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 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 proteins, which include adhesive proteins, venoms, enzymes, ion channels, stress and injury response factors and diverse uncharacterized proteins (see supplementary table S1 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.

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 table S5 for a full list of differentially expressed core matrisome genes. Larva = 18hr:4day samples; Primary Polyp = 5:16 day samples; Adult = 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.

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 dotplot 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.

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. 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 2-fold 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).

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.