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) An in silico matrisome of 1812 proteins was predicted bioinformatically. The manually curated matrisome consists of 829 proteins 287 of which were confirmed by mass spectrometry. About 25 % (213) of the ECM proteins are specific to cnidarians. (C) The curated in silico matrisome proteins were manually annotated and sorted into core matrisome, matrisome-associated and other (mostly transmembrane) proteins (see supplementary table S1 for detailed annotations). (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 other ECM 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.

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 (nem) specification shares many genes, while spirocyte specification uses a distinct gene set.

Mesoglea dynamics across life stages assessed by quantitative proteomics of isolated mesoglea.

(A) 2-log transformed median abundances of proteins across different life stages. The curated matrisome was 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. (B-C) Volcano plots showing the differential abundance of proteins in the mesoglea extracts of the three different life stages. (B) Proteins involved in BM organization including all polydoms and in Wnt/PCP signaling are upregulated during larva-to-primary polyp transition as highlighted. (C) 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.

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.