Molecular dynamics of the matrisome across sea anemone life history

Reviewer #1 (Public review):

Summary:

In this manuscript entitled "Molecular dynamics of the matrisome across sea anemone life history", Bergheim and colleagues report the prediction, using an established sequence analysis pipeline, of the "matrisome" - that is, the compendium of genes encoding constituents of the extracellular matrix - of the starlet sea anemone Nematostella vectensis. Re-analysis of an existing scRNA-Seq dataset allowed the authors to identify the cell types expressing matrisome components and different developmental stages. Last, the authors apply time-resolved proteomics to provide experimental evidence of the presence of the extracellular matrix proteins at three different stages of the life cycle of the sea anemone (larva, primary polyp, adult) and show that different subsets of matrisome components are present in the ECM at different life stages with, for example, basement membrane components accompanying the transition from larva to primary polyp and elastic fiber components and matricellular proteins accompanying the transition from primary polyp to the adult stage.

Strengths:

The ECM is a structure that has evolved to support the emergence of multicellularity and different transitions that have accompanied the complexification of multicellular organisms. Understanding the molecular makeup of structures that are conserved throughout evolution is thus of paramount importance.

The in-silico predicted matrisome of the sea anemone has the potential to become an essential resource for the scientific community to support big data annotation efforts and understand better the evolution of the matrisome and of ECM proteins, an important endeavor to better understand structure/function relationships. This study is also an excellent example of how integrating datasets generated using different -omic modalities can shed light on various aspects of ECM metabolism, from identifying the cell types of origins of matrisome components using scRNA-Seq to studying ECM dynamics using proteomics.

Weaknesses:

My concerns pertain to the three following areas of the manuscript:

(1) In-silico definition of the anemone matrisome using sequence analysis:

a) While a similar computational pipeline has been applied to predict the matrisome of several model organisms, the authors fail to provide a comprehensive definition of the anemone matrisome: In the text, the authors state the anemone matrisome is composed of "551 proteins, constituting approximately 3% of its proteome (see page 6, line 14), but Figure 1 lists 829 entries as part of the "curated" matrisome, Supplementary Table S1 lists the same 829 entries and the authors state that "Here, we identified 829 ECM proteins that comprise the matrisome of the sea anemone Nematostella vectensis" (see page 17, line 10). Is the sea anemone matrisome composed of 551 or 829 genes? If we refer to the text, the additional 278 entries should not be considered as part of the matrisome, but what is confusing is that some are listed as glycoproteins and the "new_manual_annotation" proposed by the authors and that refer to the protein domains found in these additional proteins suggest that in fact, some could or should be classified as matrisome proteins. For example, shouldn't the two lectins encoded by NV2.3951 and NV2.3157 be classified as matrisome-affiliated proteins? Based on what has been done for other model organisms, receptors have typically been excluded from the "matrisome" but included as part of the "adhesome" for consistency with previously published matrisome; the reviewer is left wondering whether the components classified as "Other" / "Receptor" should not be excluded from the matrisome and moved to a separate "adhesome" list.

In addition to receptors, the authors identify nearly 70 glycoproteins classified as "Other". Here, does other mean "non-matrisome" or "another matrisome division" that is not core or associated? If the latter, could the authors try to propose a unifying term for these proteins? Unfortunately, since the authors do not provide the reasons for excluding these entries from the bona fide matrisome (list of excluding domains present, localization data), the reader is left wondering how to treat these entries.

Overall, the study would gain in strength if the authors could be more definitive and, if needed, even propose novel additional matrisome annotations to include the components for now listed as "Other" (as was done, for example, for the Drosophila or C. elegans matrisomes).

b) It is surprising that the authors are not providing the full currently accepted protein names to the entries listed in Supplementary Table S1 and have used instead "new_manual_annotation" that resembles formal protein names. This liberty is misleading. In fact, the "new_manual_annotation" seems biased toward describing the reason the proteins were positively screened for through sequence analysis, but many are misleading because there is, in fact, more known about them, including evidence that they are not ECM proteins. The authors should at least provide the current protein names in addition to their "new_manual_annotations".

c) To truly serve as a resource, the Table should provide links to each gene entry in the Stowers Institute for Medical Research genome database used and some sort of versioning (this could be added to columns A, B, or D). Such enhancements would facilitate the assessment of the rigor of the list beyond the manual QC of just a few entries.

d) Since UniProt is the reference protein knowledge database, providing the UniProt IDs associated with the predicted matrisome entries would also be helpful, giving easy access to information on protein domains, protein structures, orthology information, etc.

e) In conclusion, at present, the study only provides a preliminary draft that should be more rigorously curated and enriched with more comprehensive and authoritative annotations if the authors aspire the list to become the reference anemone matrisome and serve the community.

(2) Proteomic analysis of the composition of the mesoglea during the sea anemone life cycle:

a) The product of 287 of the 829 genes proposed to encode matrisome components was detected by proteomics. What about the other ~550 matrisome genes? When and where are they expressed? The wording employed by the authors (see line 11, page 13) implies that only these 287 components are "validated" matrisome components. Is that to say that the other ~550 predicted genes do not encode components of the ECM? This should be discussed.

b) Can the authors comment on how they have treated zero TMT values or proteins for which a TMT ratio could not be calculated because unique to one life stage, for example?

c) Could the authors provide a plot showing the distribution of protein abundances for each matrisome category in the main figure 4? In mammals, the bulk of the ECM is composed of collagens, followed by fibrillar ECM glycoproteins, the other matrisome components being more minor. Is a similar distribution observed in the sea anemone mesoglea?

d) Prior proteomic studies on the ECM of vertebrate organisms have shown the importance of allowing certain post-translational modifications during database search to ensure maximizing peptide-to-spectrum matching. Such PTMs include the hydroxylation of lysines and prolines that are collagen-specific PTMs. Multiple reports have shown that omitting these PTMs while analyzing LC-MS/MS data would lead to underestimating the abundance of collagens and the misidentification of certain collagens. The authors may want to re-analyze their dataset and include these PTMs as part of their search criteria to ensure capturing all collagen-derived peptides.

e) The authors should ensure that reviewers are provided with access to the private PRIDE repository so the data deposited can also be evaluated. They should also ensure that sufficient meta-data is provided using the SRDF format to allow the re-use of their LC-MS/MS datasets.

(3) Supplementary tables:

The supplementary tables are very difficult to navigate. They would become more accessible to readers and non-specialists if they were accompanied by brief legends or "README" tabs and if the headers were more detailed (see, for example, Table S2, what does "ctrl.ratio_Larvae_rep2" exactly refer to? Or Table S6 whose column headers using extensive abbreviations are quite obscure). Similarly, what do columns K to BX in Supplementary Table S1 correspond to? Without more substantial explanations, readers have no way of assessing these data points.

Reviewer #2 (Public review):

This work set out to identify all extracellular matrix proteins and associated factors present within the starlet sea anemone Nematostella vectensis at different life stages. Combining existing genomic and transcriptomic datasets, alongside new mass spectometry data, the authors provide a comprehensive description of the Nematostella matrisome. In addition, immunohistochemistry and electron microscopy were used to image whole mount and de-cellularized mesoglea from all life stages. This served to validate the de-cellularization methods used for proteomic analyses, but also resulted in a very nice description of mesoglea structure at different life stages. A previously published developmental cell type atlas was used to identify the cell type specificity of the matrisome, indicating that the core matrisome is predominantly expressed in the gastrodermis, as well as cnidocytes. The analyses performed were rigorous and the results were clear, supporting the conclusions made by the authors.

Reviewer #3 (Public review):

Summary:

This manuscript by Bergheim et al investigates the molecular and developmental dynamics of the matrisome, a set of gene products that comprise the extra cellular matrix, in the sea anemone Nematostella vectensis using transcriptomic and proteomic approaches. Previous work has examined the matrisome of the hydra, a medusozoan, but this is the first study to characterize the matrisome in an anthozoan. The major finding of this work is a description of the components of the matrisome in Nematostella, which turns out to be more complex than that previously observed in hydra. The authors also describe remodeling of the extra cellular matrix that occurs in the transition from larva to primary polyp, and from primary polyp to adult. The authors interpret these data to support previously proposed (Steinmetz et al. 2017) homology between the cnidarian endoderm with the bilaterian mesoderm.

Strengths:

The data described in this work are comprehensive (but see important considerations of reviewer #1) combining both transcriptome and proteomic interrogation of key stages in the life history of Nematostella and are of value to the community.

Weaknesses:

The authors offer numerous evolutionary interpretations of their results that I believe are unfounded. The main problem with extending these results, together with previous results from hydra, into an evolutionary synthesis that aims to reconstruct the matrisome of the ancestral cnidarian is that we are considering data from only two species. I agree with the authors' depiction of hydra as "derived" relative to other medusozoans and see it as potentially misleading to consider the hydra matrisome as an exemplar for the medusozoan matrisome. Given the organismal and morphological diversity of the phylum, a more thorough comparative study that compares matrisome components across a selection of anthozoan and medusozoan species using formal comparative methods to examine hypotheses is required.
Specifically, I question the author's interpretation of the evolutionary events depicted in this statement:

"The observation that in Hydra both germ layers contribute to the synthesis of core matrisome proteins (Epp et al. 1986; Zhang et al. 2007) might be related to a secondary loss of the anthozoan-specific mesenteries, which represent extensions of the mesoglea into the body cavity sandwiched by two endodermal layers."
Anthozoans and medusozoans are evolutionary sisters. Therefore, secondary loss of "anthozoan-like mesenteries" in hydrozoans is at least as likely as the gain of this character state in anthozoans. By extension, there is no reason to prefer the hypothesis that the state observed in Nematostella, where gastroderm is responsible for the synthesis of the core matrisome components, is the ancestral state of the phylum.
Moreover, the fossil evidence provided in support of this hypotheses (Ou et al. 2022)is not relevant here because the material described in that work is of a crown group anthozoan, which diversified well after the origin of Anthozoa. The phylogenetic structure of Cnidaria has been extensively studied using phylogenomic approaches and is generally well supported(Kayal et al. 2018; DeBiasse et al. 2024). Based on these analyses, anthozoans are not on a "basal" branch, as the authors suggest. The structure of cnidarian phylogeny bifurcates with Anthozoa forming one clade and Medusozoa forming the other. From the data reported by Bergheim and co-workers, it is not possible to infer the evolutionary events that gave rise to the different matrisome states observed in Nematostella (an anthozoan) and hydra (a medusozoan).
Furthermore, I take the observation in Fig 5 that anthozoan matrisomes generally exhibit a higher complexity than other cnidarian species to be more supportive of a lineage specific expansion of matrisome components in the Anthozoa, rather than those components being representative of an ancestral state for Cnidaria. Whatever the implication, I take strong issue with the statement that "the acquisition of complex life cycles in medusozoa, that are distinguished by the pelagic medusa stage, led to a secondary reduction in the matrisome repertoire." There is no causal link in any of the data or analyses reported by Bergheim and co-workers to support this statement and, as stated above, while we are dealing with limited data, insufficient to address this question, it seems more likely to me that the matrisome expanded in anthozoans, contrasting with the authors conclusions. While the discussion raises many interesting evolutionary hypotheses related to the origin of the cnidarian matrisome, which is of vital interest if we are to understand the origin of the bilaterian matrisome, a more thorough comparative analysis, inclusive of a much greater cnidarian species diversity, is required if we are to evaluate these hypotheses.

DeBiasse MB, Buckenmeyer A, Macrander J, Babonis LS, Bentlage B, Cartwright P, Prada C, Reitzel AM, Stampar SN, Collins A, et al. 2024. A Cnidarian Phylogenomic Tree Fitted With Hundreds of 18S Leaves. Bulletin of the Society of Systematic Biologists [Internet] 3. Available from: https://ssbbulletin.org/index.php/bssb/article/view/9267

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Steinmetz PRH, Aman A, Kraus JEM, Technau U. 2017. Gut-like ectodermal tissue in a sea anemone challenges germ layer homology. Nature Ecology & Evolution 2017 1:10 [Internet] 1:1535-1542. Available from: https://www.nature.com/articles/s41559-017- 0285-5

Zhang X, Boot-Handford RP, Huxley-Jones J, Forse LN, Mould AP, Robertson DL, Li L, Athiyal M, Sarras MP. 2007. The collagens of hydra provide insight into the evolution of metazoan extracellular matrices. J Biol Chem [Internet] 282:6792-6802. Available from: https://pubmed.ncbi.nlm.nih.gov/17204477/

Author response:

We appreciate the effort the reviewers have put into evaluating our work, and will take the opportunity to revise and improve our submission. In response to the reviewer's comments, we will carefully revisit our manuscript to address the concerns they have raised. Specifically, we will ensure that our revised version is coherent with our annotations and public databases, clarify any discrepancy between the investigated proteins and gene models, and re-examine our discussion of the evolutionary implications in light of their suggestions. We are confident that these revisions will strengthen our work and provide a clearer understanding of our research findings.