Figures and data
The Sycon ciliatum skeleton features specific spicule types in distinct body regions: parallel diactines in the oscular region (upper inset), radial tubes supported by triactines and tufted with diactines (lower inset), and the atrial skeleton composed of triactines and tetractines. B: The upper oscular region shows increased spicule formation (calcein staining) in the growing zone of new radial tubes and around the osculum, where oscular diactines are predominantly produced (modified from Voigt et al., 2014). C: Spicules are formed by sclerocytes, specialized cells controlling spicule formation. Diactine formation involves two sclerocytes, triactine formation six (f = founder cell, t = thickener cell).
A: Structural similarities in Alpha-Fold predictions of galaxins (Acropora millepora) and selected calcarins (Sycon ciliatum). B: Beta hairpins in Cal7 connected by disulfide bridges of di-cysteines. C: Number of calcarins, galaxin-like, and galaxins transcripts in sponges and corals, assigned to orthogroups.
Expression of calcarins.
A-D: Cal1, Cal2, and Spiculin Expression in a regenerated Sycon ciliatum at the asconoid juvenile stage. Scale bars = 50 µm. A: Single channels and overlay showing distinct gene expression with minimal co-expression. B: Expression around the ocular region; Spiculin in sclerocytes’ apical thickener cells, Cal2 in basal founder cells, and Cal1 in triactine/tetractine founder cells. C: Sponge wall detail; Cal2 in diactine founder cells, Spiculin in thickener cells. D: Triactine/tetractine founder cells expressing Cal1, thickener cells expressing Spiculin. E: Cal1 expression in founder cells ceases when they transform into thickener cells, and Spiculin expression sets in. Cal1 continues to be expressed in actine-producing founder cells in triactines, but in the diactine actine-forming founder cell, it is replaced by Cal 2 expression in later stages. Scale bars 10 µm. F: Expression of Cal3 in founder cells and Triactinin in thickener cells attached to the preserved diactine (di) and triactine (tri) spicules (overlay of light microcopic picture). Note how thickener cells thinly ensheath the spicule. Scale bar: 50 µm. G: Cal7 expression in the founder cells of oscular diactines. Co-expression of Spiculin and Cal7 rarely occurs in transient stages of emerging thickener cells. Scale bar: 50 µm. H: Early triactine stage with six founder cells expressing Cal7. Scale bar: 50 µm. I: In later triactine formation stages, thickener cells no longer express Cal7. Scale bar: 50 µm. J: Expression of Cal4 and Cal5 in thickener cells. Scale bar: 50 µm. K: Cal6 and Spiculin expression in oscular region. Osc: oscular opening, Expression at the distal end of radial tubes of the body wall (rt). Scale bars= 100 µm, inset 20 µm.
Summary of expression changes of biomineralization genes in sclerocytes (expressing cells in blue).
In inital spicule formation stages, all sclerocytes act as founder cells. Genes with expression patterns that were described peviously (Voigt et al., 2017, 2014) are shown in grey.
Differential gene expression of 13 calcarins and other confirmed or candidate biomineralization genes.
A: Osculum region vs. sponge wall; B: Changes in relative expression during whole-body regeneration.
Arrangements of biomineralization genes (dark blue) and related genes (lighter blue).
A: Calcarins (Cal) in Sycon ciliatum; B: Galaxin (Glx) and Galaxin-like (Glx-l) proteins in the stony coral Acropora millepora; C: Membrane-bound carbonic anhydrases (CA) in Sycon ciliatum. *predicted nested genes not shown.
Alpha-Fold structure predictions of additional selected Sycon ciliatum calcarins.
Expression of biomineralization genes in radial tubes (rt).
A-D: Single channels; E: Overlay of fluorescent channels. Boxes indicate the position of details in F-I. F-I: Details with superimposed sketches to show the original position of dissolved spicules. Triactinin and Spiculin are co-expressed in thickener cells of triactines (F & G). Spiculin additionally occurs in thickener cells of diactines at the distal end of radial tubes (E). Cal1 is expressed in founder cells of both spicule types (F-I). Co-expression of Cal1 and Spiculin marks the transition stage from founder to thickener cell (H & I). Many Spiculin signals at the distal end of radial tubes are not associated with a Cal1 signal, suggesting that Cal1 expression in diactine founder cells ceases before spicule formation is complete (E). In contrast, Cal1 signal is detected in founder cells of late triactine stages (G). BF= bright field, Cal1= Calcarin1, Tria= Triactinin, Spic= Spiculin, rt= radial tube. Scale bar 100 µm. Images processed with LVCC.
A: The radial tube’s distal end shows Cal2 expression in spherical sclerocytes closely associated with the choanoderm (Image processed with LVCC). B-D: Similar expression patterns of Cal2 and Cal6 in distal radial tubes. E: Expression of Cal6 and Spiculin around the oscular opening, F: atrial wall (no diactines) of the same individual lacks Cal6-expressing cells associated with triactine and tetractine thickener cells. Cal: Calcarin, Choa: choanoderm, Pin: pinacoderm, Spic: Spiculin. Scalebar 50 µm (A), 20 µm (B-D), 100 µm (E-F).
Expression of SOM proteins in cells of young Stylophora pistillata polyps.
A. Fourteen of the known SOM proteins (Peled et al., 2020) are specifically overexpressed in calicoblast metacells. Graph obtained from https://sebe-lab.shinyapps.io/Stylophora_cell_atlas/. B. Normalized and scaled expression of 980 calicoblast cells show that several secreted SOM proteins are exclusively expressed by different calicoblast cells, suggesting a spatio-temporal expression regulation as observed in calcareous sponges.
Changes of Module Eigengene expression between low spicule formation transcriptomes and high spicule formation transcriptomes.
Most known biomineralization effector genes occur in MEmidnightblue.
Enriched biological process GO-terms (Treemap from Revigo) of genes in meta module MEmidnightblue.
Domain structure of the only glass sponge (Vazella pourtalesii) protein with a blast hit for a Galaxin query (Dataset S6).
Expression of SOM proteins in adult Stylophora pistillata corals.
A. Most calicoblast metacells did not express known SOM proteins (Peled et al., 2020).Graph obtained from https://sebe-lab.shinyapps.io/Stylophora_cell_atlas/. B. Normalized and scaled expression of the 14 SOM proteins specific to polyp calicoblasts (Fig. S4) in 896 calicoblast cells of adult corals.
Accession numbers or RNAseq data generated for the body part dataset (BioProject PRJEB78728).
Accession for RNAseq data of regeneration experiment from Soubigou et al. (3). Set I and set II are two regeneration experiments followed for 24 days. Additional experiments of the study were not used, because they did not include the first spicule-free stages.
Source of the data used in the OrthoFinder analysis.
Gene-specific primer sequences for generating ISH probes
