Temporal and spatial expression patterns of biomineralization proteins during early development in the stony coral Pocillopora damicornis

Abstract

Reef-building corals begin as non-calcifying larvae that, upon settling, rapidly begin to accrete skeleton and a protein-rich skeletal organic matrix that attach them to the reef. Here, we characterized the temporal and spatial expression pattern of a suite of biomineralization genes during three stages of larval development in the reef-building coral Pocillopora damicornis: stage I, newly released; stage II, oral-aborally compressed and stage III, settled and calcifying spat. Transcriptome analysis revealed 3882 differentially expressed genes that clustered into four distinctly different patterns of expression change across the three developmental stages. Immunolocalization analysis further reveals the spatial arrangement of coral acid-rich proteins (CARPs) in the overall architecture of the emerging skeleton. These results provide the first analysis of the timing of the biomineralization 'toolkit' in the early life history of a stony coral.

Keywords: acidic proteins; biomineralization; coral development; gene expression.

Figure 1.

Stages of the brooded P. damicornis larvae used for our comparative analyses [12]. Pictured are: (a) stages I and II, motile larvae and (b) stage III, settled and calcifying spat.

Figure 2.

Gene expression clusters from RNAseq analysis of 3882 DE genes in P. damicornis. VSD, variance stabilizing transformation. (Online version in colour.)

Figure 3.

Gene expression clusters from RNAseq analysis of 74 novel acidic proteins. VSD, variance stabilizing transformation. (Online version in colour.)

Figure 4.

Immunolabelling of P. damicornis larvae (embedded in paraffin) at the three developmental stages studied. Immunohistochemistry of four CARPs (1–4) reveals labelling at distinct intracellular locations for each protein (brown), counterstained with hematoxylin (blue) to show nuclei (insets). Sk, skeleton side; GV, gastrovascular canals; LB, lipid bodies; ME, mesentery; red arrow, aboral epidermis in stage II. Stage II sections are cut on an oblique plane that shows the basal body wall on the left, which is thicker and contains granules that stain brown; on the right is to the thinner surface body wall, which does not have stained cytoplasmic granules (insets). Inset scale bars are all 10 µm.

Figure 5.

Summary of the major findings of our study on gene differential expression (DE) and protein immunolocalization during early development in P. damicornis. (a) The identity of structural proteins with DE during the three stages of coral development, with red text indicating previously described biomineralization toolkit genes; (b) the per cent of novel acidic proteins upregulated in the DE analysis; (c) the relative expression levels of CARPs during development (mean of the triplicate samples following variant stabilizing transformation); letters above indicate the novel acidic protein expression cluster (see figure 3 and electronic supplementary material, table S1); (d) cartoon summary of immunolocalization patterns of CARPs across the three life-history stages; and (e) the proposed roles of these proteins in the animal. CARP 4 and 5 have high protein similarity and our polyclonal antibody epitope region shares 68% identity and 79% similarity; therefore, they may cross-react with the same antibody. VSD, variance stabilizing transformation.