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

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 1^st pharyngeal arch is termed the mandibular arch, the 2^nd 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.”