Ectodermal Wnt signaling, cell fate determination, and polarity of the skate gill arch skeleton
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Evaluation Summary:
In this highly innovative study, the authors use combinatorial gene expression analysis to study the development of the gill arch of the little skate. This process depends on Shh and Fgf ligand-derived endodermal cells at the endoderm-ectoderm junction, providing insight into not only the fundamental developmental mechanisms regulating brachial arch formation in cartilaginous fishes, but also highlighting a unique relationship between inhibition of Wnt and Hedgehog signaling pathways in the context of early appendage development. The work will be of interest to developmental biologists and colleagues studying Wnt and Hedgehog signaling pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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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.
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Evaluation Summary:
In this highly innovative study, the authors use combinatorial gene expression analysis to study the development of the gill arch of the little skate. This process depends on Shh and Fgf ligand-derived endodermal cells at the endoderm-ectoderm junction, providing insight into not only the fundamental developmental mechanisms regulating brachial arch formation in cartilaginous fishes, but also highlighting a unique relationship between inhibition of Wnt and Hedgehog signaling pathways in the context of early appendage development. The work will be of interest to developmental biologists and colleagues studying Wnt and Hedgehog signaling pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the …
Evaluation Summary:
In this highly innovative study, the authors use combinatorial gene expression analysis to study the development of the gill arch of the little skate. This process depends on Shh and Fgf ligand-derived endodermal cells at the endoderm-ectoderm junction, providing insight into not only the fundamental developmental mechanisms regulating brachial arch formation in cartilaginous fishes, but also highlighting a unique relationship between inhibition of Wnt and Hedgehog signaling pathways in the context of early appendage development. The work will be of interest to developmental biologists and colleagues studying Wnt and Hedgehog signaling pathways.
(This preprint has been reviewed by eLife. We include the public reviews from the reviewers here; the authors also receive private feedback with suggested changes to the manuscript. Reviewer #1, Reviewer #2 and Reviewer #3 agreed to share their name with the authors.)
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Reviewer #1 (Public Review):
The development and patterning of the pharyngeal arches of the vertebrate embryo have not been as well studied as many other more classical areas of embryonic development, such as the developing limb. However congenital malformations of pharyngeal arch derivatives are common, and elucidation of the mechanisms of pharyngeal arch development would be informative for human and animal health, as well as adding to our knowledge of biological mechanisms of patterning during organogenesis.
Using an unusual model - the development of the skate gill arch, and underpinned by a complementary analysis of pharyngeal arch patterning in the chicken embryo, this paper builds on previous work by the authors as well as more established paradigms of embryonic development and patterning, to understand the organisers and …
Reviewer #1 (Public Review):
The development and patterning of the pharyngeal arches of the vertebrate embryo have not been as well studied as many other more classical areas of embryonic development, such as the developing limb. However congenital malformations of pharyngeal arch derivatives are common, and elucidation of the mechanisms of pharyngeal arch development would be informative for human and animal health, as well as adding to our knowledge of biological mechanisms of patterning during organogenesis.
Using an unusual model - the development of the skate gill arch, and underpinned by a complementary analysis of pharyngeal arch patterning in the chicken embryo, this paper builds on previous work by the authors as well as more established paradigms of embryonic development and patterning, to understand the organisers and molecular pathways they express which contribute to gill arch/pharyngeal arch patterning.
Based on previous work that showed that the primary organiser of skate gill arch patterning - the GAER, expressed SHH, the authors used fate mapping techniques to establish the origin and subsequent morphogenesis of the GAER. They found that it has an endodermal origin. They repeated this experiment on chicken and found it to be the same.
They subsequently followed the expression of SHH and FGF8 through gill arch development, to show both the morphogenesis of the gill arch and that these genes go from a complementary gene expression to having an overlapping gene expression which is most highly in the posterior arch environment. The posterior expression and activation of the SHH and FGF pathways are also shown to be highest in the posterior gill arch- thus this is proposed as the primary mechanism by which the gill arch is 'polarised'.
Further work identified that the anterior gill arch expresses components of the Wnt signalling pathway, in a complementary way to FGF/SHH. Pharmalogical inhibition of Wnt signalling produces extra, non-polarised gill arches, suggestive of not only a loss of polarity but also a change in the distribution of gill arches- perhaps due to a modification of a Turing-type mechanism that would space the cartilages appropriately. At a molecular level, SHH expression did not change, but the activation of the SHH signalling pathway expanded. This perhaps suggests that Wnt signalling acts to restrain/inhibit SHH pathway activation, increasing and underpinning the mechanism of polarisation of the gill arch.
Fundamentally I think the work is strong overall. Each section of the paper is based on a clear platform of data and a hypothesis, which link together to really tell us about how this tissue is patterned - the organisers and the signalling pathways and the interactions between them. The fate mapping, sequencing, pharmacological inhibition, and HCR ISH are conclusive, although I presume due to using the usual skate a model the replicate numbers are quite low.
I do find the paper overly complex in interpretation and the figure quality of summary figures lacking in detail so that a non-gill arch expert can struggle to understand the findings. While the additional work in chicken pharyngeal arch is also strong, it is not overtly covered in the main body of the paper - and I think this is a mistake. I think interest here is uncovering mechanisms of vertebrate development - in which case a stronger comparison between chicks would demonstrate the similarities. I would suggest including make figures in which these species are shown together.
I also feel that there is much that could be discussed - not only about the formation of a polarised tissue but about how the gill arches are spaced - is this a Turing-type mechanism? Are there similarities that can be drawn with the limb or other systems which generate repeating structures? An interpretation of this could interest a wider group than only those that work on pharyngeal arch development.
In summary, I think this is an exciting paper using an unusual model and an understudied but important area of embryonic development which gives us an insight into how some of our commonly held dogmas may apply across different systems.
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Reviewer #2 (Public Review):
This work from the Gillis laboratory sets out to determine the embryonic origin of the gill arch epithelial ridge (GAER) in the 'little skate'. In parallel, they also assess the origin of a reminiscent tissue in the chick embryo, the posterior ectodermal margin (PEM). Using DiI lineage tracing they show both tissues arise from the pharyngeal endoderm, with some ectodermal contribution to the PEM. This work is nicely conducted with good use of marker genes to back up their conclusions, it could be presented slightly better by maximising the multiplex capacity of HCR and condensing the figures.
The authors follow their lineage tracing experiments with gene expression profiling. Here they perform RNA-seq on dissected GAER tissue and compare this to the surrounding non-GAER tissue. They validate this data with …Reviewer #2 (Public Review):
This work from the Gillis laboratory sets out to determine the embryonic origin of the gill arch epithelial ridge (GAER) in the 'little skate'. In parallel, they also assess the origin of a reminiscent tissue in the chick embryo, the posterior ectodermal margin (PEM). Using DiI lineage tracing they show both tissues arise from the pharyngeal endoderm, with some ectodermal contribution to the PEM. This work is nicely conducted with good use of marker genes to back up their conclusions, it could be presented slightly better by maximising the multiplex capacity of HCR and condensing the figures.
The authors follow their lineage tracing experiments with gene expression profiling. Here they perform RNA-seq on dissected GAER tissue and compare this to the surrounding non-GAER tissue. They validate this data with ISH using HCR, which clearly shows GAER-specific gene expression of numerous Wnt signalling genes. They build on this finding by functionally perturbing Wnt signalling and assessing the effects on the expression of Wnt genes. This data could be advanced by performing RNA-seq on Wnt modulated embryos. The authors also compared the expression of the same Wnt genes in the chick, where they demonstrate conserved spatial expression profiles in the PEM. The RNA-seq data could be further explored, looking at general pathways upregulated in the GAER and validating gene expression of novel transcription factors they identified by differential gene expression analysis.Overall, the data presented supports the conclusions made and the study is of interest and useful to the broad developmental biology field. The figures could benefit from additional annotation and could be condensed to improve the flow and clarity.
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Reviewer #3 (Public Review):
In this study, the authors aimed to provide evidence of a novel developmental mechanism regulating brachial arch formation in the little skate. More specifically, the authors leveraged previous studies establishing the role of Hedgehog signaling in early little skate brachial arch development and built upon these studies by discovering the embryonic identity of Shh-expressing cells and the role of canonical Wnt signaling in regulating proper anterior brachial arch formation. The authors nicely combined the use of the spatiotemporal expression of various Hedgehog and Fgf signaling members with transcriptomic analysis and pharmacologic experiments to assess genetic relationships. In general, this manuscript is of high quality and will appeal to a diverse array of scientific disciplines. Moreover, the …
Reviewer #3 (Public Review):
In this study, the authors aimed to provide evidence of a novel developmental mechanism regulating brachial arch formation in the little skate. More specifically, the authors leveraged previous studies establishing the role of Hedgehog signaling in early little skate brachial arch development and built upon these studies by discovering the embryonic identity of Shh-expressing cells and the role of canonical Wnt signaling in regulating proper anterior brachial arch formation. The authors nicely combined the use of the spatiotemporal expression of various Hedgehog and Fgf signaling members with transcriptomic analysis and pharmacologic experiments to assess genetic relationships. In general, this manuscript is of high quality and will appeal to a diverse array of scientific disciplines. Moreover, the relationship between Shh-Fgf8 and the importance of Wnt signaling in the context of brachial arch formation in the little skate may be more broadly applied to other cartilaginous fishes or other aquatic vertebrate species in general. As the little skate is largely an unexplored model organism, this study exemplifies the utility of the little skate and emphasizes the wide array of methods that can be implored to further identify this species' development on a molecular basis. Future studies should consider the generation of genetically modified skate species, as current functional interrogation is limited to pharmacological approaches. Although this study has been eloquently conducted, there is some extraneous information that takes away from the major conclusions of the story in addition to some gaps in experimental data that are required to clarify their findings.
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