1. Developmental Biology
  2. Evolutionary Biology

Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton

  1. Jenaid M Rees
  2. Victoria A Sleight
  3. Stephen J Clark
  4. Tetsuya Nakamura
  5. J Andrew Gillis  Is a corresponding author
  1. Department of Zoology, University of Cambridge, United Kingdom
  2. School of Biological Sciences, University of Aberdeen, United Kingdom
  3. The Babraham Institute, United Kingdom
  4. Department of Genetics, Rutgers University, United States
  5. Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, United States
https://doi.org/10.7554/eLife.79964
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  1. Jenaid M Rees
  2. Victoria A Sleight
  3. Stephen J Clark
  4. Tetsuya Nakamura
  5. J Andrew Gillis
(2023)
Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton
eLife 12:e79964.
https://doi.org/10.7554/eLife.79964
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Abstract

The gill skeleton of cartilaginous fishes (sharks, skates, rays, and holocephalans) exhibits a striking anterior–posterior polarity, with a series of fine appendages called branchial rays projecting from the posterior margin of the gill arch cartilages. We previously demonstrated in the skate (Leucoraja erinacea) that branchial rays derive from a posterior domain of pharyngeal arch mesenchyme that is responsive to Sonic hedgehog (Shh) signaling from a distal gill arch epithelial ridge (GAER) signaling centre. However, how branchial ray progenitors are specified exclusively within posterior gill arch mesenchyme is not known. Here, we show that genes encoding several Wnt ligands are expressed in the ectoderm immediately adjacent to the skate GAER, and that these Wnt signals are transduced largely in the anterior arch environment. Using pharmacological manipulation, we show that inhibition of Wnt signalling results in an anterior expansion of Shh signal transduction in developing skate gill arches, and in the formation of ectopic anterior branchial ray cartilages. Our findings demonstrate that ectodermal Wnt signalling contributes to gill arch skeletal polarity in skate by restricting Shh signal transduction and chondrogenesis to the posterior arch environment and highlights the importance of signalling interactions at embryonic tissue boundaries for cell fate determination in vertebrate pharyngeal arches.

Editor's evaluation

Building on previous work dissecting the polarisation of a gill arch epithelial ridge (GAER) of a cartilaginous fish (skate), this paper uses RNA–Sequencing, multiplexed HCR, cell fate mapping, and pharmacological manipulation to uncover the contribution of the Wnt signalling pathway to the patterning of the gill arch, highlighting the role tissues boundaries in signalling and patterning embryonic tissues.

https://doi.org/10.7554/eLife.79964.sa0

Introduction

The pharyngeal arches of vertebrates are a series of paired columns of tissue that form on either side of the embryonic head. Pharyngeal arches form as iterative outpockets of foregut endoderm contact the overlying surface ectoderm, and this meeting of endoderm and ectoderm generates columns lined laterally by ectoderm, medially by endoderm, and containing a core of mesoderm and neural crest-derived mesenchyme (Graham and Smith, 2001). In fishes, endodermal outpockets fuse with the overlying surface ectoderm, giving rise to the gill slits and the respiratory surfaces of the gills (Gillis and Tidswell, 2017). In amniotes, endodermal outpockets give rise to glandular tissues, such as the tonsils, parathyroid, and ultimobranchial glands (Grevellec and Tucker, 2010). The largely neural crest-derived mesenchyme of the pharyngeal arches gives rise to the pharyngeal skeleton – i.e., the skeleton of the jaws and gills in fishes, and of the jaw, auditory ossicles, and larynx in amniotes (Jiang et al., 2002; Kague et al., 2012; Sleight and Gillis, 2020; Couly and Le Douarin, 1990) – with mesenchymal derivatives of each arch receiving patterning and polarity information via signals from adjacent epithelia (Veitch et al., 1999; Gillis et al., 2009a, Couly et al., 2002; Brito et al., 2006). Pharyngeal arches are named according to their ancestral skeletal derivatives: the 1st pharyngeal arch is termed the mandibular arch, the 2nd pharyngeal arch is the hyoid arch, while the caudal pharyngeal arches are collectively termed the gill arches. In fishes, the gill arches give rise to a series of skeletal ‘arches’ that support the lamellae of the gills.

All jawed vertebrates belong to one of two lineages: cartilaginous fishes (sharks, skates, rays, and holocephalans) or bony vertebrates (ray- and lobe-fined fishes, with the latter including tetrapods). While the gill arch skeleton of both cartilaginous and bony fishes ancestrally consisted proximally of two principal gill arch cartilages (the epi- and ceratobranchials), the gill arch skeleton of cartilaginous fishes additionally includes a distal series of fine cartilaginous rods called branchial rays (Figure 1A–C). These rays reflect the clear anteroposterior polarity of the gill arch skeleton of cartilaginous fishes, originating along the posterior margin of the epi- and ceratobranchial cartilage, and curving posteriorly as they project into the interbranchial septum of each arch (Gillis et al., 2009b). Elasmobranch cartilaginous fishes (sharks, skates, and rays) possess five sets of branchial rays, associated with their hyoid and first four gill arches. Holocephalans, on the other hand, possess a single set of branchial rays supporting their hyoid arch-derived operculum (Gillis et al., 2011).

Figure 1
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Overview of skate gill arch skeletal anatomy.

Skeletal preparation of an S33 skate embryo in (A) ventral and (B) lateral view. In (A’) and (B’) the gill arches and branchial rays are highlighted, with branchial rays false-colored red. Note that branchial rays project from the posterior margin of the hyoid and first four gill arches. (C) A dissected gill arch with the (C’) dorsal epibranchial and ventral ceratobranchial gill arch cartilages indicated, and with branchial rays false-colored red. a–p indicates anterior–posterior axis. br: branchial ray, cb: ceratobranchial, eb: epibranchial, ga1-4: gill arches 1–4, ha: hyoid arch, ma: mandibular arch. Scale bars: 1 mm.

The branchial rays of cartilaginous fishes develop under the influence of the GAER (gill arch epithelial ridge): a Sonic hedgehog (Shh)-expressing signaling centre located within the posterior–distal epithelium of the gill arches (Gillis et al., 2009b; Gillis and Hall, 2016). As the gill arches undergo a prolonged phase of lateral expansion, Shh signaling from the GAER is asymmetrically transduced within the posterior arch environment, as evidenced by posterior expression of Ptc2 (Gillis and Hall, 2016) – a Shh co-receptor and transcriptional readout of Shh signaling (Pearse et al., 2001). This posterior transduction of Shh signaling, in turn, appears to underlie the anteroposterior polarity of the gill arch skeleton: in skate, branchial rays derive exclusively from posterior–distal (Shh-responsive/GAER-adjacent) gill arch mesenchyme, application of exogenous Shh protein within gill arches is sufficient to induce ectopic branchial ray formation, and targeted or systemic inhibition of hedgehog signaling using cyclopamine results in branchial ray deletion (Gillis et al., 2009b; Gillis and Hall, 2016). However, whether other signaling mechanisms function alongside or in conjunction with GAER Shh signaling to establish and maintain gill arch skeletal polarity remains unexplored.

Here, we show in the little skate (L. erinacea) that the ectoderm immediately adjacent to the GAER expresses several genes encoding Wnt ligands. These Wnt signals are transduced in the anterior arch environment in a pattern that is broadly complementary to the posterior transduction of GAER Shh signaling. Inhibition of Wnt signaling in developing skate embryos results in an anterior expansion of Shh signaling transduction, and in the formation of ectopic branchial rays in the anterior gill arch. We propose that Wnt signaling from the pharyngeal arch ectoderm contributes to the maintenance of anterior–posterior polarity of the skate gill arch skeleton by repressing Shh signaling and chondrogenesis to the posterior gill arch territory.

Results

Wnt gene expression in the skate GAER and GAER-adjacent epithelium

In developing skate gill arches, Shh is expressed in the GAER (Figure 2A), and branchial rays develop from a GAER-responsive domain of posterior arch mesenchyme (Gillis and Hall, 2016). To further explore the transcriptional environment of the GAER and to discover additional gene expression features that may contribute to gill arch polarity, we conducted a comparative transcriptomic and differential gene expression analysis of GAER and non-GAER regions of the first gill arch of the embryonic skate. Briefly, we manually dissected the (1) GAER and (2) non-GAER (control) regions from the first gill arch of stage (S)26 skate embryos (n=5; Figure 2B–Bi), and we performed RNA extraction, library preparation, and RNAseq analysis on these samples. For the GAER region of the first gill arch, we included the whole distal tip of the arch in our dissection to ensure that the Shh-expressing GAER and adjacent Shh-responsive (i.e. Ptc2+) tissues were captured for this analysis (Figure 2C–D). Following de novo transcriptome assembly, we tested for differential gene expression between GAER and non-GAER tissues (after first testing for differential expression of Shh in the GAER region, to confirm dissection accuracy). We used a false discovery rate of 5% and no log fold-change (LogFC) cut-off to generate a list of 401 genes that were upregulated in the GAER region. We sorted this list for genes encoding signaling pathway components and transcription factors with a log-fold change >2 (Figure 2E). Differentially expressed genes were subjected to functional enrichment analysis, and this analysis indicated enrichment of Wnt signaling pathway components in the GAER region (Figure 2—figure supplement 1 for enrichment analysis). Differentially expressed genes in the GAER region included those encoding the Wnt ligands Wnt2b, Wnt3, Wnt4, Wnt7b, and Wnt9b, the transmembrane Wnt inhibitors Kremen1 (Mao et al., 2002) and APCDD1 (Shimomura et al., 2010), and the secreted Wnt antagonist Notum (Zhang et al., 2015). We, therefore, chose to further investigate the involvement of the Wnt pathway in GAER signaling by spatially validating the expression of these genes, using the most highly expressed isoforms for mRNA in situ hybridization by chain reaction (HCR) probe design.

Figure 2 with 1 supplement see all
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Differential gene expression analysis of gill arch epithelial ridge (GAER) and non-GAER gill arch tissues in S26 skate embryos.

(A) Sonic hedgehog (Shh) is expressed in the GAER of the hyoid and first four gill arches of skate embryos. (B) Schematic of dissection from gill arch one of (Bi) the non-GAER region (control, green) and the GAER region (blue, with GAER location indicated by yellow dashed outline), as guided by (C) mRNA in situ hybridization for Shh on paraffin section. (D) The dissected GAER region includes the Shh-expressing GAER, as well as adjacent Shh-responsive (i.e. Ptc2+) tissues, as indicated by multiplexed mRNA in situ hybridization by chain reaction (HCR) for Shh and Ptc2 on paraffin section. (E, Ei) Volcano plot illustrating differential gene expression between GAER and non-GAER tissues. Genes with >2 logFC and <0.05 false discovery rate (FDR) are highlighted and assigned functional categories using color coding as per key. Genes with >2 logFC are shown in larger point sizes. Signaling molecules and transcription factors with >2 logFC and <0.05 FDR are labeled (* denotes manual annotation of sequence). Differential expression was determined using edgeR with a general linear model and likelihood ratio test, corrected for multiple testing using the Benjamin-Hochberg method to control the FDR. a–p indicates anterior–posterior axis, p–d indicates proximal–distal axis. epi: epithelium, ga1-4: gill arches 1–4, ha: hyoid arch, ma: mandibular arch, mc: mesodermal core, mes: mesenchyme. Scale bars; A: 500 μm C-D: 50 μm.

To characterize the expression of genes encoding Wnt ligands relative to Shh expression in the GAER, we used multiplexed ISH by HCR to visualize transcript localization in sections of S26 skate gill arches. We found that while Wnt7b was co-expressed with Shh in the cells of the GAER (Figure 3A), Wnt2b, Wnt3, Wnt4, and Wnt9b (Figure 3B–E) were all expressed predominantly in the epithelium immediately adjacent to the GAER. We next examined the expression of the Wnt signaling downstream target genes (Figure 4) using Shh or Wnt7b as a marker for the GAER. We found that Apcdd1 was expressed in the GAER and anterior–distal mesenchyme (Figure 4A), Notum in the anterior–distal mesenchyme (Figure 4B), and Kremen1 in the anterior (GAER-adjacent) epithelium (Figure 4C). Finally, broad transcription of Axin2 throughout distal gill arch tissues (Figure 4D) indicates that Wnt signaling within this territory is occurring through the canonical/β-catenin pathway (Lustig et al., 2002). While these findings do not allow us to attribute expression of specific transcriptional readouts to signaling by particular Wnt ligands, our spatial expression data nevertheless indicate that some Wnt signals emanating from the GAER or GAER-adjacent epithelium are transduced preferentially within the anterior arch environment, in a pattern that is largely complimentary to the posterior epithelial and mesenchymal transduction of Shh signals from the GAER.

Figure 3
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Genes encoding Wnt ligands are expressed in and around the gill arch epithelial ridge (GAER) in skate.

In S26 skates, (A, A’) Wnt7b is co-expressed with Sonic hedgehog (Shh) in the GAER. (B) Wnt2b, (C) Wnt3, (D) Wnt4 and (E), Wnt9b are predominantly expressed in the epithelium immediately adjacent to the GAER. For ease of visualization in A’–E’, Shh expression is not shown but GAER cells are outlined with a green dashed line. Arch tissues are outlined with white dashed lines. a–p indicates anterior–posterior axis. epi: epithelium, mes: mesenchyme, mc: mesodermal core. Scale bar: 50 μm.

Figure 4 with 1 supplement see all
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Transduction of canonical Wnt signaling in the anterior gill arch territory in skate.

In S26 skates, (A, A’) Apcdd1 is expressed in the gill arch epithelial ridge (GAER) and in the anterior-distal mesenchyme underlying the GAER. (B) Notum is expressed in anterior-distal arch mesenchyme. (C) Kremen1 is expressed in the GAER and in GAER-adjacent epithelium. (D) Broad Axin2 expression in the vicinity of Wnt-expressing epithelium indicates signaling through the canonical Wnt signaling pathway. ISH by in situ hybridization by chain reaction (HCR) for Sonic hedgehog (Shh) or Wnt7b was performed alongside each gene of interest as a marker of the GAER (outlined with a magenta dashed line). a–p indicates anterior–posterior axis. epi: epithelium, mes: mesenchyme, mc: mesodermal core. Scale bar: 50 μm.

Wnt and Shh expression at the ectoderm–endoderm interface of skate gill arches

The epithelium of vertebrate pharyngeal arches derives from both ectoderm and endoderm, but the specific germ layer origin of the GAER of cartilaginous fishes (endoderm vs. ectoderm) is not known. In the skate, Shh is initially expressed broadly in the anterior endodermal epithelium of each pharyngeal pouch, which subsequently gives rise to the posterior endodermal epithelium of each pharyngeal arch (Gillis and Tidswell, 2017). To test whether the GAER derives from this initially broad endodermal Shh expression domain, we labeled the pharyngeal endoderm of early skate embryos by microinjecting the lipophilic dye CM-DiI into the pharyngeal cavity at stage S18. At this stage, the pharyngeal pouches have not yet fused with the overlying surface ectoderm, allowing for specific CM-DiI labeling of the pharyngeal endodermal epithelium (Figure 5A and B). Injected embryos were then grown to S25–S29, by which time the GAER of the hyoid and gill arches is detectable by expression of Shh (Figure 5C).

Figure 5
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Endodermal origin of the skate gill arch epithelial ridge (GAER).

(A) Microinjection of CM-DiI into the pharyngeal cavity of skate embryos at S18 results in (B) specific labeling of the pharyngeal endoderm. (C) Sonic hedgehog (Shh) is a marker of the GAER (dashed yellow line) in CM-DiI-labeled embryos, and (C’, C’’) co-localization of CM-DiI and Shh expression indicates that the cells of the GAER are of endodermal origin. Dashed white outline demarcates GAER-adjacent (ectodermal) epithelium, which is never labeled with CM-DiI. (C), (C’), and (C’’) are the same section, imaged sequentially for Shh expression and CM-DiI. a–p indicates anterior–posterior axis. dpi: days post-injection, fg: foregut, nt: neural tube. Scale bars; A: 500 μm, B–C: 40 μm.

Of 29 embryos analyzed, all retained some CM-DiI label within the endodermal lining of the pharyngeal arches. In 8/29 embryos, we recovered CM-DiI labeling up to and including the Shh-expressing cells of the GAER in one or more pharyngeal arches (Figure 5C–C’’), indicating the endodermal origin of these cells. The remaining 21 embryos did not have sufficient labeling of pharyngeal epithelia to inform the origin of the GAER (likely due to label dilution during the long incubation time between injection and analysis). Importantly, however, no CM-DiI labeling was ever observed in epithelial cells immediately anterior to the GAER, suggesting that this epithelium is of ectodermal origin. Together, these observations indicate that the GAER is of endodermal origin and arises at the ectoderm–endoderm interface during pharyngeal arch development and that the Wnt-expressing epithelium immediately anterior to the GAER derives from the ectoderm.

Wnt signaling suppresses GAER Shh signal transduction in the anterior gill arch

To explore the function of Wnt signaling during skate pharyngeal arch development, we inhibited canonical Wnt signaling in skate embryos using IWR1, a small molecule tankyrase inhibitor that antagonizes Wnt signaling by stabilizing the Axin/β-catenin destruction complex (Lu et al., 2009; Kulak et al., 2015). Briefly, skate embryos were maintained in a bath of 50 μM IWR1 or vehicle-only control (DMSO) in seawater from S25 for 72 hr to assess gene expression changes in response to drug treatment (Figure 4—figure supplement 1), or for 6–8 weeks (until S31/32) to assess effects of Wnt signaling inhibition on gill arch skeletal patterning, with replacement of drug- or control-seawater every 48 hr. IWR1-treated embryos showed a marked reduction in the expression of Axin2 throughout their gill arches (Figure 4—figure supplement 1A–B), as well as a reduction in the expression of Apcdd1 in the GAER and anterior–distal gill arch mesenchyme (Figure 4—figure supplement 1C–D) and a complete loss of Notum expression in anterior–distal gill arch mesenchyme (Figure 4—figure supplement 1E–F). These findings indicate that our drug treatment effectively suppressed canonical Wnt signaling from the GAER and GAER-adjacent epithelium, including those signals transduced asymmetrically in the anterior gill arch environment.

Shh is a pro-chondrogenic signal from the GAER in skate gill arches (Gillis et al., 2009b), and this signal is transduced asymmetrically with the posterior gill arch environment (Gillis and Hall, 2016, see above Figure 2D). To test whether anteriorly transduced Wnt signaling may act to restrict Shh signal transduction to the posterior gill arch, we examined the expression of Shh and Ptc2 in skate embryos 72 hr after the onset of IWR1 treatment (Figure 6). While we observed no noticeable change in the expression of Shh or in the spatial distribution of Ptc2 transcripts in gill arch mesenchyme with IWR1 treatment, we did note a qualitative increase in Ptc2 expression within the normal mesenchymal domain and a clear anterior expansion of Ptc2 expression within the epithelium adjacent to the GAER (Figure 6A–D). To quantify this epithelial expansion, we compared the mean number of Ptc2+ nuclei anterior to the Shh-expressing GAER (using DAPI staining and counted manually) across two or three sections at equivalent positions in DMSO control (n=3) or IWR1 (n=4) treated embryos and found a significant anterior expansion of Ptc2 expression within the epithelium adjacent to the GAER in IWR1 treated embryos (p=0.036; Figure 6E–G). This points to a role for GAER-adjacent Wnt signaling in restricting the transduction of pro-chondrogenic Shh signaling in the anterior gill arch territory in skate.

Figure 6
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Wnt signaling restricts transduction of gill arch epithelial ridge (GAER) Sonic hedgehog (Shh) signaling to the posterior gill arch environment.

ISH by in situ hybridization by chain reaction (HCR) on sections of skate embryos treated for 72 hr with (A,B) DMSO or (C,D) IWR1 shows no difference in Shh expression but an expansion of Ptc2 expression within the epithelium anterior to the Shh-expressing GAER. (E) Schematic illustration of the mean number of Ptc2+ nuclei anterior to the GAER in DMSO control (n=3 embryos; mean = 6.3 cells) or (F) IWR1 treated (n=4 embryos; mean = 12.94 cells) embryos, using the mean cell count of two or three sections at equivalent positions in the gill arch from each embryo. (G) We find a significant increase in Ptc2+ nuclei in the epithelium anterior to the GAER in IWR1-treated embryos compared to control (p=0.036; t-test, t=–3.87, df = 2.74 - box and whisker plot shows the interquartile range and spread of the data). In A–F, Shh expression is indicated by a green dashed line. Images in A–D were taken using identical exposure settings. a–p indicates anterior–posterior axis. Scale bar: 20 μm.

Wnt signaling inhibits branchial ray development in the anterior gill arch environment

Finally, to test for an effect of Wnt inhibition on the gill arch skeleton, we reared skate embryos in DMSO control seawater or IWR1 from S25–S31/32 (Figure 7). IWR1-treated embryos had several distinct phenotypic changes, including smaller size, a loss of gill filaments and disrupted fin morphology (the latter consistent with previous findings of Nakamura et al., 2015; Figure 7A–B). We specifically investigated the impact of IWR1 treatment on gill arch skeletal morphology. The skate hyoid and gill arch skeleton include branchial rays that articulate exclusively with the posterior margin of the gill arch cartilages (Gillis et al., 2009a – see also Figure 1). These rays also invariably develop on the posterior side of the interbranchial muscle plate – a histologically and molecularly distinct derivative of the paraxial mesodermal core that extends down the middle of each pharyngeal arch (Daniel, 1934; Graham, 2008; Hirschberger et al., 2021). Skate embryos reared in DMSO control seawater showed no gill arch skeletal defects (n=4 embryos; Figure 7Ai), while embryos reared in IWR1 seawater possessed conspicuous ectopic branchial rays in the anterior gill arch territory (n=7/8 embryos; Figure 7Bi). These ectopic branchial rays occurred along the entire dorsoventral axis of the arch and ranged from short cartilage rods located in the anterior–distal gill arch to nearly full-length branchial rays that extended much of the way toward the gill arch cartilages. In section, branchial rays of skate embryos reared in DMSO control seawater always developed, as per normal, on the posterior side of the mesodermally-derived interbranchial muscle plate of each gill arch: this was evident from histochemical staining and from HCR for the chondrocyte markers Sox9 and Col2a1 (Figure 8A–B). Conversely, in skate embryos reared in IWR1 seawater, ectopic branchial rays were consistently located anterior to the interbranchial muscle plate (Figure 8C–D). The induction of ectopic branchial rays with IWR1 treatment suggests that Wnt signaling contributes to the maintenance of anteroposterior polarity of the skate gill arch skeleton by repressing chondrogenesis in the anterior gill arch and may do so by restricting transduction of GAER Shh signaling to the posterior gill arch territory.

Figure 7
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Ectopic branchial ray formation upon inhibition of Wnt signaling.

(A) In control (DMSO) skate embryos, branchial rays (magnified in Ai) articulate exclusively with the posterior margin of the gill arches. (B) Embryos reared from S25 to S31/32 in the Wnt inhibitor IWR1 possessed ectopic branchial rays (magnified in Bi) that were embedded within the connective tissue of the anterior gill arch (ectopic rays present in n=7/8 embryos examined; indicated by a red asterisk). a–p indicates anterior–posterior axis. Scale bars: 500 μm.

Figure 8
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Repression of cartilage development in the anterior gill arch by Wnt signaling.

(A) Section through the gill arch of control (DMSO) S31 skate embryo, showing that branchial rays develop exclusively on the posterior side of the interbranchial muscle plate, as is evident from histochemical staining and (B, Bi, Bii) ISH by in situ hybridization by chain reaction (HCR) for the chondrocyte markers Sox9 and Cola2a1. (C) Section through the gill arch of a S31 embryo reared in the canonical Wnt inhibitor IWR1 reveals that ectopic branchial rays form anterior to the interbranchial muscle plate, as evident from histological analysis and (D, Di, Dii) ISH by HCR for Sox9 and Col2a1. Red asterisks indicate ectopic branchial rays, and the interbranchial muscle plate is outlined with a dashed line. a–p indicates anterior–posterior axis. Scale bars: 50 μm.

Discussion

The molecular mechanisms governing spatial regulation of cell fate decisions are crucial to the establishment and maintenance of anatomical polarity within developing tissues and organs. Here, we show that the GAER signaling centre that forms on the hyoid and gill arches of cartilaginous fishes is of endodermal origin, arising precisely at the boundary of surface ectoderm and pharyngeal endoderm, and that pro-chondrogenic Shh signals from the GAER are transduced preferentially within the posterior gill arch environment. We further show that the ectoderm immediately anterior and adjacent to the GAER is a signaling centre that expresses several Wnt family genes and that Wnt signals from this centre are transduced preferentially in the anterior arch environment. Pharmacological inhibition of canonical Wnt signaling in skate embryos leads to an expansion of Shh signal transduction within the anterior territory of the gill arch, and to the formation of ectopic anterior branchial ray cartilage. These findings suggest that Wnt–Shh signaling antagonism at the pharyngeal ectodermal–endodermal tissue boundary is a key molecular regulator of cell fate determination and anatomical polarity with the developing gill arch skeleton of the skate (Figure 9).

Figure 9
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Wnt and Sonic hedgehog (Shh) signaling and cell fate determination in skate gill arches.

Shh signaling from the endodermal-derived gill arch epithelial ridge (GAER) is transduced within the posterior-distal gill arch (ga) environment, where it promotes the differentiation of cartilaginous branchial rays (br). Wnt signals from ectoderm adjacent to the GAER are transduced within the anterior–distal gill arch environment, where they repress Shh signal transduction and inhibit cartilage formation in the anterior gill arch. a–p indicates the anterior–posterior axis and p–d indicates the proximal–distal axis.

Previous analyses have reported the expression of several Wnt genes in developing craniofacial tissues of bony vertebrates (Summerhurst et al., 2008), including the expression of WNT2B and WNT9B in the pharyngeal arch ectoderm of the chick (Geetha-Loganathan et al., 2009), Wnt9b in the first and second pharyngeal arches in zebrafish (Jezewski et al., 2008), Wnt4a in zebrafish pharyngeal ectoderm (Choe et al., 2013), and Wnt9a and Wnt5b in zebrafish oral epithelium (Dougherty et al., 2013; Rochard et al., 2016). While β-catenin-independent (i.e. non-canonical) Wnt signaling plays a crucial role in epithelial remodeling during the early formation of pharyngeal endodermal pouches in zebrafish (Choe et al., 2013) and in maturation and morphogenesis of the zebrafish oropharyngeal skeleton (Rochard et al., 2016; Ling et al., 2017), canonical Wnt signaling has been implicated in patterning of the pharyngeal arch-derived skeleton in multiple taxa. Blocking canonical Wnt signaling causes disrupted facial cartilage formation, including reductions of Meckel’s and ceratohyal cartilages in zebrafish (Alexander et al., 2014) and loss of ectodermal canonical Wnt signaling in mouse causes loss of facial bones with hypoplasia of all facial prominences (Reid et al., 2011). Furthermore, in mouse and human, cleft palate phenotypes may arise as a consequence of mutations in Wnt3 and Wnt9b (Niemann et al., 2004; Juriloff et al., 2006), and perturbation of Wnt/β-catenin signaling has been implicated in CATSHL syndrome, a human developmental disorder involving craniofacial bone malformation and mispatterning of the pharyngeal arches (Sun et al., 2020). Our finding of a role for canonical Wnt signaling in patterning the gill arch skeleton of skate may, therefore, reflect broadly conserved roles for this pathway in pharyngeal skeletal development across jawed vertebrates, though with the skate’s possession of branchial rays offering a unique anatomical readout of anterior–posterior polarity defects that arise within the pharyngeal arch-derived skeleton in response to perturbations.

Our findings are also consistent with previously reported complex and context-dependent roles for Wnt signaling in the regulation of cell fate determination events during vertebrate skeletogenesis. In some instances, Wnt signaling functions to promote skeletogenesis: for example, in chick, misexpression of WNT5A/5B promotes early chondrogenesis of limb bud mesenchyme in vitro and delays the terminal differentiation of growth plate chondrocytes in vivo (Church et al., 2002), and in mouse, canonical WNT signaling from cranial ectoderm induces specification of osteoblast progenitors of cranial dermal bone within underlying mesenchyme (Goodnough et al., 2016). However, in other contexts, Wnt signaling functions to inhibit chondrogenic differentiation or homeostasis. In chick, signaling through WNT3, 4, 7 A, 14 or FZ7 have all been shown to inhibit chondrogenesis in vitro or in vivo (Rudnicki and Brown, 1997), while in mouse conditionally induced haploinsufficiency of the gene encoding the β-catenin degradation complex component APC (i.e. activation of canonical Wnt signaling) results in loss of resting zone chondrocytes and their clonal progeny in developing growth plates (Hallett et al., 2021). In the limb bud, genes encoding WNT ligands are expressed in the ectoderm (Kengaku et al., 1998; Parr et al., 1993; Geetha-Loganathan et al., 2005; Summerhurst et al., 2008), where they function synergistically with fibroblast growth factor signaling from the apical ectodermal ridge to inhibit chondrogenesis of limb bud mesenchyme and to promote soft connective tissue fates (ten Berge et al., 2008). Downstream effectors of Wnt signaling are expressed out-of-phase with Sox9+ digit progenitors in the mouse limb bud, with Wnt signaling functioning to repress digit chondrogenesis as part of a Turing-like mechanism that underlies periodic patterning of the distal limb endoskeleton (Raspopovic et al., 2014). Our findings, therefore, demonstrate a striking parallel between developing skate gill arches and tetrapod limb buds, with Wnt signals emanating from ridge-adjacent epithelia functioning to inhibit chondrogenic differentiation, thereby ensuring differentiation of cartilaginous appendages in a spatially controlled manner.

We have previously shown that Shh signaling from the GAER is required for branchial ray chondrogenesis in skate (Gillis and Hall, 2016), and that the application of exogenous Shh protein to the anterior gill arch is sufficient to induce ectopic branchial rays (Gillis et al., 2009b). However, it is unclear whether Shh from the GAER is a direct inducer of chondrogenesis in the posterior arch mesenchyme, or rather induces chondrogenesis indirectly, via a secondary signal (i.e. emanating from the Shh-responsive epithelium or mesenchyme). We have observed that inhibition of Wnt signaling in skate embryos has no effect on the expression of Shh in the GAER, but rather results in an anterior expansion of Shh signal transduction in the gill arch ectoderm, which in turn, correlates with ectopic chondrogenesis in the anterior gill arch. This observation indicates that the pro-chondrogenic influence of GAER Shh signaling may be indirect, occurring via a secondary Shh-depending epithelial signal whose expression is typically posteriorly restricted by Wnt signaling from GAER-adjacent ectoderm. The Shh and Wnt signaling pathways share multiple downstream components (reviewed in Ding and Wang, 2017). For example, components of the canonical Wnt signaling pathway can positively regulate Shh signaling via GSK3β, by phosphorylating the downstream component SUFU and promoting the release of SUFU from Gli (Takenaka et al., 2007), and by β-catenin affecting Gli1 transcriptional activity via TCF/LEF (Maeda et al., 2006). It has also been found that Gli3 may function as a downstream effector of the Wnt pathway and that Wnt signaling represses Shh activity in the dorsal neural tube through the regulation of Gli3 expression (Alvarez-Medina et al., 2008). Finally, the Shh signaling pathway may regulate Wnt signaling through Gli1 and Gli2, with these factors positively regulating the expression of the secreted Wnt inhibitor frizzled-related protein-1 (sFRP-1) (He et al., 2006). These mechanisms, or others, could account for the apparent cross-regulation between Wnt and Shh signaling that we have observed during the growth and patterning of skate pharyngeal arches.

Finally, it remains to be determined whether antagonistic ectodermal Wnt and endodermal Shh signaling is a derived mechanism of cell fate determination and pharyngeal skeletal patterning in cartilaginous fishes or a conserved feature of the pharyngeal arches of jawed vertebrates. In bony vertebrates, a Shh-expressing posterior endodermal margin (PEM) promotes the proliferative expansion of the second (hyoid) pharyngeal arch (Wall and Hogan, 1995; Richardson et al., 2012; Rees and Gillis, 2022). In bony fishes, the hyoid arch gives rise to an operculum that is supported by a series of dermal bones, and this operculum functions as a protective cover for the gills that arise from the posterior pharyngeal arches, whereas in amniotes, the hyoid arch expands caudally and ultimately fuses with the cardiac eminence to enclose the cervical sinus and close the surface of the neck (Richardson et al., 2012). Amniotes lack homologs of the opercular series of bony fishes or the branchial rays of cartilaginous fishes, respectively, but fossil evidence points to the operculate condition of bony fishes as ancestral for jawed vertebrates (Dearden et al., 2019). Whether juxtaposed Wnt- and Shh-expressing epithelia influence fate determination (e.g. dermal bone vs. connective tissue) and patterning within the opercular series of bony fishes is not known. Further comparative work between bony and cartilaginous fishes with disparate pharyngeal skeletal architectures and tissue compositions will permit the inference of ancestral functions and interactions of epithelial signals during vertebrate pharyngeal arch skeletal development.

Materials and methods

Embryo harvesting

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Skate (L. erinacea) embryos were obtained from the Marine Resources Center at the Marine Biological Laboratory in Woods Hole, MA, U.S.A., were reared as described in Gillis et al., 2012 and staged according to Ballard et al., 1993 and Maxwell et al., 2008. Skate embryos were euthanized with an overdose of MS-222 (1 g/L in seawater), and all embryos were fixed in 4% paraformaldehyde (Thermofisher) as per Gillis et al., 2012.

Sectioning and histochemical staining

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Embryos were embedded in paraffin and sectioned at 7 μm as described in O’Neill et al., 2007. Sectioned embryos were stained with Masson’s Trichrome as per Witten and Hall, 2003 or Mayer’s Hematoxylin and Eosin by clearing in two rinses of Histosol (National Diagnostics), rehydration through serial dilutions of ethanol, staining for 15 min in Mayer’s Haematoxylin (Sigma), rinsing in running tap water for 20 min, rinsing briefly in 95% ethanol, and staining in 0.1% w/v Eosin Y (Sigma) in 95% ethanol for 2 min. Slides were then washed briefly in 95% ethanol, washed briefly with 100% ethanol, cleared with Histosol, and mounted with permount (Sigma).

Lineage tracing

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Endodermal lineage tracing was performed by microinjection of CellTracker CM-DiI (1,1'-dioctadecyl-3,3,3'3'-tetramethylindocarbocyanine perchlorate; Thermofisher) into the pharyngeal cavity of the stage (S)18 skate embryos with a pulled glass needle. CM-DiI was prepared as per Gillis et al., 2012. Labeled embryos were then grown to S25–29, euthanized, fixed, embedded, and sectioned as described above, and CM-DiI localization was assessed relative to the GAER based on mRNA in situ hybridization for Shh (see below).

mRNA in situ hybridization

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Chromogenic mRNA in situ hybridization (ISH) was performed on paraffin sections and in whole mount as described in O’Neill et al., 2007, with modifications according to Gillis et al., 2012. An ISH probe against skate Shh (GenBank EF100667) was generated by in vitro transcription using standard methods. Following ISH, whole mounts were rinsed, post-fixed in 4% paraformaldehyde, and graded into 75% glycerol for imaging, while slides were rinsed in PBS, post-fixed in 4% paraformaldehyde, and coverslipped with DAPI-Fluoromount G (SouthernBiotech). Third-generation mRNA ISH by chain reaction (HCR) was performed as per the Choi et al., 2018 protocol for formaldehyde-fixed, paraffin-embedded sections, with modifications as per Criswell and Gillis, 2020. Probes, buffers, and hairpins were purchased from Molecular Instruments (Los Angeles, California, USA). HCR probe set lot numbers are as follows: skate Shh (Lot PRA753), Ptc2 (Lot PRA754), Wnt2b (Lot PRE300), Wnt3 (Lot PRG814), Wnt4 (Lot PRE301), Wnt7b (Lot PRE302), Wnt9b (Lot PRE303), Notum (Lot PRG817), Kremen1 (Lot PRG816), Axin2 (Lot PRG818), Apcdd1 (Lot PRG815), Col2a1 (Lot PRB574), and Sox9 (Lot PRB571). All mRNA ISH experiments were replicated, at minimum, in biological triplicate.

Pharmacological manipulations

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For systemic inhibition of canonical Wnt signaling, experimental and control skate embryos were reared at 15 °C in Petri dishes containing 50 μM IWR1 (Selleck chemicals) in artificial seawater or artificial seawater containing an equivalent volume of vehicle (DMSO) only, respectively. IWR1 has been used previously to inhibit canonical Wnt signaling in skate embryos by Nakamura et al., 2015 and in shark embryos by Thiery et al., 2022. For these experiments, IWR1 was diluted to a working concentration from a 25 mM stock solution in DMSO. Skate embryos were reared in IWR1- or vehicle-containing seawater from S25 until S31/32, with drug or control seawater changes every 48 hr. Once embryos reached the desired stage, they were euthanized, fixed, and processed for whole mount skeletal preparation according to the protocol of Gillis et al., 2009a or paraffin histology as described above.

RNA-Seq analysis of differentially expressed genes in the GAER region

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GAER or non-GAER (‘control’) regions of gill arch 1 were manually dissected from skate embryos at S26 using tungsten needles and flash frozen in lysis buffer using liquid nitrogen. In all cases, GAER and control sample pairs were collected from the same arch within the same embryos, or from opposite arches within the same embryo. RNA was extracted from each sample using the RNAaqueous-Micro Kit. cDNA was synthesized from extracted RNA according to the Smart-seq2 protocol (Picelli et al., 2014) and libraries were prepared using the Nextera XT kit (Illumina). Prior to sequencing, barcoded libraries were pooled in equal proportions and checked for quality, insert size, and quantity using Qubit 2.0, and Agilent2100. Sequencing of the 20 libraries generated was conducted by Novogene on an Illumina Hi-Seq-XTen generating 150 bp paired-end reads. The Cambridge Service for Data-Driven Discovery (CSD3) high performance computer was used for cleaning, normalization, quality assessment, assembly, abundance quantification, and annotation, as per Hirschberger et al., 2021. Raw reads were cleaned to remove adapter contamination using Trim Galore v0.4.0 (parameters: --paired -q 20 –nextera), for quality (Phred score 20) and minimum read length (149 bp) using ea-utils tool fastq-mcf (parameters: -q 20 l 100) (Aronesty, 2011). Prior to assembly, all libraries were normalized using Trinity v2.6.6 in silico read normalization (Haas et al., 2013) (parameters: --JM 100 G --max_cov 75). Trinity v2.6.6 was used to assemble the normalized reads (parameters: --seqType fq --max_memory 250 G --CPU 50 min_contig_length 450) (Nishimura et al., 2017; Langmead et al., 2009; Marçais and Kingsford, 2011). Basic Local Alignment Search Tool (BLAST) was used to compare the assembly to the Uniprot protein database with an e-value cut-off of e-20 to search for sequence similarity (Altschul et al., 1990). Transcript abundance was estimated using Salmon, an alignment-free quantification method, via Trinity v2.6.6 utilities (Haas et al., 2013; Patro et al., 2017). A matrix was built for each of the libraries of gene-level abundance estimates (raw counts) using the Trinity abundance_estimates_to_matrix.pl script. MDS principal component analysis was used to check for batch effects and outliers. Individual embryo origin was a batch effect and so was accordingly accounted for in the additive general linear model. Differentially expressed genes between control and GAER tissue samples were identified using the edgeR_3.28.0 package in R version 3.6.2 (Robinson et al., 2010) using a generalized linear model likelihood ratio test (Chen, 2008) using a false discovery rate of 5% and no log fold-change (LogFC) cut-off. See supplementary information for assembly statistics, differential expression analysis, and all R scripts used.

Significantly upregulated genes in the S26 GAER tissue (logFC >2, FDR <0.05) were extracted and annotated using blastx sequence similarity searches against the Swissprot/Uniprot database (taxid: Gallus gallus). Annotation IDs were inputted into the STRING (v11.5) webserver and tested for predicted protein-protein interactions. In the protein-protein interaction analysis, line thickness was set to show confidence, and a medium confidence threshold was set. The interaction network was then tested for functional enrichment against a series of databases (Gene Ontology Biological Process, KEGG Pathways, WikiPathways, Pfam Domains, and InterPro Domains) using the default whole genome as the background. Results from the functional enrichment were downloaded and Rstudio (GGplot2) was used to generate dot plots to visualize the most significantly enriched terms.

Differentially upregulated genes with a log-fold change over two were manually screened for genes involved in known signaling pathways and transcription factors to generate a list for candidate gene validation by ISH. Wnt family members were highly upregulated in the GAER region so were validated as candidate markers for this region through third-generation ISH. Transcripts for probes in skate were based on the most highly expressed isoforms.

Imaging and figure preparation

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Images were taken on a Zeiss Axioscope.A1 compound microscope with a Zeiss colibri 7 fluorescence LED light source using a Zeiss Axiocam 305 color or 503 mono camera and ZenPro software or a Leica M165FC stereomicroscope, a Leica DFC7000 T camera and LAS X software. All figures were assembled using Fiji and Adobe creative cloud, with some images flipped or colors inverted for clarity and consistency where needed.

Data availability

All data and R scripts for analysis have been deposited on Figshare: 10.6084/m9.figshare.19615779. RNA sequencing data are available at NCBI-SRA under BioProject ID: PRJNA825354. Biosample accessions: SAMN27512544, SAMN27512545, SAMN27512546, SAMN27512547, SAMN27512548, SAMN27512549, SAMN27512550, SAMN27512551, SAMN27512552, SAMN27512553, SAMN27512554, SAMN27512555, SAMN27512556, SAMN27512557, SAMN27512558, SAMN27512559, SAMN27512560, SAMN27512561, SAMN27512562, SAMN27512563. Probes were purchased from Molecular Instruments (Los Angeles, California, USA). This included the following: for skate Shh (Lot PRA753), Ptc2 (Lot PRA754), Wnt2b (Lot PRE300), Wnt3 (Lot PRG814), Wnt4 (Lot PRE301), Wnt7b (Lot PRE302), Wnt9b (Lot PRE303), Notum (Lot PRG817), Kremen1 (Lot PRG816), Axin2 (Lot PRG818), Apcdd1 (Lot PRG815), Col2a1 (Lot PRB574) and Sox9 (Lot PRB571).

The following data sets were generated
    1. Gillis JA
    (2023) NCBI BioProject
    ID PRJNA825354. Ectodermal Wnt signaling, cell fate determination and polarity of the skate gill arch skeleton.
    https://www.ncbi.nlm.nih.gov/bioproject?term=PRJNA825354
    1. Rees JM
    (2023) figshare
    Scripts and data on Wnt signalling, cell fate determination and anteroposterior polarity of the skate gill arch skeleton.
    https://doi.org/10.6084/m9.figshare.19615779.v2

References

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    1. Daniel JF
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    Berkeley,Calif: University of California press.
    https://doi.org/10.5962/bhl.title.10180
    1. Graham A
    (2008) Deconstructing the pharyngeal metamere
    Journal of Experimental Zoology. Part B, Molecular and Developmental Evolution 310:336–344.
    https://doi.org/10.1002/jez.b.21182
    1. Maeda O
    2. Kondo M
    3. Fujita T
    4. Usami N
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    9. Sekido Y
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    Enhancement of GLI1-transcriptional activity by beta-catenin in human cancer cells
    Oncology Reports 16:91–96.

Decision letter

  1. Megan G Davey
    Reviewing Editor; University of Edinburgh, United Kingdom
  2. Didier YR Stainier
    Senior Editor; Max Planck Institute for Heart and Lung Research, Germany
  3. Megan G Davey
    Reviewer; University of Edinburgh, United Kingdom
  4. Ruth Williams
    Reviewer; Stowers Institute for Medical Research, United States
  5. Shawn A Hallett
    Reviewer; University of Michigan-Ann Arbor, United States

Our editorial process produces two outputs: (i) public reviews designed to be posted alongside the preprint for the benefit of readers; (ii) feedback on the manuscript for the authors, including requests for revisions, shown below. We also include an acceptance summary that explains what the editors found interesting or important about the work.

Decision letter after peer review:

Thank you for submitting your article "Wnt signalling, cell fate determination and anteroposterior polarity of the skate gill arch skeleton" for consideration by eLife. Your article has been reviewed by 3 peer reviewers, including Megan G Davey as the Reviewing Editor and Reviewer #1, and the evaluation has been overseen by Didier Stainier as the Senior Editor. The following individuals involved in the review of your submission have agreed to reveal their identity: Ruth Williams (Reviewer #2); Shawn A. Hallett (Reviewer #3).

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

Essential revisions:

1) The paper requires significant re–writing in order to improve its accessibility, including combining figures and reducing the number of figures; shortening and improving the introduction; improving figure annotation (scale bars, labelling, etc) and improving summary figures; addition of key missing references i.e. Wnt signaling in the developing zebrafish craniofacial complex (10.1038/s41598–021–85415–y, 10.1016/j.ydbio.2016.11.016, 10.1242/dev.137000); justification of the selection of Ptch2 and Dusp6 as downstream markers of Shh and Fgf8; improved recording of statistical methods and acknowledgment of how tissues were correctly dissected. Please reference the extensive suggestions from all the reviewers.

2) While I do not think that additional wet experimental work beyond that supporting the current data is required, there is wet work that should be undertaken to support current data and which may benefit the authors and allow them to reduce the complexity of the figures. Including–

– Expression data validating the molecular profile of "mesoderm" cells should be included.

– Histologic evidence that the tissues they are isolating are truly what they appear to be. For example, following manual dissection of similarly staged samples, remaining tissue should be stained with markers for the GAER versus non–GAER.

– Multiplexed HCR RNA in situ hybridisation, in order to fully explore co–localisation of genes expressed in the GAER and non–GAER tissues i.e. SHH and FGF8.

– In addition, to support the transcriptomic approach, it would be an improvement to see the expression of other components of the FGF and WNT signalling pathways, such as Wnt2b, Wnt3, Wnt4, and Wnt9b, FGF receptors, 1/2/3.

– Multiplexed HCR RNA in situ hybridisation to underpin findings from the transcriptomics, for example, gene expression of tissue–specific transcription factors would also significantly lift the paper, but it is acknowledged that tissues, time, and expense may prevent this.

3) If available for the little skate, the authors should perform KEGG analysis and gene ontology of data from the transcriptomic data set.

Reviewer #1 (Recommendations for the authors):

I like your paper but it was overly complex. Not many people understand the branchial arch formation and even less the nuances of gill arch anatomy and the difference between cartilaginous and non–cartilaginous fishes. I feel your Intro is really bogged down in the complexities of cartilaginous and non–cartilaginous fishes and gill arches – whereas your premise is really pretty simple. "These signalling pathways work together to make a conserved patterned structure". Basically, spell out what the interesting point of the paper is, and frame the question. And why skate? At present, it is overly complex.

To improve the paper I think an intro with some back–to–basics would help considerably. If you need the reader to understand the differences between the ancestral condition, cartilaginous Vs bony fish gill arches, then perhaps consider an intro drawing showing this, highlighting the pattern you are interested in. I would also add an intro figure outlining your hypothesis – perhaps something like Figure 5Bi or Fig10, showing the signalling centres, rather than the factors they express. Do you actually need a drawn figure which compares chicken and skate tissues and signalling centres?

Also, I think there are some incorrect sentences in places, which make the paper confusing, I assume they are just editing mistakes but do make the paper tricky to read e.g.

"All jawed vertebrates belong to one of two lineages: cartilaginous fishes (sharks, skates, rays, and holocephalans) or bony fishes (ray– and lobe–fined fishes…)" But not all jawed vertebrates are fish (?)

250 The epithelium of vertebrate pharyngeal arches derives from both ectoderm and endoderm, and the specific tissue origin of the GAER of cartilaginous fishes is not known."

But isn't a cartilaginous fish a vertebrate? And the GAER is the epithelium of a branchial arch–derived structure– so should be from ectoderm and endoderm? I appreciate this might not have been shown but your sentence is confusing. Should it be "The epithelium of vertebrate pharyngeal arches derives from both ectoderm and endoderm? While this suggests the specific tissue origin of the GAER of cartilaginous fishes is probably ectoderm and endoderm this has not been shown."?

Figure1 is beautiful – but really needs annotation. In A', I presume we are looking at the gill arch? In B' Would be better to annotate which are the branchial rays, the epibranchial cartilage, and the ceratobranchial cartilages. And how the branchial arches are polarised. Obvious to you, I'm sure. But not so much to the rest of us.

Figure3 – label the endoderm for clarity.

I think the fate mapping data and gene expression data for the chick PEM would be better in the main text to better support your conclusion that "These findings demonstrate that the skate GAER and chick PEM share embryonic tissue origins and expression patterns of signalling molecules through development and are therefore homologous and an ancestral feature of the pharyngeal arche(s) of jawed vertebrates.", although it does make me wonder why, if the structures are homologous that you didn't use the chick more…or at least test your Wnt hypothesis in the chick with IWR1 treatment.

I would recommend publication but I think you should re–write in a style that is easier to understand and perhaps reorganise your data to show chicken and ray side by side.

Reviewer #2 (Recommendations for the authors):

Very nice work. An interesting read. I just have a few specific comments about the presentation of the figures below. If feasible, it would be very nice to see HCR used to its potential for multiplexing, this would condense the figures and therefore improve flow, and indeed the overall length – which at 10 figures is off–putting from the outset.

I've also commented that RNA–seq on treated embryos, more specific Wnt modulation, and equivalent experiments in chicks would be nice. I understand these are expensive, and in the case of the treatment regime, time–consuming, so perhaps something for a 'research advance' on this work.

And just a general comment/thought, going forward, it would be interesting to find out if there is a tissue–specific enhancer for Shh in the GAER, similar to that in the limb bud and pectoral fin. This would provide a valuable tool to drive tissue–specific gene manipulation and also isolate this specific cell population.

Figure 1: Please help the reader by orientating what is shown in B. Where is this in A? Please provide a scale bar here too. In panel C, the legend is not clear, do the asterisks label the hyoid and gill arches, or just gill arches – but 5 asterisks are present and only 4–gill arches are mentioned in the legend? Maybe label the hyoid differently to be clear. Also, is Shh expressed in a lower jaw structure? In panel D, please annotate A/P and D/V orientation. Ideally, an additional gill arch marker should be shown to demonstrate the posterior regionalisation of Shh. HCR would also give a better resolution.

Figure 2: Please label the A–P axis on the figures, as mentioned in the legend. This is actually lacking throughout the figures and would be very helpful to include.

Figure 3/4: It would look much nicer if Shh and Fgf8 co–expression at S22 and S29 were also shown by HCR in figure 4, making one concise in situ figure instead of two. Also, co–expression of all 4 markers Shh, Fgf8, Ptc2, and Dusp6 could be shown in one HCR experiment. On the HCR images, please show the outline of the gill arches with a white dotted line, as figure 2C.

Figure 2 supplement 1: I think it would read better if the chick lineage tracing section came immediately after the skate lineage tracing. In panel A, it would be useful to indicate the location of a whole embryo in this section is taken at. Ideally, Shh HCR should be shown on the same embryo with CCFSE staining, or is this not possible of CCFSE–treated embryos? I think this figure could be merged with the main figure 2. For example, panel E can be removed and only E' is needed. E", Ei, and Ei' are not necessary, this labelling is not logical and they are not referred to in the legend. In main figure 2, panel C is not necessary, so I think these can be easily combined.

It seems only 24–31% of embryos injected with DiI had the same labelling in the GAER, is this proportion expected with DiI labelling?

Figure 3 supplement 1: It seems this is more a supplemental figure for figure 2 since it pertains more to the lineage tracing. I think the U panel of HCRs could join a new combined figure of Figure 2 and Figure 2 supplement 1.

In figure 3 supplement 1, panels B, G, L, and Q need to have more structures labelled, as described in the legend. These in situs would be so much clearer by multiplexed HCR.

Figure 5: Did you do gene ontology analysis for the upregulated genes? What were the general pathways that were potentially perturbed, do you see Wnt signalling for example? Also, can you tie this in better with the in situs shown in previous figures, eg do you see Shh, Dusp6, Ptc2, and Fgf8 upregulated in the GAER?

Figure 6: Did you observe any general phenotype on the IWR1 and DMSO treated embryos? I see now in figure 8, that the embryos are very deformed. Perhaps you can comment on the general phenotype and broad range effects of the IWR1 treatment.

Please show the Shh expression in green, not just the green dotted line. If this clouds the magenta signal of the Wnt molecules please show separate colours in different panels.

Did you use HCR to validate any of the transcription factors?

It would be most useful to conduct RNA–seq on GAER dissections from IWR1 treated embryos. But not essential for revision.

Did you attempt a more specific disruption of Wnt signalling by way of CRISPR mutation? Also, did you examine the effects of disrupted Wnt signalling on the chick PEM?

Figure 7: Please label A–P. How many sections were examined from each embryo? How were Ptc+ cells counted, how did you define a cell, and did you use a membrane stain or Dapi? In the figure legend, please quote the t–test result correctly, eg what are the degrees of freedom and t statistics.

Figure 9: This is a very interesting specific phenotype in an otherwise broadly affected 'mutant'. Were Sox9 and Col2a1 expression examined at earlier stages? Please quote the embryo stage in the legend.

Figure 10: This is a very nice schematic summary of the key findings presented here, but please annotate further eg A/P, D/V, label GAER, and surrounding tissues. It would be very useful to include this schematic in figure 1 to give the reader better orientation of the tissues from the outset, then use the same schematic in figure 10 to show your findings. A similar schematic of the chick PEM would also help to demonstrate the similarities between these tissues.

Reviewer #3 (Recommendations for the authors):

– Although it may be useful to orient the reader, Figure 1 is largely extraneous and unnecessary as there have been several studies cited that provide the same expression data.

– Figure 3. Although histologically one can discern between the epidermal–mesodermal boundary, some expression data validating the molecular profile of "mesoderm" cells should be included.

– Figure 4. Additional justification in the text should be included to inform the reader of the selection of Ptch2 and Dusp6 as downstream markers of Shh and Fgf8, respectively.

– Figure 4. The authors should assess expression domains of the FGF receptors, 1/2/3, as they have been shown to be critically important in chondrogenesis in other models and to discern the target of Fgf8.

– Figure 4. Additional literature review highlighting the potential differential roles of other FGF ligands in brachial arch formation should be included in the text and potentially assessed on an mRNA level histologically.

– Figure 5. How do the authors differentiate cell types during their isolation method in preparation for transcriptomic analysis? To be sure of the accuracy of their methods, the authors should provide histologic evidence that the tissues they are isolating are truly what they appear to be. For example, following manual dissection of similarly staged samples, remaining tissue should be stained with markers for the GAER versus non–GAER.

– Figure 5. If available for the little skate, the authors should perform KEGG analysis on transcriptomes of their data set.

– Figure 6. If mRNA transcripts of Wnt2b, Wnt3, Wnt4, and Wnt9b are truly restricted to non–GAER regions, the authors should provide more conclusive evidence, for example by co–staining these sections with Shh. The current diagram seems slightly arbitrary, as the GAER location may vary across tissue sections.

– Figure 9. It would be interesting to further elucidate the state of the Sox9/Col2a1–expressing. A more extensive analysis of gene expression profiles would be important to further explain the ectopic brachial arch phenotype (e.g. Pthrp, Ihh, Col10).

– Discussion. Several papers have explored the role of Wnt signaling in the developing zebrafish craniofacial complex (10.1038/s41598-021-85415-y, 10.1016/j.ydbio.2016.11.016, 10.1242/dev.137000). These should be considered and included.

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

Thank you for resubmitting your study entitled "Ectodermal Wnt signalling, cell fate determination and polarity of the skate gill arch skeleton" for further consideration by eLife. Your revised article has been evaluated by Didier Stainier (Senior Editor) and a Reviewing Editor.

The reviewers are all in agreement that the manuscript has been improved and merits acceptance based on scientific merit but there are some remaining issues that need to be addressed. These are primarily to make the paper accessible. My feeling is that Figure 1 is overly complex and, as it is primarily an introduction figure, could be improved and/or reduced and combined with Figure 2.

Figure1 problems–

The panels are not in the same orientation, which makes it confusing to the reader.

– Figure 1A is a very stunning picture– but it does not explain the gill arch anatomy in relation to the brachial ray anatomy. It would be better to have a drawn picture I think.

– Figure 1A'. Not mentioned in the figure legend.

– Figure 1B– this is the Anterior–posterior polarity the paper is focused on, but to the uninitiated, what the polarity is in the figure, is unclear and isn't easy to associate with the text. There is no A–P marked in this panel. Is this figure really the same orientation as A and A'? I don't understand the orientation of these samples at all, doesn't the axis of the gill arch run medial–lateral?

– Figure1C. Again a different orientation. what is lateral and what is medial?

– Figure 1D – the asterisk is not defined in the figure legend (it is in the body of the text). But if the asterisk marks the SHH domain in the lateral part of the gill arch, what is purple in the medial part of the gill arch? Why is the GAER not annotated here? Why is M–L not annotated? What about the HA?

Figure F – Is this a summary of the gill arch? This is not stated in the figure or the text. 'distal' is added as a term. Does distal=lateral? The text for F is incomplete "…where is promotes differentiation of cartilaginous branchial'…branchial what?

Figure 2 is a much better figure. I think that Figures 1 and 2 should be combined, keeping Figure 1B, C, E–E' and all of figure2. For example, Figure 2C. is a (better) version of 1D. My main confusion now is that in Figure 2, M–L seems to be reversed from Figure 1. Is this correct?

These comments also relate to Fig7 and 9 which either do not annotate A–P or have M–L in a different orientation.

Reviewer 2 also comments that they are unsure about Figure 5A– the DiI labelling. I think this is Figure 7? But as the figures are not numbered we cannot be sure. please check and revise if incorrect.

Other confusing points in the paper– is the gill arch the same as the pharyngeal arch? These terms are used interchangeably in the paper, for example in the first two sentences. But I am not sure if they are equivalent.

Other confusing points in the paper- is the gill arch the same as the pharyngeal arch? These terms are used interchangeably in the paper, for example in the first two sentences. But I am not sure if they are equivalent.

The other very positive comments are below-

Reviewer #1 (Recommendations for the authors):

The manuscript is much improved for removing the chick data. The figures flow better and the annotation is also much improved (although they were not numbered in the document provided here, and I think the Dil labelling figure is not in the correct position).

The data provides a good characterization of gene expression in the GAER region and non-GAER tissue which, at least for Wnt factors has been beautifully validated by multiplexed HCR.

Lineage tracing provides novel insight into the embryonic origin of these tissues.

Overall, I think the manuscript is significantly improved and the data offers a useful resource for other researchers as well as providing a platform for further, more extensive analysis of signaling events from the GAER underlying gill development, which I look forward to reading about!

Reviewer #3 (Recommendations for the authors):

The authors have significantly improved the clarity and quality of the writing and data, respectively, included herein, by highlighting key references and further expanding on their transcriptomic analyses in the introduction and discussion sections. Importantly, by placing an emphasis on Wnt and Hedgehog signaling, the authors have eloquently demonstrated a critical role of these pathways in establishing gill arch skeletal polarity in skates. I have no further suggestions at this time.

https://doi.org/10.7554/eLife.79964.sa1

Author response

Essential revisions:

1) The paper requires significant re–writing in order to improve its accessibility, including combining figures and reducing the number of figures; shortening and improving the introduction; improving figure annotation (scale bars, labelling, etc) and improving summary figures; addition of key missing references i.e. Wnt signaling in the developing zebrafish craniofacial complex (10.1038/s41598–021–85415–y, 10.1016/j.ydbio.2016.11.016, 10.1242/dev.137000); justification of the selection of Ptch2 and Dusp6 as downstream markers of Shh and Fgf8; improved recording of statistical methods and acknowledgment of how tissues were correctly dissected. Please reference the extensive suggestions from all the reviewers.

As discussed above, we have significantly revised and streamlined the text, figures, and figure annotations throughout, in response to the detailed feedback of all three reviewers. All three reviewers made suggestions of different ways in which the manuscript could be improved. We’ve taken guidance from all three reviews, but in some instances, changes requested by one reviewer were at odds with suggestions from another (cutting vs combining and expanding figures, sections of text to expand/reduce, etc). Rather than going through a point-by-point list of the many individual edits that we’ve made (and given the scale of the revision), we hope that you will consider this revised submission in total and will recognised the many places in which reviewers’ comments greatly helped to improve the clarity of the text, figures and message.

In our revised discussion, we have included additional references (Lines 403-414), including some of the ones recommended by Shawn Hallett in his review. There are hundreds of papers dealing with Wnt signalling and skeletal development, and we can’t really review them all in this paper. We have opted to focus largely on discussing the role of Wnt signalling in cell fate determination (rather than the role of non-canonical Wnt signalling in morphogenesis and convergent extension). We have been selective about which new references to include to keep with the focus of our paper. But we have also taken your suggestion and included additional new references relating to the Sox9-Wnt-BMP Turing-like mechanism that underlies amniote digit patterning, and we discuss parallels with the anti-chondrogenic role for Wnt signalling in skate branchial rays that we report here (Lines 449-456).

We have added a reference to Pearse et al. (2001), to support the use of Ptc2 as a transcriptional readout of Shh signalling (Lines 71-74). We no longer discuss Fgf8 signalling in this manuscript, and so have not added additional references relating to Dusp6.

In the legend to Figure 6 (formerly Figure 7), we have improved reporting of our statistical methods. We have specified that we counted Ptc2+ nuclei (based on DAPI staining) and compared the means of 2-3 sections from equivalent positions in the arches of DMSO control (n=3 embryos) and IWR1-treated (n=4 embryos) animals. We also now include the t value and the degrees of freedom in the legend.

2) While I do not think that additional wet experimental work beyond that supporting the current data is required, there is wet work that should be undertaken to support current data and which may benefit the authors and allow them to reduce the complexity of the figures. Including–

– Expression data validating the molecular profile of "mesoderm" cells should be included.

We presume that the mesoderm referred to here is the interbranchial muscle plate, which derives from the paraxial mesodermal core that runs down the center of the pharyngeal arch. This mesoderm-derived muscular sheet is readily identifiable histologically and also exhibits distinct gene expression features that have been reported elsewhere (most recently, in skate, by us in Hirschberger et al., 2021, Mol. Biol. Evol. 38: 4187). In our figures, these muscle fibres are clearly recognisable with regular histochemical staining (in revised Figure 8), and we don’t really think that we could easily include additional panels to illustrate gene expression features of this muscle sheet into that plate. But we have added new citations of a classic anatomical text, a review article on pharyngeal arch tissue structure and our study on gene expression in pharyngeal arch tissues in skate to further support and clarify the nature and location of this tissue within the arch (Lines 347-350).

– Histologic evidence that the tissues they are isolating are truly what they appear to be. For example, following manual dissection of similarly staged samples, remaining tissue should be stained with markers for the GAER versus non–GAER.

Our manual microdissection of embryonic skate gill arches was guided by arch morphology, and by our extensive experience working with this tissue. We first used sharpened tungsten needles to dissect the posterodistal GAER region (which appears as a morphologically distinct pseudostratified epithelial ridge along the leading edge of the expanding arch), and then removed a region of non-GAER tissue from the anterior face of the same arch. By the time these dissections were finished, the arch had been heavily dissected, and so we did not save or fix the remaining tissue. But to confirm that we did effectively capture the GAER region, we did check that the GAER tissue did significantly differentially express Shh, relative to the non-GAER tissue (admittedly, Shh was not among the top hits, likely because the GAER is only ~5 cells wide, and so makes up a relatively small proportion of the total cells of our sample – but it was still differentially expressed in the GAER region). We add a new statement to this effect from Lines 238-242.

But more importantly, in the manuscript we have spatially validated several additional factors that emerged from our analysis as differentially expressed in the GAER region of the gill arch, and we confirmed that all of these genes are, indeed, expressed in the vicinity of the GAER (as predicted by our differential expression analysis). We feel that these observations quite strongly support the accuracy of our dissections. In this study, we used our differential gene expression analysis to guide us to signalling pathways that might be interacting with GAER Shh signalling to polarise skate gill arch appendages – this led us to the Wnt signalling pathway, and we then opted to focus the result of our spatial and experimental work that pathway. And everything that we observed is consistent with activity of this pathway within the GAER region of tissue indicated in our dissection schematic in revised Figure 2.

– Multiplexed HCR RNA in situ hybridisation, in order to fully explore co–localisation of genes expressed in the GAER and non–GAER tissues i.e. SHH and FGF8.

We no longer deal with FGF signalling in this revised manuscript. In our initial figures, we had originally performed HCR for Wnt ligands and Wnt signalling readouts with a GAER marker (either Shh or Wnt7a). We opted not to show the GAER marker, so as not to obscure the signal of the other markers. But our dashed lines in the original figures were based on actual expression of a GAER marker (i.e., they were not just approximations of the location of the GAER). But in response to reviewer feedback, we now include the signal for all markers in revised Figures 3 and 4.

– In addition, to support the transcriptomic approach, it would be an improvement to see the expression of other components of the FGF and WNT signalling pathways, such as Wnt2b, Wnt3, Wnt4, and Wnt9b, FGF receptors, 1/2/3.

We focus in this paper on the Wnt signalling pathway, and we do include expression data for Wnt2b, Wnt3, Wnt4 and Wnt9b (in revised Figure 3). We no longer deal with FGF signalling in this manuscript, as our dataset relating to this was incomplete and only of peripheral relevance to the focus of our revised manuscript.

– Multiplexed HCR RNA in situ hybridisation to underpin findings from the transcriptomics, for example, gene expression of tissue–specific transcription factors would also significantly lift the paper, but it is acknowledged that tissues, time, and expense may prevent this.

Our lab has just completed a move from the UK to the USA, and our lab is not yet up and running for molecular work. Additionally, the lead author on this work is currently on maternity leave. “-Omic” datasets like the one presented in this paper offer many avenues for follow-up study. In this case, we used our differential gene expression analysis to guide us to signalling pathways that might be interacting with GAER Shh signalling to polarise skate gill arch appendages – again, this analysis led us to the Wnt signalling pathway, and we opted to specifically focus the result of our spatial and experimental work on genes encoding Wnt ligands and downstream effectors. We hope that you’ll consider these data as sufficient for a paper, and we hope that the additional data from these analyses will eventually lead to further papers on other aspects of molecular patterning of skate pharyngeal arches.

3) If available for the little skate, the authors should perform KEGG analysis and gene ontology of data from the transcriptomic data set.

Thank you for this suggestion. We have now performed this analysis on our RNAseq data and found an enrichment of Wnt signalling components in the GAER region of the gill arch. We have now included this as an addition to the Methods section (Lines 196-206), a brief statement in the Results (Lines 251-254), and as a new supplemental figure and legend (Figure 2 Supplement 1).

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

The reviewers are all in agreement that the manuscript has been improved and merits acceptance based on scientific merit but there are some remaining issues that need to be addressed. These are primarily to make the paper accessible. My feeling is that Figure 1 is overly complex and, as it is primarily an introduction figure, could be improved and/or reduced and combined with Figure 2.

Figure1 problems–

The panels are not in the same orientation, which makes it confusing to the reader.

– Figure 1A is a very stunning picture– but it does not explain the gill arch anatomy in relation to the brachial ray anatomy. It would be better to have a drawn picture I think.

– Figure 1A'. Not mentioned in the figure legend.

– Figure 1B– this is the Anterior–posterior polarity the paper is focused on, but to the uninitiated, what the polarity is in the figure, is unclear and isn't easy to associate with the text. There is no A–P marked in this panel. Is this figure really the same orientation as A and A'? I don't understand the orientation of these samples at all, doesn't the axis of the gill arch run medial–lateral?

– Figure1C. Again a different orientation. what is lateral and what is medial?

– Figure 1D – the asterisk is not defined in the figure legend (it is in the body of the text). But if the asterisk marks the SHH domain in the lateral part of the gill arch, what is purple in the medial part of the gill arch? Why is the GAER not annotated here? Why is M–L not annotated? What about the HA?

Figure F – Is this a summary of the gill arch? This is not stated in the figure or the text. 'distal' is added as a term. Does distal=lateral? The text for F is incomplete "…where is promotes differentiation of cartilaginous branchial'…branchial what?

Figure 2 is a much better figure. I think that Figures 1 and 2 should be combined, keeping Figure 1B, C, E–E' and all of figure2. For example, Figure 2C. is a (better) version of 1D. My main confusion now is that in Figure 2, M–L seems to be reversed from Figure 1. Is this correct?

These comments also relate to Fig7 and 9 which either do not annotate A–P or have M–L in a different orientation.

Reviewer 2 also comments that they are unsure about Figure 5A– the DiI labelling. I think this is Figure 7? But as the figures are not numbered we cannot be sure. please check and revise if incorrect.

Other confusing points in the paper– is the gill arch the same as the pharyngeal arch? These terms are used interchangeably in the paper, for example in the first two sentences. But I am not sure if they are equivalent.

We completely agree with the outstanding issues that you have flagged up, and we have addressed these issues as follows:

1. We would like to provide the readers with an anatomical overview of skate gill skeletal anatomy, so that this is not being encountered for the first time in Figure 7. But we recognise that our original Figure 1 was overly complex. We have replaced our original Figure 1 with a new and simpler figure. The revised Figure 1 shows the skate gill skeleton in ventral and lateral view, as well as a dissected gill arch. The focus of this simpler figure is the distribution of branchial rays, and how these rays relate to the proximal gill arches. We have also oriented panels A and B so that the AP axis is consistent in both. We have attached the revised figure and legend below for your review.

2. As recommended, we have incorporated the original Figure 1C and E-E’' into Figure 2. We also now consistently indicate AP and PD axes (here, and in all subsequent figures). Branchial rays and gill arches are analogous to the tetrapod limb and girdle, and so we have opted to discuss and label the axes of these appendages in a similar way. We have attached the revised figure and legend below for your review:

We also revised the first paragraph of our Results section to accommodate these new figure panels:

“In developing skate gill arches, Shh is expressed in the GAER (Figure 2A), and branchial rays develop from a GAER-responsive domain of posterior arch mesenchyme (Gillis and Hall, 2016). To further explore the transcriptional environment of the GAER and to discover additional gene expression features that may contribute to gill arch polarity, we conducted a comparative transcriptomic and differential gene expression analysis of GAER and non-GAER regions of the first gill arch of the embryonic skate. Briefly, we manually dissected the (1) GAER and (2) non-GAER (control) regions from the first gill arch of stage (S)26 skate embryos (n=5; Figure 2B–Bi), and we performed RNA extraction, library preparation, and RNAseq analysis on these samples. For the GAER region of the first gill arch, we included the whole distal tip of the arch in our dissection to ensure that the Shh-expressing GAER and adjacent Shh-responsive (i.e., Ptc2+) tissues were captured for this analysis (Figure 2C-D). Following de novo transcriptome assembly, we tested for differential gene expression between GAER and non-GAER tissues (after first testing for differential expression of Shh in the GAER region, to confirm dissection accuracy). We used a false discovery rate of 5% and no log fold-change (LogFC) cut-off to generate a list of 401 genes that were upregulated in the GAER region. We sorted this list for genes encoding signalling pathway components and transcription factors with a log-fold change >2. (Figure 2E). Differentially expressed genes were subjected to functional enrichment analysis, and this analysis indicated enrichment of Wnt signalling pathway components in GAER region (Figure 2 Supplement 1 for enrichment analysis). Differentially expressed genes in the GAER region included those encoding the Wnt ligands Wnt2b, Wnt3, Wnt4, Wnt7b and Wnt9, the transmembrane Wnt inhibitors Kremen1 (Mao et al., 2002) and APCDD1 (Shimomura et al., 2010), and the secreted Wnt antagonist Notum (Zhang et al., 2015). We therefore chose to further investigate involvement of the Wnt pathway in GAER signalling by spatially validating the expression of these genes, using the most highly expressed isoforms for mRNA in situ hybridization by chain reaction (HCR) probe set design.”

3. We have reoriented the panels in Figures 7 and 8 so that anterior is always to the left, and (where relevant) we have indicated the AP and PD axes with labels in Figures 7, 8 and 9.

4. There were a few incorrect figure references in the text (a holdover from the figure numbering in our original submission).

We have gone through the text and corrected these.

5. “Other confusing points in the paper- is the gill arch the same as the pharyngeal arch? These terms are used interchangeably in the paper, for example in the first two sentences. But I am not sure if they are equivalent.”

Vertebrate pharyngeal arches are a series of paired structures that form on either side of the embryonic head. These arches are named according to their ancestral skeletal derivatives: The first of these arches is called the mandibular arch, the second is called the hyoid arch, and the caudal pharyngeal arches are collectively called the gill arches. Unfortunately, these skeletal derivatives of pharyngeal arches are also generally referred to as “arches”, and this can lead to some confusion when referring to embryonic precursors and their derivatives. To try and clarify this, we have revised the first paragraph of our introduction as follows:

“The pharyngeal arches of vertebrates are a series of paired columns of tissue that form on either side of the embryonic head. Pharyngeal arches form as iterative outpockets of foregut endoderm contact the overlying surface ectoderm, and this meeting of endoderm and ectoderm generates columns lined laterally by ectoderm, medially by endoderm, and containing a core of mesoderm and neural crest-derived mesenchyme (Graham and Smith, 2001). In fishes, endodermal outpockets fuse with the overlying surface ectoderm, giving rise to the gill slits and the respiratory surfaces of the gills (Gillis and Tidswell, 2017). In amniotes, endodermal outpockets give rise to glandular tissues, such as the tonsils, parathyroid and ultimobranchial glands (Grevellec and Tucker, 2010). The largely neural crest-derived mesenchyme of the pharyngeal arches gives rise to the pharyngeal skeleton – i.e., the skeleton of the jaws and gills in fishes, and of the jaw, auditory ossicles, and larynx in amniotes (Jiang et al., 2002, Kague et al., 2012, Sleight and Gillis, 2020, Couly and Le Douarin, 1990) – with mesenchymal derivatives of each arch receiving patterning and polarity information via signals from adjacent epithelia (Veitch et al., 1999, Gillis et al., 2009a, Couly et al., 2002, Brito et al., 2006). Pharyngeal arches are named according to their ancestral skeletal derivatives: the 1st pharyngeal arch is termed the mandibular arch, the 2nd pharyngeal arch is the hyoid arch, while the caudal pharyngeal arches are collectively termed the gill arches. In fishes, the gill arches give rise to a series of skeletal “arches” that support the respiratory lamellae of the gills.”

https://doi.org/10.7554/eLife.79964.sa2

Article and author information

Author details

  1. Jenaid M Rees

    Department of Zoology, University of Cambridge, Cambridge, United Kingdom
    Contribution
    Conceptualization, Data curation, Formal analysis, Funding acquisition, Investigation, Visualization, Methodology, Writing – original draft
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0001-9434-5445

  2. Victoria A Sleight

    School of Biological Sciences, University of Aberdeen, Aberdeen, United Kingdom
    Contribution
    Data curation, Formal analysis, Supervision, Investigation, Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  3. Stephen J Clark

    The Babraham Institute, Cambridge, United Kingdom
    Contribution
    Methodology
    Competing interests
    No competing interests declared

  4. Tetsuya Nakamura

    Department of Genetics, Rutgers University, Piscataway, United States
    Contribution
    Methodology, Writing – review and editing
    Competing interests
    No competing interests declared

  5. J Andrew Gillis

    1. Department of Zoology, University of Cambridge, Cambridge, United Kingdom
    2. Josephine Bay Paul Center for Comparative Molecular Biology and Evolution, Marine Biological Laboratory, Woods Hole, United States
    Contribution
    Conceptualization, Formal analysis, Supervision, Funding acquisition, Investigation, Methodology, Project administration, Writing – review and editing
    For correspondence
    agillis@mbl.edu
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0003-2062-3777

Funding

Wellcome Trust (214953/Z/18/Z)

  • Jenaid M Rees

Royal Society (UF130182)

  • J Andrew Gillis

Royal Society (URF\R\191007)

  • J Andrew Gillis

Royal Society (RG140377)

  • J Andrew Gillis

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication. For the purpose of Open Access, the authors have applied a CC BY public copyright license to any Author Accepted Manuscript version arising from this submission.

Acknowledgements

With thanks to Dr Kate Criswell and Dr Christine Hirschberger for advice, and to the University of Cambridge Wellcome PhD. Programme in Developmental Mechanisms. The authors were funded by a Wellcome PhD studentship (214953/Z/18/Z) to JMR, and by a Royal Society University Research Fellowship (UF130182 and URF\R\191007) and Royal Society Research Grant (RG140377) to JAG.

Senior Editor

  1. Didier YR Stainier, Max Planck Institute for Heart and Lung Research, Germany

Reviewing Editor

  1. Megan G Davey, University of Edinburgh, United Kingdom

Reviewers

  1. Megan G Davey, University of Edinburgh, United Kingdom
  2. Ruth Williams, Stowers Institute for Medical Research, United States
  3. Shawn A Hallett, University of Michigan-Ann Arbor, United States

Version history

  1. Received: May 3, 2022
  2. Preprint posted: May 20, 2022 (view preprint)
  3. Accepted: March 3, 2023
  4. Version of Record published: March 20, 2023 (version 1)

Copyright

© 2023, Rees 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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  1. Jenaid M Rees
  2. Victoria A Sleight
  3. Stephen J Clark
  4. Tetsuya Nakamura
  5. J Andrew Gillis
(2023)
Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton
eLife 12:e79964.
https://doi.org/10.7554/eLife.79964

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