Molecular dynamics of the matrisome across sea anemone life history
- University of Heidelberg, Centre for Organismal Studies, Department of Evolutionary Neurobiology, Germany
- Department of Neurosciences and Developmental Biology, Faculty of Life Sciences, University of Vienna, Austria
- European Molecular Biology Laboratory, Germany
- Institute of Neuroanatomy, Medical University of Innsbruck, Austria
- Institute of Histology and Embryology, Medical University of Innsbruck, Austria
Figures
Figure 1 with 3 supplements
Analysis of the Nematostella matrisome.
(A) Mesoglea from larvae, primary polyps, and adults was decellularized and analyzed by mass spectrometry. In parallel, an in silico matrisome was predicted using a computational approach and curated manually. (B, C) 1812 potential ECM proteins were predicted bioinformatically. The manually curated list of ECM factors consists of 829 proteins. The curated ECM proteins were sorted into core matrisome and matrisome-associated groups, which together constitute the Nematostella matrisome (551 proteins). The remaining non-matrisomal proteins are categorized as ‘adhesome’ that include transmembrane receptors, and ‘other’ ECM domain-containing proteins, which include adhesive proteins, venoms, enzymes, ion channels, stress and injury response factors, and diverse uncharacterized proteins (see Supplementary file 1 for detailed annotations and sub-categories). In total, 287 ECM proteins were confirmed by mass spectrometry analysis, 210 of which belong to the matrisome and 47 to the ‘adhesome’. (D) Comparison of the Nematostella matrisome size with published matrisomes of other species. While the complexity of the Nematostella core matrisome is comparable to that of vertebrates, the number of ECM-associated proteins is disproportionally lower. The Drosophila core matrisome is characterized by significant secondary reduction. (E) Laminin antibody stains the bilaminar structure of the BL (magenta) at the base of the epithelial cell layers, while the pan-Collagen antibody (yellow) detects the central IM. Scale bar, 10 μm. The three life stages of Nematostella before (F–H) and after (I–K) decellularization. The mesoglea is stained with Laminin antibody to demonstrate its structural preservation and by DAPI (cyan) to visualize residual nuclei and nematocysts. The decellularized mesoglea retains morphological structures such as tentacles (t) and mesenteries (m). Scale bars: F, G, I, J, 100 µm; H, K, 1 mm.
Figure 1—figure supplement 1
Protein complexity in mesoglea samples of Nematostella and Hydra resolved by one-dimensional SDS-PAGE.
Mesogleas were prepared from adult animals and dissolved in lithium dodecylsulfate buffer containing 1 M DTT. After heating at 90°C for 30 min, 130 mg of each sample was loaded on a 4–12% gradient gel. The molecular mass of marker proteins (M) is as indicated. Uncropped gel images are provided in Figure 1—figure supplement 1—source data 1 and 2.
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Figure 1—figure supplement 1—source data 1
Uncropped gel images of Hydra (left) and Nematostella (right) mesoglea preparations.
- https://cdn.elifesciences.org/articles/105319/elife-105319-fig1-figsupp1-data1-v1.zip
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Figure 1—figure supplement 1—source data 2
Uncropped and labeled gel images of Hydra (left) and Nematostella (right) mesoglea preparations.
- https://cdn.elifesciences.org/articles/105319/elife-105319-fig1-figsupp1-data2-v1.pdf
Figure 1—figure supplement 2
Epitope sequences of laminin and collagen antibodies.
(A) The laminin antibody was raised against a peptide sequence of the Nv laminin gamma-1 chain indicated by the boxed area. (B) The collagen antibodies were raised against an epitope representing a consensus sequence of fibrillar collagens (NvPanCol) and unique sequences in NvCol4b and NvCol2c as indicated. Mark that in the consensus sequence, the central cysteine residue was replaced by a serine to prevent disulfide formation.
Figure 1—figure supplement 3
Ultrastructure of the mesoglea in Nematostella larvae (A–F) and primary polyps (G–P).
(A) Electron microscopy of the larvae with ectoderm (ec), gastroderm (ga), and an only 0.5-μm-thick mesoglea (asterisk) in between. (B) The IM of cryofixed larval mesoglea displays scarce, quite thin filaments, embedded in amorphous compounds. The basement membrane (BM: arrows) appears rather delicate at this developmental stage, measuring about 70 nm. (C) The mesoglea thickens into a triangular shape at the septa branching points. (D) Cross section of a larva stained with DAPI (cyan) and laminin antibody (magenta). DAPI hardly enters the gastroderm. The mesoglea and developing septa are shown by laminin staining. (E) The triangular expansion of the mesoglea at the septa branch is lined with laminin staining while the central region does not show any signal. (F) Electron micrograph of the septa branch. The BM is overlaid in magenta. The central region of the septa triangle is filled with loose fibrillar material. (Scale bars: A, F=1 μm, B=0.5 μm, D=100 μm, E=10 μm). (G, H) EM of the primary polyp mesoglea (asterisk) and adjacent epithelia (ec, ga). The mesoglea is now about 1.5 μm thick with numerous, haphazardly arranged about 13 nm thick and 6 nm thin fibrils in the IM. The BM (arrows) has thickened, thus, matured as well, and forms distinct, dense, about 130-nm-thick meshworks lining the epithelia. (I) Whole mount staining of a tentacle bud stage stained with DAPI and laminin antibody. (J) At the aboral end, the BM is expanded (asterisk) into the ectodermal layer forming a knot-like structure. (K-L’) Scheme and electron micrographs of the ECM fiber orientation at different sites of the primary polyps’ body (ec highlighted in rose, ga in blue, ECM in gray/green). (L) The filaments/fibrils in the region of the body column are loosely oriented along the oral-aboral (o–a) axis following the overall orientation of the mesoglea. (L’) At the aboral end, the fibrils are densely packed forming a plug-like structure parallel to the o-a axis (overlaid in green). Scale bars: G=1 μm; H=0.5 μm; I=100 μm, J=10 μm. L, L’=1 μm. (M–P) Immunoelectron microscopy of mesoglea compounds, performed on thawed cryosections. (M) Laminin immunogold label (arrows) along the plasma membrane of the ECM lining muscle cells (m). (N) Col4 label located at the BM. (O) PanCol is predominantly found throughout the IM. (P) Col2c label in the IM. Scale bars: M-P=0.25 μm.
Figure 2 with 2 supplements
Single cell atlas of core matrisome genes.
(A) Dimensional reduction cell plot (UMAP) highlighting cell clusters showing over-abundant expression of the core matrisome, matrisome-associated, and adhesome/other gene sets. Expression values correspond to gene module scores for each set of genes. (B) Dotplot expression profiles of upregulated genes of the core matrisome across cell type partitions, separated across phases of the life cycle. Illustrated are the top 5 genes with expression in at least 20% of any cell state cluster, calculated to be upregulated with a p-value of ≤ 0.001. See Supplementary file 5 for a full list of differentially expressed core matrisome genes. Larva (red colour scale) = 18 hr:4 day samples; Primary Polyp (orange colour scale) = 5:16 day samples; Adult (blue colour scale) = tissue catalog from juvenile and adult specimens. (C) Nematostella collagens. Domain organization of matrisome proteins containing a collagen triple helix as core element. The proteins are categorized into fibrillar and basement membrane collagens, short-chain collagens, and nematocyte-specific minicollagens.
Figure 2—figure supplement 1
Expression profiles of core matrisome genes.
Dotplot expression profiles across all cell-state clusters, separated across phases of the life cycle. Cell states are grouped and colored according to tissue-type partitions, and genes are grouped by category. Expression scale is the same as in Figure 2. Abbreviations for cell state identities are explained in the Supplementary file 8.
Figure 2—figure supplement 2
Single-cell atlas of collagen genes.
Dot plot expression profiles of all identified collagen-coding genes across cell type partitions, separated across phases of the life cycle. Cell states are grouped and colored according to tissue-type partition. Expression scale is the same as in Figure 2. Abbreviations for cell identities are explained in the Supplementary file 8.
Figure 3 with 1 supplement
Cell-type specificity of cnidocyte-expressed ECM genes.
(A) The distribution of cnidocyte-expressed genes categorized as ‘ubiquitous’ (blue: 41), ‘shared’ (red: 27), ‘mature-specific’ (green: 38), or ‘specification-specific’ (purple: 88). (B) Expression of the module scores of each gene subset across the main tissue-type data partitions, illustrated on UMAP dimensional reduction. (C) Sequential gene expression activation illustrated on a dot plot of top 5 differentially expressed genes (p-value ≤ 0.001) for each cnidocyte cell state. Nematocyte specification shares many genes, while spirocyte specification uses a distinct gene set. Nep.8, nematocyst-expressed protein 8 categorized as venom protein.
Figure 3—figure supplement 1
Single-cell atlas of cnidocyte-expressed matrisome genes.
Dot plot expression profiles of matrisome genes expressed within the cnidocytes, plotted across all cell-type states. Genes are grouped according to degree of overlapping expression with other cell types, with ‘ubiquitous’ expression on top, followed by ‘shared’ expression, and then cnidocyte-specification specific genes, and mature cnidocyte-specific genes.
Figure 4 with 3 supplements
Mesoglea dynamics across life stages assessed by quantitative proteomics of isolated mesoglea.
(A) Boxplot representation of normalized log2 TMT reporter ion intensities for different protein subgroups of the matrisome. ‘All’ represents all proteins in each respective dataset. A horizontal line indicates median TMT intensity in the complete dataset. (B) 2-log transformed median abundances of proteins across different life stages. The curated ECM proteins were filtered for proteins with a twofold change in any of the life stages and a false discovery rate of 0.05 using a moderated t-test (limma). The heatmap shows the 2-log transformed median abundance of 4 samples per life stage. Most proteins are upregulated in only one of the life stages. Notably, BM factors including all polydoms are upregulated in the primary polyp. Most ECM protein categories can be clearly divided into adult-specific and primary polyp-specific proteins underscoring the differential composition of the mesoglea at different life stages. (C, D) Volcano plots showing the differential abundance of proteins in the mesoglea extracts of the three different life stages. (C) Proteins involved in BM organization including all polydoms and in Wnt/PCP signaling are upregulated during larva-to-primary polyp transition as highlighted. (D) The adult mesoglea compared to primary polyps is characterized by an enrichment of elastic fibril components and matricellular glycoproteins involved in wound response and regeneration. gray = non-matrimonial background, orange = insignificant, magenta = differentially abundant matrisome proteins. (E) Domain organization of bilaterian and cnidarian polydoms. The Nematostella matrisome contains an expanded group of polydoms and polydom-like proteins, including three cnidarian-type polydom paralogs, four shorter polydom-like sequences, and a polydom-related protein, which contains only the core Sushi-HYR-TKE motif. Domain symbols: vWFA (light blue), EGF-like (purple), Sushi/SCR/CCP (orange), Hyalin repeat (red), Pentraxin (yellow), CUB (light green), Tyrosine-protein kinase ephrin (dark green), PAN/Apple (olive green), Ricin B-like (pink), Thrombospondin type-1 repeat (brown), Coagulation factor 5/8 (dark purple), Ig-like (dark grey).
Figure 4—figure supplement 1
Mesoglea protein abundance overview in specific developmental stages.
Boxplot overview of data normalization steps for mesoglea samples. Raw TMT reporter ion intensities (left) were first cleaned for batch effects (middle) and further normalized using variance stabilization normalization (vsn - right). Due to low starting material in early cell stages, protein concentration was not adjusted before mass spec measurement and only accounted for in the normalization step to achieve equal protein amounts.
Figure 4—figure supplement 2
Domain organization of metalloprotases in the Nematostella matrisome.
Metalloproteases comprise an N-terminal signal-peptide for secretion (S), a propeptide or prodomain conferring latency (PRO), and a catalytic zinc-dependent metallopeptidase domain (CD). Additional domains were listed using Interproscan-5.57 codes: (Kringle) Kringle (IPR000001), (CD) Catalytic domain (IPR024079), (PGBD) Peptidoglycan binding-domain (IPR002477), (MAM) MAM-domain (IPR000998), (PRO) Propeptide, (ShKT) ShKT domain (IPR003582), (M12B-GON) M12B-GON-ADAMTS (IPR012314), (WAP) WAP-type 'four-disulfide core' domain (IPR008197), (LDL) Low-density lipoprotein (IPR002172), (EGF) EGF-like (IPR000742), (Ricin) Ricin B-like (IPR035992), (TSR) Thrombospondin type-1 repeat (IPR000884), (mem-prox) ADAM17, membrane-proximal domain (IPR032029), (M12B-PRO) M12B-propeptide (IPR002870), (TM) Transmembrane domain, (Ig) Immunoglobulin-like (IPR013783), (Serine-protease) Serine protease (IPR001254), (PLAC/PLAT/LH2) PLAC / PLAT/LH2 (IPR001304/IPR001024), (vHL) von Hippel-Lindau domain (IPR036208), (CUB) CUB domain (IPR000859), (CLEC) C-lectin like (IPR001304), (Hemopexin) Hemopexin-like repeat (IPR018487), (CRD) ADAMTS/ADAMTS-like, Cysteine-rich domain (IPR045371I), (FA58C) Coagulation factor 5/8(IPR000421), (Fib) Fibrinogen-like (IPR036056), (FN) Fibronectin type II (IPR036943), (Disintegrin) Disintegrin domain (IPR001762), and ADAMTS/ADAMTS-like (IPR010294). All metalloproteases were identified based on manual sequence and domain analysis. The identified proteases were categorized as MASP-like (1), M28-like (1), ADAM-like (4), MMP (6), ADAMTS-like (15), and Astacin (27). The latter can be further divided into three subgroups: Meprin-like (9), BMP/Tolloid-like (2), and other Astacins (16). Based on the identified hierarchical phylogenetic orthogroups, we identified sequences that are specific to cnidarians, as indicated.
Figure 4—figure supplement 3
Expression profile of Nematostella polydoms.
All Nematostella polydoms are expressed from GD.1 neurogland cells supposed to have a digestive function. Polydom-like-2 and Polydom-related show additional expression within the uncharacterized secretory cell type. S2.tll.2&3.
Figure 5
Matrisome complexity across metazoan phyla.
Matrisome sizes of published and newly generated in silico matrisomes of representative cnidarians and other metazoan species were plotted against their respective orthogroup count. Only proteins from orthogroups shared with at least one published matrisome were counted. Anthozoans generally show a higher matrisome complexity than medusozoan species populating a transitory region between bilaterians and non-bilaterians in the evolutionary trajectory.
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Biological sample (Nematostella vectensis) | Larvae (3 dpf), primary polyps (10 dpf), adults (≥1 year) | Mark Q. Martindale, Whitney Lab; Putnam et al., 2007 | Cultured in lab conditions; used for mesoglea isolation, immunostaining, proteomics. Mixed sex animals used in all experiments | |
| Antibody | Anti-Laminin (rabbit polyclonal) | This paper | Custom polyclonal antibody produced by eurogentec, epitope in Nv Laminin γ1 chain (1:100 for IF, 1:2 for EM) | |
| Antibody | Anti-Collagen IV (guinea pig polyclonal) | This paper | Custom polyclonal antibody produced by eurogentec, epitope in NvCol4b (1:10 for EM) | |
| Antibody | Anti-Collagen II-like (rat polyclonal) | This paper | Custom polyclonal antibody produced by eurogentec, epitope in NvCol2c (1:10 for EM) | |
| Antibody | Anti-Pan-Collagen (rat polyclonal) | This paper | Custom polyclonal antibody produced by eurogentec, consensus fibrillar collagen motif (1:100 for IF, 1:2 for EM) | |
| Antibody | Alexa Fluor 488 goat anti-rat IgG (H+L) (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11006 RRID:AB_2534074 | Secondary antibody (1:400) |
| Antibody | Alexa Fluor 568 goat anti-rabbit IgG (H+L) (goat polyclonal) | Thermo Fisher Scientific | Cat# A-11011 RRID:AB_143157 | Secondary antibody (1:400) |
| Antibody | Goat anti-rabbit IgG (10 nm colloidal gold) (goat polyclonal) | British Biocell | Cat# EM.GAR10/1 RRID:AB_2715527 | Immunogold EM (1:150) |
| Antibody | Goat anti-rat IgG (10 nm colloidal gold) (goat polyclonal) | British Biocell | Cat# EM.GAT10/1 RRID:AB_2715527 | Immunogold EM (1:150) |
| Antibody | Nanogold-IgG Goat anti-Guinea Pig IgG (goat polyclonal) | Nanoprobes | Cat# 2054 RRID:AB_3711173 | Immunogold EM (1:150) |
| Antibody | Nanogold-IgG Goat anti-Rat IgG (H+L) (goat polyclonal) | Nanoprobes | Cat# 2007 RRID:AB_3711173 | Immunogold EM (1:150) |
| Chemical compound, drug | Chymotrypsin, sequencing grade | Promega | Cat# V1061 | Mass spectrometry |
| Chemical compound, drug | Cysteine | Sigma-Aldrich | Cat# C7352 | Egg dejellying |
| Chemical compound, drug | DAPI | Sigma-Aldrich | Cat# D9542 | Nuclear stain |
| Chemical compound, drug | Dithiothreitol | Sigma-Aldrich | Cat# D9779 | Mesoglea preparation |
| Chemical compound, drug | EGTA | Sigma Aldrich | Cat# 324626 | Electron microscopy |
| Chemical compound, drug | Formaldehyde solution min. 37% | Merck KGaA | Cat# 252549 | Electron microscopy |
| Chemical compound, drug | Glutaraldehyde | Sigma-Aldrich | Cat# G5882 | Electron microscopy |
| Chemical compound, drug | HEPES | Sigma-Aldrich | Cat# H3375 | Mesoglea preparation |
| Chemical compound, drug | HQ-Silver | Nanoprobes Yaphank | Cat# 2012 | Electron microscopy |
| Chemical compound, drug | Magnesium chloride | Sigma-Aldrich | Cat# M8266 | Immunocytochemistry |
| Chemical compound, drug | N-lauryl-sarcosinate | Sigma-Aldrich | Cat# L5125 | Mesoglea decellularization |
| Chemical compound, drug | OASIS HLB µElution Plate | Waters | Cat# 186001828BA | Mass spectrometry |
| Chemical compound, drug | Osmium tetroxide | Sigma Aldrich | Cat# O5500 | Electron microscopy |
| Chemical compound, drug | Precast 4–12% gradient gels | Carl Roth | Cat# 3673.2 | SDS-PAGE |
| Chemical compound, drug | TMT10plex Isobaric Label Reagent | ThermoFisher | Cat# 90110 | Mass spectrometry |
| Chemical compound, drug | Trichloroacetic acid | Sigma-Aldrich | Cat# T6399 | Mesoglea preparation |
| Chemical compound, drug | Triton X-100 | Sigma-Aldrich | Cat# T8787 | Immunocytochemistry |
| Chemical compound, drug | Tween-20 | Roche | Cat# 11332465001 | Immunocytochemistry |
| Chemical compound, drug | Uranyl acetate | Electron Microscopy Sciences | Cat# 541-09-3 | Electron microscopy |
| Software, algorithm | BLAST | Altschul et al., 1990 | RRID:SCR_004870 | Database searches |
| Software, algorithm | Custom Python scripts | This paper | RRID:SCR_024202 | For domain and orthogroup analysis |
| Software, algorithm | DeepLoc-2 | Thumuluri et al., 2022 | RRID:SCR_026503 | Protein localization prediction |
| Software, algorithm | Fiji (ImageJ) | Schindelin et al., 2012 | RRID:SCR_003070 | Image processing |
| Software, algorithm | IsobarQuant | Franken et al., 2015 | RRID:SCR_016732 | MS data analysis |
| Software, algorithm | limma (R package) | Ritchie et al., 2015 | RRID:SCR_010943 | Proteomics analysis |
| Software, algorithm | Mascot | Matrix Science | RRID:SCR_014322 | MS data analysis |
| Software, algorithm | NIS elements Imagine software | Nikon Instruments Inc. | Image processing. https://www.microscope.healthcare.nikon.com/products/software/nis-elements | |
| Software, algorithm | OrthoFinder v2.5.4 | Emms and Kelly, 2019 | RRID:SCR_017118 | Orthogroup prediction |
| Software, algorithm | Seurat package | Stuart et al., 2019 | RRID:SCR_016341 | Single cell expression analysis |
| Software, algorithm | SignalP-6.0 | Teufel et al., 2022 | RRID:SCR_015644 | Signal peptide prediction |
| Software, algorithm | SMART | Schultz et al., 1998 | RRID:SCR_005026 | Protein domain analysis |
| Other | Single-cell RNA atlas | Cole et al., 2024 | Expression data | |
| Other | Nikon A1R Confocal Laser Scanning Microscope | Nikon, Tokyo, Japan | RRID:SCR_020317 | Confocal microscopy |
| Other | Nikon Eclipse 80i microscope | Nikon, Tokyo, Japan | RRID:SCR_015572 | Fluorescence microscopy |
| Other | Gemini C18 column (3 μm, 110 Å, 100 × 1.0 mm) | Phenomenex | Cat# 00D-4439-A0 | Mass spectrometry |
| Other | Agilent 1200 Infinity high-performance liquid chromatography system | Agilent | RRID:SCR_018018 | Mass spectrometry |
Additional files
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Supplementary file 1
In silico matrisome prediction of the Nematostella mesoglea including results of differential abundance analysis using moderated t-test (limma) for each developmental comparison (Adult - Larvae, Larvae - Primary Polyp, Adult - Primary Polyp).
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp1-v1.xlsx
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Supplementary file 2
Full mass spectrometry data of the Nematostella mesoglea.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp2-v1.xlsx
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Supplementary file 3
Raw data of mesoglea and BM thickness as well as fibril thickness measurements in electron micrographs of larva and primary polyp cryosections.
Related to Figure 1—figure supplement 3.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp3-v1.xlsx
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Supplementary file 4
Gene expression profiles indicating the average expression (avg.exp), the percentage of the cells with gene expression (pct.exp), and the relative expression levels (avg.exp.scaled) for all ECM genes across all cell state identities (cell.state.id) in Cole et al., 2024.
pSC, putative germ cell, PGC, primary germ cell, NPC, neuroglandular progenitor cell. GD, digestive gland cell, NGD, digestive neuroglandular cell. Related to Figure 2.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp4-v1.xlsx
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Supplementary file 5
Single cell expression profiles of core matrisomal genes.
Related to Figure 2. Output of the FindAllMarkers function of the Seurat package (vs.4.4.0) run with default parameters using all core matrisome genes across all clusters annotated in Cole et al., 2024 (cell.state.id). gene: core matrisome gene tested, p_val: calculated p-value of the expression difference; avg_log2FC: calculated average log2 fold change between cluster 1 (cell.state.id) and the rest of the dataset; pct.1: percentage of cells in cluster 1 (cell.state.id) with gene expression; pct.2: percentage of cells within the remaining clusters with gene expression; p_val_adj: adjusted p-value.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp5-v1.xlsx
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Supplementary file 6
Matrix of average expression values for all cnidocyte-specific genes (rows) across all cell state identities in Cole et al., 2024 (columns).
Related to Figure 3.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp6-v1.xlsx
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Supplementary file 7
Quantitative mass spectrometry data of mesoglea samples from different life stages.
Related to Figure 4.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp7-v1.xlsx
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Supplementary file 8
README for Supplementary files 1–7.
- https://cdn.elifesciences.org/articles/105319/elife-105319-supp8-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/105319/elife-105319-mdarchecklist1-v1.pdf
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Molecular dynamics of the matrisome across sea anemone life history
eLife 14:RP105319.
https://doi.org/10.7554/eLife.105319.3
