the specific issues raised in the two reviews below – especially the issues raised by reviewer #2. With respect to the writing, we appreciate that in the current publishing environment there is an almost irresistible temptation to over-state one's conclusions. eLife is trying to restore some balance to the world of scientific writing. We welcome self-critical comments and we believe that such comments actually enhance the reader's ability to judge the science fairly.

I will offer two examples of scientific writing that I think illustrates the preceding point. They are from two papers on ubiquitin that form the core of the discoveries for which the authors shared the Nobel Prize.

Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

Hershko A, Ciechanover A, Heller H, Haas AL, Rose IA.

Proc Natl Acad Sci U S A. 1980 Apr;77(4):1783-6.

In the second paragraph of the Discussion section, after summarizing the evidence that leads the authors to propose the conjugation of APF-1 (=ubiquitin) to proteins as a way of marking them for degradation, the authors write: "Evidence that APF-1-proteins are intermediates in the breakdown of denatured protein as proposed in Figure 6 is indirect and inconclusive at this time."

Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

Ciechanover A, Heller H, Katz-Etzion R, Hershko A.

Proc Natl Acad Sci U S A. 1981 Feb;78(2):761-5.

In the last paragraph of the Discussion section, after describing their discovery and characterization of ubiquitin (=APF-1) ligase, the authors write "Evidence suggesting the role of the APF-1-activating enzyme in conjugation and in ATP-dependent protein breakdown is not conclusive at present."

Reviewer #1:

The authors have added new experiments that address some of the criticisms but they have not been able to address others. In response to the criticism that their gain-of-function experiments show that Wnt signaling CAN direct planar polarity but not that it DOES direct endogenous polarity, they include new data showing that the polarization of endogenous Vangl2 is altered by localized WNT overexpression, and that morpholino knockdown of Wnt11b eliminates endogenous Vangl2 polarization. This, combined with the gain-of-function data showing that Wnt expressing cells can repolarize Vangl2 and Pk3 over several cell diameters, is compelling evidence for a role for Wnts in orienting PCP in the frog gastrula non-neural ectoderm. On the other hand, the authors tried but failed to detect polarization of any sub-cellular structure apart from PCP core components themselves. They argue that microtubule or centrosomal asymmetry may be a much later consequence of PCP, not detectable at these early stages. They have also addressed some relatively minor comments I had about quantification of the data. In sum, the paper is improved by demonstrating a requirement for Wnts in endogenous Vangl2 localization, but remains focused on the polarity proteins themselves and not their morphological or cellular consequences. As I was already quite positive about the paper, which I found to be the clearest demonstration to date of a role for Wnts in directing epithelial PCP, I feel that it is now appropriate for publication in eLife.

Reviewer #2:

This resubmission contains all of the same data regarding the epidermal ectoderm from the first submission, unchanged except for the additional statistical analysis, plus one new figure about polarity in the neuroectoderm. As such, my opinion about the interpretive difficulties concerning the epidermal ectoderm remains unchanged. The main response of the authors was to change what they choose to say about the epidermal ectoderm data. For this part of the manuscript, the message has been changed (mostly) from the claim that a Wnt signal is instructive for planar polarization, to the well justified statement that a Wnt signal can be instructive for planar polarization.

The new data concern a separate and possibly distinct planar polarization event, so it is not clear how this is supposed to support the findings in the first four figures as opposed to representing a different example. This new data, which was presumably added to address concerns about a true loss of function phenotype, demonstrates that Wnt11b is required for planar polarity of Vangl2 in the neuroectoderm. On the other hand, it does not show that this signal works at a distance, since descendants of the 8 ventral blastomeres into which the MO was injected contribute to the neural plate, and expression of Wnt11b, perhaps at a low level, throughout the neural plate is not ruled out. On the other hand, they do show that ectopic Wnt5a can non-autonomously repolarize neural plate over several cell diameters. They acknowledge that this might occur by either paracrine signaling and propagation, or by graded signaling of the ligand. Concerning this new data, the authors are a bit more circumspect, but shockingly, the final conclusion of the paper again incorrectly claims that Wnt's are instructive for PCP: "Whereas our results demonstrate the instructive role of Wnt proteins in PCP…"

If we conflate the three different paradigms (ectodermal ectoderm, Wnt11b in neural ectoderm and Wnt5a in neural ectoderm), the data would probably make the best argument yet that a Wnt signal acts instructively to determine PCP, the main weaknesses being that the localized expression of Wnt needs to be more carefully examined, and one would like to see gain and loss of function for the same Wnt (or combination of Wnts) in the same tissue. However, I don't feel it's sufficiently rigorous to combine these systems to come to a conclusion as important as the claim that a Wnt IS instructive for PCP.

If the authors wish to try to produce a more definitive conclusion, I'd advise them to build a careful analysis of Wnt11b in the neural plate, and not just add the minimal data presented here as an addendum. If they wish to moderate their overall conclusion to Wnts can instruct planar polarity (two misleading statements would need to be changed, see below), that would be well supported by the data provided. This course would require a somewhat detailed discussion of the distinction between these conclusions. On the other hand, that would be a far less novel result, and one could debate whether it would be of sufficient impact for eLife.

The two unjustified statements:

"Based on these experiments, Wnt5a appears to act as an instructive cue for Pk3 polarization."

This implies that this is what it normally does.

"Whereas our results demonstrate the instructive role of Wnt proteins in PCP, whether such function involves mechanical or chemical signaling remains to be clarified in future studies."

This is clearly not supported by the present data.

It's also relevant to comment on one of the responses that the authors provided in the rebuttal.

"Several lines of evidence argue for Wnt proteins acting on ectodermal PCP over a long distance during vertebrate gastrulation. First, the effect of ectopic Wnt proteins on the GFP-Pk3/Vangl2 complex is detectable across many cell diameters."

So too is clonal knockdown of fz in a fly wing detected several cell diameters away. But this would still clearly be a paracrine signal that is then propagated via other means beyond the clone border. This is therefore not strong evidence for a direct long range action of a ligand.

"Second, Wnt5a can reorient endogenous Vangl2, which is normally polarized throughout the neuroectoderm along the anteroposterior axis (new Figure 5A–D)."

Doesn't address the paracrine vs. long range issue.

"Third, Wnt11b depletion in the posterior region of the embryo disrupts Vangl2 polarity in the neural plate (new Figure 5E,F), supporting its role in long-range anteroposterior PCP."

This may be the strongest argument, but still the experimental design is flawed (as noted above), as the vegetal blastomeres of eight cell embryos contribute to neural plate, so the knockdown is presumably distributed throughout the region, and local expression is not ruled out. The authors therefore correctly state "Nevertheless, currently we cannot discriminate between long-range diffusion and local effects of Wnt proteins propagated by a signal relay system or cell division (Alexandre et al., 2014; Farin et al., 2016; Zecca et al., 1996)."

Finally, if the authors wish to build a case around Wnt11b. How do they reconcile their story with "Wnt11 has been argued to act permissively in convergent extension during zebrafish gastrulation (Heisenberg et al., 2000)"? Of course, then one is comparing fish and frogs.

https://doi.org/10.7554/eLife.16463.012

Author response

All of the reviewers were impressed with the importance of your work. Overall, the experiments appear to be carefully executed. However, there is a general consensus that the data should be more cautiously interpreted and that various experiments that could strengthen (or weaken) the conclusions are feasible.

In particular, the experiments are really a demonstration of what is possible with an experimental perturbation, but not necessarily what actually happens in vivo, which would require a true loss-of-function experiment. Describing it as such is important. Also, the connection between polarized PCP protein distribution and some biological structure (e.g. cytoskeleton or cilia) is not demonstrated here. Finally, there are technical caveats related to over-expressing a Frizzled CRD or dominant Wnt that should be critically addressed.

We would like to encourage you to resubmit a revised manuscript that addresses the specific issues raised in the reviews and summarized above. We realize that several of the suggestions may be beyond the scope of the present work (e.g. doing a true loss of function analysis), but we have not removed them from the reviews since they convey the reaction to the work over-all.

Reviewer #1:

The planar cell polarity pathway (PCP) polarizes cells in the plane of an epithelium. Core components of the pathway include Fzd and Dsh family members, however the requirement for a Wnt ligand in planar polarization is variable: essential for some PCP processes and apparently dispensable for others. Even in contexts where a Wnt is essential, the loss of function experiments that demonstrate this do not distinguish between a permissive versus an instructive role in specifying the direction of polarization. This brief manuscript demonstrates that the polarization of PCP components occurs in response to a Wnt ligand. Using the robust Vangl2-dependent localization of Pk3 to anterior cell boundaries in the Xenopus non-neural ectoderm as a direct readout of PCP signaling in dominant-negative and gain-of-function experiments, they show a) that planar polarization depends on Wnt signaling and b) that localized expression of a Wnt ligand can repolarize epithelial cells several cell diameters away from the source. This is not the only mechanism by which PCP can be established; in other contexts, PCP can arise in response to tension, flow, or by unknown but Wnt-independent means. Nevertheless, the paper shows clearly that a directional Wnt signal can establish PCP and as such is an important addition to the literature. With the relatively minor revisions suggested below I feel that the paper is appropriate for publication in eLife.

Criticisms:

1) The quantification and statistics were done on a per-cell basis, with each N being the orientation of Pk3 in a single cell. However, it is not reasonable to expect that the behavior of one cell in an embryo is independent of the behavior of a neighboring cell in the same embryo. This is particularly true when we are dealing with planar polarity. Therefore, the data should be re-analyzed on a per-embryo basis, with each N being the average Pk3 polarization angle in a single embryo.

As requested by the referee, we modified Figures 1, 3 and 4 to present the results as polar plots summarizing mean cell orientation per embryo. In addition, we kept the original rose plots to preserve raw data.

2) In the Results and Discussion the paper says: "Moreover, this effect of Wnt5a persisted to the neurula stage, resulting in PCP reversal in 77% of embryos”. How was this eventual polarity reversal demonstrated? Was it based on something other than the orientation of Pk crescents? This claim of tissue polarity reversal should be substantiated using a separate readout such as primary cilium orientation.

In order not to over-interpret our results, we revised the text to provide more precise description of the experiment (Results and Discussion, fifth paragraph). Prompted by the referee, we searched for other markers that indicate tissue polarization in our system and examined microtubules and centrosome polarity. Confocal live imaging of CLIP170-GFP, a microtubule plus-end-binding protein, allowed us to analyze microtubule orientation in response to Wnt5a, but we did not see a significant effect (new Figure 3—figure supplement 1, Video 1). The analysis of EB1-GFP, Ensconsin-GFP and γ-tubulin immunostaining also failed to reveal a detectable effect of Wnt5a (data not shown). Whereas Wnt signaling may trigger an early response in core PCP proteins, tissue polarity may not become visible morphologically until later developmental stages. Nevertheless, we were successful in demonstrating the Wnt5a-mediated reorientation of Vangl2 that is highly enriched in the neuroectoderm. We show that endogenous Vangl2 accumulates at the distal ends of the cells that respond to Wnt5a (new Figure 5A–D), supporting our conclusions obtained for exogenous Pk3 and Vangl2 constructs.

Reviewer #1 (Additional data files and statistical comments):

No additional data required. Statistics should be re-done on a per-embryo (rather than per-cell) basis.

As stated above, both original image analysis and new data are now shown in modified Figures 1, 3, 4, following the referee’s request.

Reviewer #2:

The central contention of this manuscript is that Wnt's provide a long range directional signal for PCP in the gastrula stage Xenopus ectoderm. The authors use coexpression of Pk3-GFP and Vangl2 as markers of GFP, and dominant negative Wnt and FzECD constructs to indicate a requirement for Wnt's, and Wnt ectopic expression to demonstrate the ability to determine directionality. The notion that Wnt's act as long range directional signals for PCP has been unsettled in all systems in which it has been addressed for as long as the idea has been floated. The evidence presented here that this is the case in the gastrula stage ectoderm is as good as any in any system so far, which is to say intriguing, but not entirely compelling. It has been clear for some time that Wnts can direct polarity. The key question is whether they do direct polarity, and it is here that this study, like several previous ones, falls just short of being entirely convincing. I'm on the fence about whether the arguments presented here warrant publication in a leading journal, but at a minimum there are several things that should be done to shore up the argument.

The interaction between Pk3 and Vangl2 is consistent with our understanding of PCP signaling and is not particularly novel, and the deletion analysis to identify required Pk3 domains is a bit of a distraction. Why Vangl2 should be limiting for Pk3 localization at these stages is an interesting and more puzzling question in light of recently published results. The failure to observe asymmetry of exogenous Pk3-GFP if endogenous Pk3 is asymmetrically localized is best explained if the Pk3-GFP is present in large excess, so that asymmetry that would otherwise be evident is swamped out by randomly localized and unassociated Pk3. The less interesting possibility is that endogenous Pk3 is not localized asymmetrically, but can be induced to do so in the presence of the necessary other players.

This contrasts with results from Butler and Wallingford, who showed that Pk2, Vangl1 and Dvl1 all localize asymmetrically when independently expressed at later stages. The puzzle here is the contrasting dynamics. Butler's results suggest a much later acquisition of polarity, as their probes were symmetric as late as stage 19, and only subsequently showed increasing asymmetry by the time of MCC differentiation. From these results, we would infer that there is little or no activity of the PCP components at the gastrula stage.

If that is the case, this raises the question of whether the authors are examining what is essentially the potential to polarize, or in other words, a synthetic polarization that can be created by expressing needed components (and that can then perhaps respond to endogenous or exogenous Wnt's), but that doesn't reflect an endogenous polarization that normally occurs. For example, we have no evidence that Pk3 is needed for this polarity, and there is no independent readout of polarization. In the GRP, Pk3 is normally at the basal bodies, though it can be induced to apicolateral asymmetry, and its role and localization in radial intercalation of MCCs (Sokol lab) are both potentially consistent with different mechanisms. Given these observations, it is plausible that the authors have created a synthetic PCP system that can respond to Wnt's, but that doesn't normally exist. The authors offer no other readout for PCP at this time.

As the referee acknowledges in the next paragraph, our data are consistent with the observations of Chien et al., 2015, arguing for the existence of PCP at late gastrula stages. At present, we have no explanation for the discrepancy between our findings and those of Butler and Wallingford (2015). This could be due to the functional difference between PCP proteins or the difference in developmental stages. We argue that Pk3 is a relevant PCP protein, based on its expression analysis and its requirement for Vangl2 polarity and ciliogenesis (Chu et al., 2016). Moreover, our new experiments that assess endogenous Vangl2 in the neuroectoderm provide further support to our claim that the distribution of GFP-Pk3 reflects endogenous PCP (new Figure 5A–D).

On the other hand, asymmetry, or at least asymmetric activity, of Vangl2, Celsr1, Fzd3 was inferred by FRAP at this time by Chien et al. (although these assays also required the co-expression of exogenous components), and this was associated with polarized apical microtubules. However, these authors concluded that tissue strain provided the directionality to polarization, in contrast to the current contention that Wnt signaling directs polarization. If Chien et al. are correct, it may be that microtubules determine the direction of the PCP orientation, as opposed to being responsive to PCP protein polarization. Furthermore, these authors showed that microtubule orientation is responsive to strain, which might be providing the directional signal. If the authors suggest that microtubules are reading Wnt dependent polarity, it would stretch the imagination to conceive that strain reorganizes the pattern of Wnt expression. A more harmonious conclusion would be that if Wnt's are involved, they are involved in local cell to cell signaling rather than long range signaling.

Therefore, whether PCP signaling normally occurs, what if anything it is doing, and the upstream directional signal are not entirely established. Furthermore, Pk3 may or may not be involved, though even if not, it could be a legitimate marker of PCP.

The relevance of the Pk3/Vangl2 complex as a PCP marker in reading a Wnt signal would be bolstered if the authors could show that the polarized microtubules observed at this time depend on Pk3 or Vangl2. Even better, the authors should look at microtubules in their loss and gain of function experiments to determine their potential responsiveness to these inputs. If microtubules reorganize in response to Wnt's, they should then demonstrate that the requirement for normal microtubule orientation, proposed to be Wnt dependent, is lost upon knockdown of PCP components. If microtubules do not depend on Wnt endogenous or exogenous Wnt signals, then I would not feel confident that endogenous PCP signaling is occurring at gastrula stages.

The results of Chien et al. do not exclude a role for Wnt signaling in the establishment of PCP. Consistent with their report, either Wnt11b or other Wnts that are present at the vegetal/posterior pole may be responsible for the tension produced by the blastopore ring during gastrulation. Alternatively, one can envisage that both mechanical forces and Wnt signaling provide input to PCP.Our additional analysis of microtubule orientation in gastrula ectoderm cells does not support the idea that Wnt5a orients Pk3/Vangl2 complexes by regulating microtubule alignment (Figure 3—figure supplement 1). More detailed studies are required to elucidate the relationship between Wnt signaling and mechanical strains during gastrulation, which is beyond the scope of the current work.

Based on the currently provided results, the authors' statement "In this study, we use the complex of Pk3 and Vangl2 as a sensor of epidermal PCP to demonstrate that the polarity of this complex is directly defined by Wnt ligands during gastrulation" should instead say: "the polarity of this complex CAN BE directly defined by…".

We amended our statement as suggested by the referee.

Another important concern is the conclusion that can or cannot be drawn from the methods used to interfere with endogenous Wnt signaling. As discussed above, these are critical to the distinction between CAN and DO. The mechanisms by which the Fz8ECD and the dominant negative Wnt's act is not certain. While they may bind and block Wnt activity, it is also hypothesized that PCP involves direct interaction between Fz and Vangl proteins, and these reagents could potentially interfere with such an interaction to disrupt PCP. The authors could potentially address this concern if they could show that RNAi of the suspect Wnt's, either alone or simultaneously, would disrupt asymmetry of their PCP markers.

We used multiple Wnt antagonists including Ror2-TM, a construct interfering with Ror2 function, and tried to avoid overstating our conclusions. Prompted by the referee, we knocked down candidate wnt5a and wnt11b gene functions with previously characterized morpholino oligonucleotides. Depletion of Wnt11b disrupted Vangl2 polarity, supporting its role in PCP (Figure 5E,F). Despite this promising observation, we feel that further analysis of the requirement of Wnt ligands in PCP is beyond the scope of our manuscript.

Last, the authors acknowledge that they don't truly distinguish between a long range Wnt signal providing a directional signal, and a paracrine signal involved in cell to cell signaling, as proposed by Adler many years ago, that would not provide a directional signal. The localized Wnt expression patterns (particularly since they are very low resolution old in situ studies) are not enough to force the former conclusion. Does this not undermine their thesis?

As is, I'm left to conclude that PCP signaling might be occurring at gastrula stage, this polarization might do something that matters, and Wnt's might provide a long range signal for this polarization. It would be good to feel a little more confident.

Several lines of evidence argue for Wnt proteins acting on ectodermal PCP over a long distance during vertebrate gastrulation. First, the effect of ectopic Wnt proteins on the GFP-Pk3/Vangl2 complex is detectable across many cell diameters. Second, Wnt5a can reorient endogenous Vangl2, which is normally polarized throughout the neuroectoderm along the anteroposterior axis (new Figure 5A–D). Third, Wnt11b depletion in the posterior region of the embryo disrupts Vangl2 polarity in the neural plate (new Figure 5E,F), supporting its role in long-range anteroposterior PCP. However, given the history of the attempts to resolve this issue in the Drosophila model in the past 20 years, we admit that further studies are required to determine whether Wnt proteins act locally or by diffusing over long distance in our system.

Reviewer #3:

The results presented support a role for Wnts in early PCP establishment in the Xenopus epidermis. The authors contend that this is an instructive role, and such a finding would stand in contrast to the recent report by Chien et al. (2015 Curr Biol 25, 2774-2784) that suggests mechanical cues are responsible for early PCP orientation in the Xenopus epidermis (although the authors don't make this point).

The results of Chien et al. do not exclude a role for Wnt signaling in the establishment of PCP as we also stated in our response to referee 2. Either Wnt11b or other Wnts that are present at the vegetal/posterior pole may be required for mechanical strains produced during gastrulation. Alternatively, both mechanical forces and Wnt signaling may regulate PCP by different mechanisms.

However, as it stands, I think that while the authors make a case that Wnts can re-orient polarity, they do not provide good evidence that this normally happens and/or whether it happens when Wnts are expressed at physiological levels. It is equally likely that the observed results could be a dominant negative effect caused by Wnt binding to a Fzd CRD domain and blocking interactions with Vangl, similar to what Wu et al. 2013 see in Drosophila upon Wnt over expression.

The authors need better evidence to convince me that they have made any advance upon the previous view that Wnts might only play a permissive role in this context.

Our new loss-of-function experiments demonstrate the involvement of Wnt11b in establishing anteroposterior PCP (Figure 5E,F), supporting the idea that Wnt-dependent PCP happens normally in vivo. Moreover, the activity of ectopic Wnt5a in our experiments is comparable to that of the endogenous cue that leads to Vangl2 polarity in the neuroectoderm (Figure 5A–D), but the physiological levels of active Wnt proteins are hard to assess. Although the referee suggested that Wnt5a acts in our experiments by blocking Frizzled-Vangl2 interaction, we consider it unlikely, because the extracellular domain of Frizzled 8 (ECD8), which is expected to compete with Frizzled for Vangl2 binding, did not orient the Pk3/Vangl2 complexes like Wnt5a did (Figure 3—figure supplement 2). This point has been added to the amended discussion. Overall, we argue for the instructive role of Wnts in PCP based on the ability of Wnt5a-expressing clones to orient the Pk3/Vangl2 complex regardless of their position relative to the body axis (Figure 3). This model is further supported by the effect of ectopic Wnt5a on endogenous Vangl2 in the neuroectoderm (Figure 5A–D).

Other major comments:

1) This is not the first evidence for instructive role of Wnts in vertebrates, as Wnt5a has previously been shown to have an instructive role in the mouse limb bud, in a study that also impressively showed direct evidence of a Wnt-induced activity gradient (Gao et al. (2011) Dev Cell 20, 163-176).

Gao et al. (2011) visualized graded polarization of Vangl2 in the mouse limb and have demonstrated its disruption in Wnt5a -/- embryos. Although the authors propose an instructive role for Wnt5a, this hypothesis has not been supported by an in vivo gain-of-function assay, such as the one provided by our study.

2) Although the "fluorescent sensor for planar polarity in Xenopus" may be new (as indicated in the "Impact statement"), it is not really novel for a GFP fusion to be used to reveal polarity, as even in Xenopus this has been previously demonstrated (Butler and Wallingford (2015) Development 142, 3429-3439).

We agree that fluorescent GFP fusions are common tools for cell polarity studies. Nevertheless, we wish to emphasize that our constructs are the first to reveal PCP in early non-neural ectoderm. The sentence noted by the referee has been rephrased to clarify the meaning.

3) A structure-function dissection of Pk3 domains required for polarization is described, without any reference to the similar results for Pk2 described in Butler et al. 2015.

We apologize for inadvertently omitting this reference. This point has now been corrected.

I'm also concerned about how much can be concluded if endogenous protein is not first removed in a structure-function experiment like this.

Since we know little about how Pk3 is polarized, our initial observations may be useful even in the presence of endogenous Pk3. Future studies are needed to study the role of endogenous Pk3 and other Prickle family members in PCP.

[Editors' note: further revisions were requested prior to acceptance, as described below.]

We would like to encourage you to make some additional text modifications to the manuscript that addresses the specific issues raised in the two reviews below – especially the issues raised by reviewer #2. With respect to the writing, we appreciate that in the current publishing environment there is an almost irresistible temptation to over-state one's conclusions. eLife is trying to restore some balance to the world of scientific writing. We welcome self-critical comments and we believe that such comments actually enhance the reader's ability to judge the science fairly.

I will offer two examples of scientific writing that I think illustrates the preceding point. They are from two papers on ubiquitin that form the core of the discoveries for which the authors shared the Nobel Prize.

Proposed role of ATP in protein breakdown: conjugation of protein with multiple chains of the polypeptide of ATP-dependent proteolysis.

Hershko A, Ciechanover A, Heller H, Haas AL, Rose IA.

Proc Natl Acad Sci U S A. 1980 Apr;77(4):1783-6.

In the second paragraph of the Discussion section, after summarizing the evidence that leads the authors to propose the conjugation of APF-1 (=ubiquitin) to proteins as a way of marking them for degradation, the authors write: "Evidence that APF-1-proteins are intermediates in the breakdown of denatured protein as proposed in Figure 6 is indirect and inconclusive at this time."

Activation of the heat-stable polypeptide of the ATP-dependent proteolytic system.

Ciechanover A, Heller H, Katz-Etzion R, Hershko A.

Proc Natl Acad Sci U S A. 1981 Feb;78(2):761-5.

In the last paragraph of the Discussion section, after describing their discovery and characterization of ubiquitin (=APF-1) ligase, the authors write "Evidence suggesting the role of the APF-1-activating enzyme in conjugation and in ATP-dependent protein breakdown is not conclusive at present."

Reviewer #1:

The authors have added new experiments that address some of the criticisms but they have not been able to address others. In response to the criticism that their gain-of-function experiments show that Wnt signaling CAN direct planar polarity but not that it DOES direct endogenous polarity, they include new data showing that the polarization of endogenous Vangl2 is altered by localized WNT overexpression, and that morpholino knockdown of Wnt11b eliminates endogenous Vangl2 polarization. This, combined with the gain-of-function data showing that Wnt expressing cells can repolarize Vangl2 and Pk3 over several cell diameters, is compelling evidence for a role for Wnts in orienting PCP in the frog gastrula non-neural ectoderm. On the other hand, the authors tried but failed to detect polarization of any sub-cellular structure apart from PCP core components themselves. They argue that microtubule or centrosomal asymmetry may be a much later consequence of PCP, not detectable at these early stages. They have also addressed some relatively minor comments I had about quantification of the data. In sum, the paper is improved by demonstrating a requirement for Wnts in endogenous Vangl2 localization, but remains focused on the polarity proteins themselves and not their morphological or cellular consequences. As I was already quite positive about the paper, which I found to be the clearest demonstration to date of a role for Wnts in directing epithelial PCP, I feel that it is now appropriate for publication in eLife.

The seventh paragraph of the Results and Discussion described our experiments that attempted to document morphological consequences of Wnt-dependent PCP. So far, these experiments remain inconclusive. To acknowledge this deficiency, we introduced the following sentence in the last but one paragraph of the discussion. “Although our results demonstrate that Wnt proteins can instruct Pk3 polarization in our specific experimental conditions, the immediate morphological manifestations of this activity remain obscure”

Reviewer #2:

This resubmission contains all of the same data regarding the epidermal ectoderm from the first submission, unchanged except for the additional statistical analysis, plus one new figure about polarity in the neuroectoderm. As such, my opinion about the interpretive difficulties concerning the epidermal ectoderm remains unchanged. The main response of the authors was to change what they choose to say about the epidermal ectoderm data. For this part of the manuscript, the message has been changed (mostly) from the claim that a Wnt signal is instructive for planar polarization, to the well justified statement that a Wnt signal can be instructive for planar polarization.

The new data concern a separate and possibly distinct planar polarization event, so it is not clear how this is supposed to support the findings in the first four figures as opposed to representing a different example. This new data, which was presumably added to address concerns about a true loss of function phenotype, demonstrates that Wnt11b is required for planar polarity of Vangl2 in the neuroectoderm. On the other hand, it does not show that this signal works at a distance, since descendants of the 8 ventral blastomeres into which the MO was injected contribute to the neural plate, and expression of Wnt11b, perhaps at a low level, throughout the neural plate is not ruled out. On the other hand, they do show that ectopic Wnt5a can non-autonomously repolarize neural plate over several cell diameters. They acknowledge that this might occur by either paracrine signaling and propagation, or by graded signaling of the ligand. Concerning this new data, the authors are a bit more circumspect, but shockingly, the final conclusion of the paper again incorrectly claims that Wnt's are instructive for PCP: "Whereas our results demonstrate the instructive role of Wnt proteins in PCP…"

If we conflate the three different paradigms (ectodermal ectoderm, Wnt11b in neural ectoderm and Wnt5a in neural ectoderm), the data would probably make the best argument yet that a Wnt signal acts instructively to determine PCP, the main weaknesses being that the localized expression of Wnt needs to be more carefully examined, and one would like to see gain and loss of function for the same Wnt (or combination of Wnts) in the same tissue. However, I don't feel it's sufficiently rigorous to combine these systems to come to a conclusion as important as the claim that a Wnt IS instructive for PCP.

If the authors wish to try to produce a more definitive conclusion, I'd advise them to build a careful analysis of Wnt11b in the neural plate, and not just add the minimal data presented here as an addendum. If they wish to moderate their overall conclusion to Wnts can instruct planar polarity (two misleading statements would need to be changed, see below), that would be well supported by the data provided. This course would require a somewhat detailed discussion of the distinction between these conclusions. On the other hand, that would be a far less novel result, and one could debate whether it would be of sufficient impact for eLife.

The two unjustified statements:

"Based on these experiments, Wnt5a appears to act as an instructive cue for Pk3 polarization."

This implies that this is what it normally does.

"Whereas our results demonstrate the instructive role of Wnt proteins in PCP, whether such function involves mechanical or chemical signaling remains to be clarified in future studies."

This is clearly not supported by the present data.

In the Discussion, we did not mean to imply that Wnt signals are proven to act instructively. Our results only show how Wnt proteins behave in our specific experimental conditions. They do not establish that the Wnt-dependent instructive mechanism directs PCP during normal development, but simply suggest a possible scenario of what could happen. The misleading sentences noted by the referee were deleted and corrected, and the above distinction has been made clear in the revised Discussion at the end of the manuscript.

It's also relevant to comment on one of the responses that the authors provided in the rebuttal.

"Several lines of evidence argue for Wnt proteins acting on ectodermal PCP over a long distance during vertebrate gastrulation. First, the effect of ectopic Wnt proteins on the GFP-Pk3/Vangl2 complex is detectable across many cell diameters."

So too is clonal knockdown of fz in a fly wing detected several cell diameters away. But this would still clearly be a paracrine signal that is then propagated via other means beyond the clone border. This is therefore not strong evidence for a direct long range action of a ligand.

"Second, Wnt5a can reorient endogenous Vangl2, which is normally polarized throughout the neuroectoderm along the anteroposterior axis (new Figure 5A–D)."

Doesn't address the paracrine vs. long range issue.

"Third, Wnt11b depletion in the posterior region of the embryo disrupts Vangl2 polarity in the neural plate (new Figure 5E,F), supporting its role in long-range anteroposterior PCP."

This may be the strongest argument, but still the experimental design is flawed (as noted above), as the vegetal blastomeres of eight cell embryos contribute to neural plate, so the knockdown is presumably distributed throughout the region, and local expression is not ruled out. The authors therefore correctly state "Nevertheless, currently we cannot discriminate between long-range diffusion and local effects of Wnt proteins propagated by a signal relay system or cell division (Alexandre et al., 2014; Farin et al., 2016; Zecca et al., 1996)."

The experiment has been done with 8-cell embryos in order to deplete Wnt11b as much as possible in its vegetal expression domain (Ku and Melton, 1993). The reviewer correctly pointed out that we do not rule out the effect on local signal production. This is now acknowledged in the revised text (Results and Discussion, eighth paragraph). Overall, our work did not intend to address how far Wnt signals are diffusing. Although our observations are consistent with long-range effects, we make no claims or conclusions regarding this issue in the paper.

Finally, if the authors wish to build a case around Wnt11b. How do they reconcile their story with "Wnt11 has been argued to act permissively in convergent extension during zebrafish gastrulation (Heisenberg et al., 2000)"? Of course, then one is comparing fish and frogs.

The argument that Wnt11 acts permissively in zebrafish embryos was based on the partial rescue of silberblick mutants with ectopic Wnt11. We propose that this effect is incomplete because Wnt11 is not properly localized. In other words, the instructive role of Wnt11 may be necessary for the full rescue. This alternative interpretation is fully compatible with our observations. This explanation was introduced into revised Discussion (last paragraph).

https://doi.org/10.7554/eLife.16463.013

Article and author information

Author details

  1. Chih-Wen Chu

    Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, United States
    Contribution
    C-WC, Conception and design, Acquisition of data, Analysis and interpretation of data, Drafting or revising the article
    Competing interests
    The authors declare that no competing interests exist.
  2. Sergei Y Sokol

    Department of Developmental and Regenerative Biology, Icahn School of Medicine at Mount Sinai, New York, United States
    Contribution
    SYS, Conception and design, Analysis and interpretation of data, Drafting or revising the article
    For correspondence
    sergei.sokol@mssm.edu
    Competing interests
    The authors declare that no competing interests exist.
    ORCID icon 0000-0002-3963-9202

Funding

National Institutes of Health (HD078874)

  • Sergei Y Sokol

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

We thank Kyeongmi Kim for the HA-tagged Xenopus Vangl2 construct, Brian Mitchell, Chris Kintner, Randy Moon, Chenbei Chang, Paris Skourides, and Andrey Zaraisky, for plasmids. We also thank Chi Pak for comments on the manuscript, Vladimir Gelfand for advice on microtubule plus end tracking and members of the Sokol laboratory for discussions. Confocal microscopy was performed at the Microscopy CORE at the Icahn School of Medicine at Mount Sinai. This study was supported by NIH grants to SS.

Ethics

Animal experimentation: This study was carried out in strict accordance with the recommendations in the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. The protocol 04-1295 was approved by the institutional animal care and use committee (IACUC) of the Icahn School of Medicine at Mount Sinai.

Reviewing Editor

  1. Jeremy Nathans, Reviewing Editor, Johns Hopkins University School of Medicine, United States

Publication history

  1. Received: March 29, 2016
  2. Accepted: September 21, 2016
  3. Accepted Manuscript published: September 23, 2016 (version 1)
  4. Version of Record published: October 7, 2016 (version 2)

Copyright

© 2016, Chu et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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