Comparative Approaches in Vertebrate Cartilage Histogenesis and Regulation: Insights from Lampreys and Hagfishes

Round 1

Reviewer 1 Report

The authors review and compare the histogenesis and gene regulations of cartilage development between jawed and jawless vertebrates. The propose that vertebrate cartilage types develop via the influence of a spectrum of signaling pathways that induce distinct cell morphologies. This spectrum is also shared by non-chondrogenic cells and may exist as a larger continuum. The differences in these pathways can be used as markers in the diagnostic and treatment of musculoskeletal disorders. They provide insights and future directions for the research in the evolution of vertebrate chondroid tissues and the development of cartilaginous tissues in lampreys and hagfishes.

General Comments:

 This review article is well written, and the figures are very informative. There are some inconsistencies in the usage of “lamprey” vs. “lampreys” and “hagfish” vs. “hagfishes”.

 Specific Comments:

1. Introduction
   

Line 41: “…likely segmental duplications as well [11-15] This suggests that the novel…”

Need a period behind [11-15].

Line 42: “… may have been partly drive by the evo-…”

Replace “drive” to “driven”.

Line 51: “…a medial joint which helped facilitate respiration as well as the capture of prey [17-21]…”

Need a period behind [17-21].

Line 55: “…of vertebrate chondrocytes, and related cell types. GRN. Unfortunately, …”

“GRN” seems to come from nowhere. Please delete GRN.

Line 65: “…features of vertebrate skeletal tissue, 2) identifying features that are likely…”

Please replace”,” with “;”.

2.1. History of Cyclostome Cartilage Research

Line 162: Delete “with”.

Lines 167-168: “…contrasted with gnathostome cartilage in, …”

Delete “in”.

2.3. Cyclostome cartilage-like tissues: hagfish pseudo-cartilage and lamprey mucocartilage

Line 274: …into a loose mesenchyme, then re-differentiates “into” adult hyaline cartilage type [37].

3.1. All gnathostome cartilages develop using a conserved gene regulatory network

Line 333: …putative sox9 target sites in the cis-regulatory regions of col2a1, col9a1, acan, “and” the…

Lines 356-358: Please spell out HH and ECM in the figure legend when they appear the first time. 

4.3. The core skeletal GRN of lamprey is similar to that in gnathostomes

Line 612: “…, there are most components remain unclear.”

Please revise.

Lines 618-619: … the sea lamprey “Petromyzon marinus” and the Arctic lamprey “Lethenteron camtschaticum” [123, 124].

4.4. Larval lampreys have a diversity of cartilage types

Line 650: “…discoidal or polygonal chondrocytes E,G] [123].”

[Figure 6E,G]?

Line 662: “…(C, E, G)…”

Please use consistent format in the figure legend.

5. Future Areas of Interest in Cyclostome Cartilage Research

Line 710: “…genomic resources and genetic tools [cite CRISPR paper]…”

Reference?

6. Conclusion

Lines 830-831: …confirm that they “are” distinct tissue types, …

Author Contributions: There is no text about author contributions.

References: Please be consistent about the format.

Author Response

Reviewer #1 Comments:

 

Line 41: “…likely segmental duplications as well [11-15] This suggests that the novel…”

Need a period behind [11-15].

 

A period was added after “[11-15]”

 

Line 42: “… may have been partly drive by the evo-…”

Replace “drive” to “driven”.

 

“Drive” was changed to “driven”

 

 

Line 51: “…a medial joint which helped facilitate respiration as well as the capture of prey [17-21]…”

Need a period behind [17-21].

 

A period was added after “[17-21]”

 

 

Line 55: “…of vertebrate chondrocytes, and related cell types. GRN. Unfortunately, …”

“GRN” seems to come from nowhere. Please delete GRN.

 

“GRN” was deleted

 

Line 65: “…features of vertebrate skeletal tissue, 2) identifying features that are likely…”

Please replace”,” with “;”.

 

The comma was replaced with a semicolon

 

 

2.1. History of Cyclostome Cartilage Research

Line 162: Delete “with”.

 

“with” was deleted

 

 

Lines 167-168: “…contrasted with gnathostome cartilage in, …”

Delete “in”.

 

“in” was deleted

 

 

2.3. Cyclostome cartilage-like tissues: hagfish pseudo-cartilage and lamprey mucocartilage

 

Line 274: …into a loose mesenchyme, then re-differentiates “into” adult hyaline cartilage type [37].

 

“into an” was added

 

 

3.1. All gnathostome cartilages develop using a conserved gene regulatory network

 

Line 333: …putative sox9 target sites in the cis-regulatory regions of col2a1, col9a1, acan, “and” the…

 

“and” was added

 

Lines 356-358: Please spell out HH and ECM in the figure legend when they appear the first time. 

 

“hedgehog” and “extracellular matrix” were added before the aforementioned acronyms, and “HH” and “ECM” are now in parentheses

 

4.3. The core skeletal GRN of lamprey is similar to that in gnathostomes

 

Line 612: “…, there are most components remain unclear.”

Please revise.

 

“there are” was removed from this sentence

 

 

Lines 618-619: … the sea lamprey “Petromyzon marinus” and the Arctic lamprey “Lethenteron camtschaticum” [123, 124].

 

The species names for the sea lamprey and Arctic lamprey were italicized

 

 

4.4. Larval lampreys have a diversity of cartilage types

 

 

Line 650: “…discoidal or polygonal chondrocytes E,G] [123].”

[Figure 6E,G]?

 

“E,G]” was corrected to [Figure 6E,G] as was intended

 

 

Line 662: “…(C, E, G)…”

Please use consistent format in the figure legend.

 

This part of the legend was formatted to (C,E,G) to match

 

 

5. Future Areas of Interest in Cyclostome Cartilage Research

 

Line 710: “…genomic resources and genetic tools [cite CRISPR paper]…”

Reference?

 

The reference was added

 

 

6. Conclusion

 

Lines 830-831: …confirm that they “are” distinct tissue types, …

 

“are” was added

 

 

Author Contributions: There is no text about author contributions.

 

A section entailing author contributions has been added

 

 

 

 

 

References: Please be consistent about the format.

 

References have been corrected and formatted (i.e, some references which were caps lock were changed and citation styles were corrected)

Author Response File: Author Response.docx

Reviewer 2 Report

This review/survey addresses the many still poorly resolved issues of homology at the molecular (GRN and function), histological, and morphological levels for vertebrate cartilages as informed by the living cyclostomes (lampreys and hagfish).  I take little issue with the overall scientific interpretations of this manuscript, and feel the authors have produced a relatively even-handed perspective on what remains phylogenetically critical, yet poorly sampled datasets. 

The paper is a bit meandering, and would be improved by careful editing across the document for style, flow, and clarity to the standards of the conclusions and the molecular sections.  Note that is VERY clear when there is a change in author, and this should be worked on to improve the narrative.

I suggest the authors consider the inclusion of specific recommendations of future research directions that leverage new technologies, such as RNAseq.  It feels a bit as though the authors may be resting upon the assumptions that future advancements will be made simply by tried techniques plus greater availability of specimens.

Below are some small comments, most of which point to the need for careful editing of the document.

Line 41-42: “may have been partly drive by the evolution…” needs editing or clarification.  Also note on same line, an animal cannot technically have a “novel histology”, as histology is the study of tissue anatomy in section.  I understand this is the more informal use of the word, but it would be ideal to explain to the reader the actual microanatomy that is novel here.

Line 55: There is a random “GRN.”

Line 61: Perhaps a brief expansion of the data supporting cyclostomes as a clade, and whether there remains dissent, would be useful for the reader before moving on.

Line 116-118: Sentence awkwardly worded.  Consider separating.

Line 121-124: Awkward and redundant wording, unclear.

Line 132:  Do you mean “do not join medially to encompass the notochord”?  There is an and instead of to as written.  Unclear to this reviewer what condition is described.

Line 136-139: “Considerable/considerably” used twice.  Please watch overuse of adjectives throughout manuscript.  There are a number of places, particularly setups and conclusions for sections, that are unproductively wordy.

Line 143-145: Statement of controversy requires citation.

Line 151-153: Differences relative to what?  Each other?  Fossil jawless forms?  Jawed vertebrates?  This is unclear.  What do you mean by changes in ecology?  Extant lamprey and hagfish niches relative to fossil ones?  Please be more precise and detailed here and cite as appropriate.

Line 162: “tried with to”

Line 168: “cartilage in, but rarely”

Line 274: Word missing between re-differentiates and adult?

Line 322: This sentence seems like a truism and is not very informative.

Line 323: Focused understanding – missing “on”

Line 334: Missing an “are”

Lines 329-352:  Consider some reorganization with an eye towards matching the visual flow of Figure 3, which is easy to follow. 

Note: It is apparent with there is a shift in author voice.  For example, Section 3.1 lacks the awkward phrasing of the prior section.  Editing is required so readers can focus on the message.

Line 498: Request a reference for the use of cyanogen bromide, such as first methods paper to use, for unfamiliar readers.  Perhaps a very brief explanation of what this technique offers?

Section 4.1 Is insightful, but again change in writing style in 4.2 is noticeable.

Line 623:  How would a developing cartilage be “occasionally” dominated by versican?  This wording is unclear.

Line 626-628:  Please summarize value of these findings specifically, this statement is vague.

Line 630: “Further elucidated upon” is unclear

Line 690-691: Awkwardly worded sentence.

Line 693: Paper would be improved by phylogenetic context earlier in the setup, not just at these later points.  This would allow the reader to think about the long branch lengths of cyclostomes and what they might mean for their unique character states relative to both gnathostomes and invertebrates.  If not a figure, then a slightly more complete discussion of the hypothesized branches of jawless/jawed, of cyclostomes, with time estimates bracketed from fossils and molecules. 

Line 695-697:  Assertion is hard to follow as written.

Line 700: “It will therefore be important to compare…” This is an interesting point, but the authors fall short in not suggesting ways in which these comparisons might be made.  For example, how might RNAseq be leveraged to test cross taxa, cross tissue and across development hypotheses about the core cartilage GRN in cyclostomes?  What promise does the future hold?

Author Response

Reviewer #2 Comments:

 

Line 41-42: “may have been partly drive by the evolution…” needs editing or clarification.  Also note on same line, an animal cannot technically have a “novel histology”, as histology is the study of tissue anatomy in section.  I understand this is the more informal use of the word, but it would be ideal to explain to the reader the actual microanatomy that is novel here.

 

We have changed the structure of this sentence to “novel composition of vertebrate cartilage and its deployment” to emphasize that the specific structure of the cartilage had changed

 

Line 55: There is a random “GRN.”

 

“GRN” was deleted

 

Line 61: Perhaps a brief expansion of the data supporting cyclostomes as a clade, and whether there remains dissent, would be useful for the reader before moving on.

 

We have added two additional parts to this section. First, we added a part which mentions the historical issue of cyclostome monophyly vs paraphyly and how classical morphological analyses gravitated towards the latter whereas modern work suggest the former. Second, we added some fossil references to a stem lamprey (Priscomyzon) and stem hagfish (Myxinikela) which we believe is useful information going forward in the review, introducing not only how conservative each cyclostome lineage is but also how ancient these morphological changes are and thus how different these developmental changes might be.

 

Line 116-118: Sentence awkwardly worded.  Consider separating.

 

This sentence has been split.

 

Line 121-124: Awkward and redundant wording, unclear.

 

This sentence has been changed to remove redundant wording.

 

Line 132:  Do you mean “do not join medially to encompass the notochord”?  There is an and instead of to as written.  Unclear to this reviewer what condition is described.

 

We have added an explanation at the end of this section explaining that the vertebral column of gnathostomes fuses medially and envelops the notochord.

 

Line 136-139: “Considerable/considerably” used twice.  Please watch overuse of adjectives throughout manuscript.  There are a number of places, particularly setups and conclusions for sections, that are unproductively wordy.

 

We have changed the instance of “considerably” to “noticeably”, and have changed as many instances of double adjectives / adverbs as we could find.

 

Line 143-145: Statement of controversy requires citation.

 

We have added a citation to a recent paper which suggests that the ammocoete form of lamprey may be a derived state.

 

Line 151-153: Differences relative to what?  Each other?  Fossil jawless forms?  Jawed vertebrates?  This is unclear.  What do you mean by changes in ecology?  Extant lamprey and hagfish niches relative to fossil ones?  Please be more precise and detailed here and cite as appropriate.

 

We have added clarification to this section that we must consider shared and divergent characteristics between cyclostomes as well as those differences that there might have with gnathostomes as well. Furthermore, we have changed the wording of “ecology” with respect to lampreys and hagfishes, as the true ecology of stem cyclostomes isn’t well understood.

 

Line 162: “tried with to”

 

“with” was removed

 

 

Line 168: “cartilage in, but rarely”

 

“in” was removed

 

 

Line 274: Word missing between re-differentiates and adult?

 

“into an” was added to this sentence

 

 

Line 322: This sentence seems like a truism and is not very informative.

 

We agree that this sentence was not informative and has thus been removed.

 

Line 323: Focused understanding – missing “on”

 

“on” has been added

 

Line 334: Missing an “are”

 

“are” has been added

 

Lines 329-352:  Consider some reorganization with an eye towards matching the visual flow of Figure 3, which is easy to follow. 

 

We have changed the location of the Figure 3 reference to the instance where we mention TGFβ signaling and its effect on mesenchyme condensation, the first steps of chondrogenesis. We have also changed the wording of the introduction to better suit this.

 

Note: It is apparent with there is a shift in author voice.  For example, Section 3.1 lacks the awkward phrasing of the prior section.  Editing is required so readers can focus on the message.

 

The start of this paragraph has been changed to feel like a more natural transition from the introductory paragraphs above.

 

Line 498: Request a reference for the use of cyanogen bromide, such as first methods paper to use, for unfamiliar readers.  Perhaps a very brief explanation of what this technique offers?

 

We have added a citation to the use of cyanogen bromide to the first paper which used it on lamprey cartilage. We have also added a brief explanation to the usage of CnBR and its role in degrading methionine-rich proteins like collagen.

 

Section 4.1 Is insightful, but again change in writing style in 4.2 is noticeable.

 

We have changed the introductory sentences in 4.2 to more naturally transition from the conclusion of 4.1

 

Line 623:  How would a developing cartilage be “occasionally” dominated by versican?  This wording is unclear.

 

We have changed the wording of this sentence so that aggrecan is the predominant lectican in gnathostome cartilage, but that versican is also occasionally present

 

Line 626-628:  Please summarize value of these findings specifically, this statement is vague.

 

We have added a final sentence on this paragraph that summarizes the ECM findings in lamprey cartilage, specifically that lecticans were present in the ancestral vertebrate cartilage but we cannot be sure whether type II collagen was present, at least during early development.

 

Line 630: “Further elucidated upon” is unclear

 

We have added “develop of the” to explain what is being elucidated

 

Line 690-691: Awkwardly worded sentence.

 

We have re-worded parts of this sentence to clarify what we are suggesting, mainly that homologizing parts of the lamprey larval and adult skeleton will help us further understand cyclostome and vertebrae skeletal evolution.

 

Line 693: Paper would be improved by phylogenetic context earlier in the setup, not just at these later points.  This would allow the reader to think about the long branch lengths of cyclostomes and what they might mean for their unique character states relative to both gnathostomes and invertebrates.  If not a figure, then a slightly more complete discussion of the hypothesized branches of jawless/jawed, of cyclostomes, with time estimates bracketed from fossils and molecules. 

 

We have added phylogenetic context in the introduction to help establish, by the time the reader gets to Section 5, that: 1) lampreys and hagfishes split hundreds of millions of years ago; and 2) these differences in morphology are ancient as well.  

 

 

Line 695-697:  Assertion is hard to follow as written.

 

We have reworded this sentence to help clarify that we can’t assume homology of pre-metamorphic and post-metamorphic lamprey cartilages

 

Line 700: “It will therefore be important to compare…” This is an interesting point, but the authors fall short in not suggesting ways in which these comparisons might be made.  For example, how might RNAseq be leveraged to test cross taxa, cross tissue and across development hypotheses about the core cartilage GRN in cyclostomes?  What promise does the future hold?

 

We have added additional sentences to this section to talk about recent advances in techniques which can help us answer these questions. Specifically, we talk about how scRNA-seq will help us capture a more comprehensive view of gene expression using less specimens overall, which will be hugely advantageous for hagfishes. We also talk about how advances in gene editing and loss-of-function studies in lamprey can help us further test the interaction of genes involved in cartilage development. Overall, these new techniques will make cyclostome research much more feasible.

Author Response File: Author Response.docx