Spicule formation in calcareous sponges: Coordinated expression of biomineralization genes and spicule-type specific genes

Abstract

The ability to form mineral structures under biological control is widespread among animals. In several species, specific proteins have been shown to be involved in biomineralization, but it is uncertain how they influence the shape of the growing biomineral and the resulting skeleton. Calcareous sponges are the only sponges that form calcitic spicules, which, based on the number of rays (actines) are distinguished in diactines, triactines and tetractines. Each actine is formed by only two cells, called sclerocytes. Little is known about biomineralization proteins in calcareous sponges, other than that specific carbonic anhydrases (CAs) have been identified, and that uncharacterized Asx-rich proteins have been isolated from calcitic spicules. By RNA-Seq and RNA in situ hybridization (ISH), we identified five additional biomineralization genes in Sycon ciliatum: two bicarbonate transporters (BCTs) and three Asx-rich extracellular matrix proteins (ARPs). We show that these biomineralization genes are expressed in a coordinated pattern during spicule formation. Furthermore, two of the ARPs are spicule-type specific for triactines and tetractines (ARP1 or SciTriactinin) or diactines (ARP2 or SciDiactinin). Our results suggest that spicule formation is controlled by defined temporal and spatial expression of spicule-type specific sets of biomineralization genes.

Figure 1. Spicule types and spicule formation in S. ciliatum.

(A) Isolated spicules (fluorescence of calcein staining overlayed, showing spicule growth during 18 h12): Di(s) = oscular slender diactines, Di (c) = curved diactines of the distal end of radial tubes, Tri = triactines of the radial tubes and the atrial skeleton, Tet = tetractines of the atrial skeleton. (B) In vivo formation of spicules by sclerocytes (f = founder cell, t = thickener cell). (C) Movement of founder (f) and thickener (t) cells during diactine and triactine formation. (A) and (C) modified from ref. , (C) partially redrawn from ref. .

Figure 2. Expression patterns of biomineralization genes.

(A) Overview over oscular region (atrial side) with SciCA2 expression (blue), SciTriactinin expression (red) and an overlay of a μCT image to show the position of the dissolved spicules. (B) SciNCBT-like1 expression in diactine, triactine and tetractine sclerocytes. Double-ISH with SciTriactinin-specific probes suggests expression in founder cells at later stages of spicule formation. (C) SciAE-like1 expression in diactine, triactine and tetractine sclerocytes. Double-ISH with SciTriactinin-specific probes suggests expression in founder cells at later stages of spicule formation. (D) SciTriactinin expression is specific to triactine and tetractine thickener cells. SciCA1 is only expressed in early stages, SciTriactinin in later stages. Double ISH with SciCA2 reveals that at later stages of triactine and tetractine formation SciCA2 expression only occurs in founder cells. (E) Expression of SciDiactinin in diactine forming sclerocytes. Inset: two close diactine-forming sclerocytes of early diactine formation. (F) SciSpiculin expression in thickener cells of triactines (tetractines not shown) and diactines. Double ISH with SciCA2 reveals that founder cells of diactines are not expressing SciSpiculin, but SciCA2 (radial tubes and oscular diactines). Abbreviations: dia = diactines, sx = sextet of sclerocytes, early stage of triactine (and tetractine) formation (compare Fig. 1C), tri = triactines; tet = tetractines.

Figure 3. Phylogeny of SLC4 proteins.

Maximum Likelihood tree (Phyml, LG + I + G + F), bootstrap values (BS) of 200 replicates and posterior probability (PP) of Bayesian analysis (MrBayes, LG + I + G + F, 5 million generations, burnin = 25% of sampled trees) are provided at the nodes (BS/PP; “*”= 100 BS or PP > 0.96; “** ” = BS of 100 and PP > 0.97; “ <=” support values below 50/0.5, “−” = node not present in Bayesian analysis), value on scale bar = substitutions/site. Biomineralization-specific proteins of S. ciliatum and Stylophora pistillata (SLC4γ) are underlined. The two biomineralization SLC4-proteins of S. ciliatum belong to the NCBT-like and the AE-like group, respectively.

Figure 4. Amino acid sequences of the ARPs SciTriactinin (ARP1), SciDiactinin (ARP2) and SciSpiculin (ARP3).

Aspartic acid and asparagine residues are highlighted by white letters on black background, serine by grey boxes. N-terminal signal peptides are marked by lined boxes, a short shared motif of SciTriactinin and SciDiactinin is marked by a yellow. Potential glycosylation sites are labelled with *(blue = O-linked glycosylation sites, black: N-linked glycosylation sites). Grey letters show parts that were not sequenced from cDNA due to position of the forward primers. For SciTriactinin and SciDiactinin, protein predictions from transcriptome data are presented, the SciSpiculin sequence is provided from combined clone sequence and transcriptome data.

Figure 5. Spatial and temporal expression of seven biomineralization genes.

(A) Schematic view of S. ciliatum body parts that were compared in the RNA-Seq analysis. The green colour depicts spicule formation, which is increased in apical regions, and was deduced from calcein-staining experiments. (B) Expression levels of biomineralization genes and remaining SLC4 proteins in top, middle and bottom parts of S. ciliatum, the colour scale is from blue (lowest) through white (medium) to red (highest). Expression levels were calculated with expected_count from RSEM package, normalized between datasets with the DESeq package and then log 10 transformed. Statistically significantly (padj ≤ 0.1) overexpressed genes in top vs. middle or top vs. bottom comparisons are marked by asterisks. (C) Summary of biomineralization gene expression in founder cells and (prospective) thickener cells of different spicule types, based on observations of the double ISH experiments. In both, tri- and tetractines on the one hand, and diactines on the other hand, the founder cells and prospective thickener cells show initially identical expression patterns. The most dramatic change of expression occurs in later stages in thickener cells, which of the seven genes only expresses SciSpiculin (all spicule types) and SciTriactinin (only triactine- and tetractine-specific thickener cells).