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

Mollusca is a morphologically diverse metazoan phylum, exhibiting an
immense variety of calcium carbonate shells. Biomineralization of the
shell involves 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 molluscan model systems, the marine slipper snails Crepidula
fornicata and Crepidula atrasolea, to determine the evolutionary lineage
of 185 Crepidula SMPs. We found that 95% of the adult C. fornicata shell
proteome belongs to conserved metazoan and molluscan orthogroups, with
molluscan-restricted orthogroups containing half of all SMPs in the shell
proteome. The low number of C. 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 SMPs across
evolutionary lineages for spatial-temporal analysis using in situ
hybridization chain reaction (HCR) during shell development in C.
atrasolea. We found that 12 out of the 18 SMPs we analyzed are expressed
in the shell tissue. These transcripts 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.