Spatial-temporal expression analysis of lineage-restricted shell matrix proteins reveals shell field regionalization and distinct cell populations in the slipper snailCrepidula atrasolea

Abstract

AbstractMolluscs are one of the most morphologically diverse clades of metazoans, exhibiting an immense diversification of calcium carbonate structures, such as the shell. Biomineralization of the calcified shell is dependent on shell matrix proteins (SMPs). While SMP diversity is hypothesized to drive molluscan shell diversity, we are just starting to unravel SMP evolutionary history and biology. Here we leveraged two complementary model mollusc systems,Crepidula fornicataandCrepidula atrasolea, to determine the lineage-specificity of 185CrepidulaSMPs. We found that 95% of the adultC. fornicatashell proteome belongs to conserved metazoan and molluscan orthogroups, with molluscan-restricted orthogroups containing half of all SMPs in the shell proteome. The low number ofC. fornicata-restricted SMPs contradicts the generally-held notion that an animal’s biomineralization toolkit is dominated by mostly novel genes. Next, we selected a subset of lineage-restricted SMPs for spatial-temporal analysis usingin situhybridization chain reaction (HCR) during larval stages inC. atrasolea. We found that 12 out of 18 SMPs analyzed are expressed in the shell field. Notably, these genes are present in 5 expression patterns, which define at least three distinct cell populations within the shell field. These results represent the most comprehensive analysis of gastropod SMP evolutionary age and shell field expression patterns to date. Collectively, these data lay the foundation for future work to interrogate the molecular mechanisms and cell fate decisions underlying molluscan mantle specification and diversification.