Proteomic and Transcriptomic Analyses in the Slipper SnailCrepidulafornicataUncover Shell Matrix Genes Expressed During Adult and Larval Biomineralization

Abstract

The gastropod shell is a composite composed of minerals and shell matrix proteins (SMPs). SMPs have been identified by proteomics in many molluscs, but few have been studied in detail. Open questions include 1) what gene regulatory networks regulate SMP expression, 2) what roles individual SMPs play in biomineralization, and 3) how the complement of SMPs changes over development. These questions are best addressed in a species in which gene perturbation studies are available; one such species is the slipper snail, Crepidula fornicata. Here, SEM and pXRD analysis demonstrated that the adult shell of C. fornicata exhibits crossed lamellar microstructure and is composed of aragonite. Using high-throughput proteomics we identified 185 SMPs occluded within the adult shell. Over half of the proteins in the shell proteome have known biomineralization domains, while at least 10% have no homologs in public databases. Differential gene expression analysis identified 20 SMP genes that are up-regulated in the shell-producing mantle tissue. Over half of these 20 SMPs are expressed during development with two, CfSMP1 and CfSMP2, expressed exclusively in the shell gland. Together, the description of the shell microstructure and a list of SMPs now sets the stage for studying the consequences of SMP gene knockdowns in molluscs.

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

“…From this analysis in C. atrasolea , we described shell field modularity and identified 3 shell field cell populations. We also found 12 SMPs that are expressed in the adult stage of C. fornicata (Batzel et al, 2022), that are also expressed during larval shell development in C. atrasolea , suggesting shared SMP expression in adult and larval mantle epithelia. Leveraging data from both species, we provide a comprehensive analysis of larval SMPs and build a foundation of knowledge in the genetically tractable molluscan model, C. atrasolea .…”

“…1A). In a previous study focusing on Crepidula fornicata (Batzel et al 2022), we identified over 185 SMPs in the adult shell and 20 SMPs differentially expressed in the adult mantle tissue. Of these 20 differentially expressed adult SMPs, at least 10 showed shell field expression during larval development in traditional chromogenic in-situ hybridization.…”

“…In order to determine the evolutionary origins of SMPs and their placement into orthogroups, we performed orthology inferences on proteins from C. fornicata and C. atrasolea proteomes, as well as proteomes of 85 additional species spanning the metazoan phylogeny. Orthology inference was performed using Orthofinder (Emms and Kelly 2019); the 185 SMPs previously identified in the shell proteome of C. fornicata (Batzel et al 2022) were included in the analysis as part of the C. fornicata proteome. Briefly, Orthofinder defines the pairwise similarities between all sequences in the provided proteomes based on length-corrected alignment scores, then uses the resulting similarities to cluster the sequences into orthogroups; genes contained in each orthogroup are considered orthologs (Emms and Kelly 2019).…”

“…In the literature, “shell field” and “larval mantle epithelium” both refer to the larval tissues responsible for shell formation. Larval SMP expression patterns have been examined in molluscan species (Werner et al 2013; Gaume et al 2014; Herlitze et al 2018; Batzel et al 2022), but with minimal cellular resolution due to the limitations of traditional, colorimetric in-situ hybridization. To detect additional SMP expression patterns and SMP-expressing cell populations, a finer cellular and subcellular resolution of signal is required.…”

“…First, we establish C. fornicata and C. atrasolea as complementary models for studying molluscan biomineralization. We used orthology inference to determine the evolutionary age and level of lineage-restriction of 185 SMPs from the adult shell of C. fornicata (Batzel et al 2022). Next, we examined the larval expression patterns of 18 conserved SMPs in C. atrasolea using hybridization chain reaction (HCR) and confocal imaging.…”