The adult shell matrix protein repertoire of the marine snail Crepidula is dominated by conserved genes that are also expressed in larvae

Abstract

Mollusca is a morphologically diverse phylum, exhibiting an immense variety of calcium carbonate structures. Proteomic studies of adult shells often report high levels of rapidly-evolving, 'novel' shell matrix proteins (SMPs), which are hypothesized to drive shell diversification. However, relatively little is known about the phylogenetic distribution of SMPs, or about the function of individual SMPs in shell construction. To understand how SMPs contribute to shell diversification a thorough characterization of SMPs is required. Here, we build tools and a foundational understanding of SMPs in the marine gastropod species Crepidula fornicata and Crepidula atrasolea because they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development in C. atrasolea for the first time. Next, we leveraged previous findings in C. fornicata combined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% of C. fornicata's SMPs belong to molluscan orthogroups, with 27% restricted to Gastropoda, and only 5% restricted at the species level. The low percentage of species-restricted SMPs underscores the importance of broad-taxon sampling and orthology inference approaches when determining homology of SMPs. From our transcriptome analysis, we found that the majority of C. fornicata SMPs that were found conserved in C. atrasolea were expressed in both larval and adult stages. We then selected a subset of SMPs of varying evolutionary ages for spatial-temporal analysis using in situ hybridization chain reaction (HCR) during larval shell development in C. atrasolea. Out of the 18 SMPs analyzed, 12 were detected in the larval shell field. These results suggest overlapping larval vs. adult SMP repertoires. Using multiplexed HCR, we observed five SMP expression patterns and three distinct cell populations within the shell field. These patterns support the idea that modular expression of SMPs could facilitate divergence of shell morphological characteristics. Collectively, these data establish an evolutionary and developmental framework in Crepidula that enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification.

Keywords: Biomineralization; Crepidula; Shell development; Shell matrix proteins.

Fig. 1

Crepidula fornicata and Crepidula atrasolea are complementary model systems for biomineralization. Phylogeny of calcium carbonate biomineralization throughout the Metazoa (A; Gilbert et al. 2022) and the Mollusca (B; Kocot et al. 2020). Triangles indicate taxa that produce calcium carbonate skeletons; squares indicate taxa that have a published biomineral proteome as of 2023; circles indicate taxa that have conducted in situ hybridization for shell matrix proteins (SMPs). Comparison of indirect larval development in C. fornicata vs. direct development in C. atrasolea (C). Adult shells of the two species (D). Organism silhouettes are from PhyloPic (www.phylopic.org) with the exception of Crepidula and Polyplacophora

Fig. 2

Characterization and timing of shell development in Crepidula atrasolea. Dark field and confocal images of fixed C. atrasolea embryos from approximately 8 days post-fertilization (dpf) to approximately 15 dpf at 20 °C. Fixed embryos were stained with wheat germ agglutinin (WGA) and imaged at the 20x objective on a confocal microscope. Cartoons highlight changes in shell morphology during development. In mid ovoid staged embryos (A), WGA stains nuclear membranes throughout the embryo. WGA begins to mark the shell field starting at the late ovoid stage (B). WGA continues to mark the developing shell throughout the organogenesis stages (C-E). WGA also marks the velar lobes, larval kidney, and foot in veliger staged embryos (F-K) Hoechst is shown in gray and WGA in yellow. fr, foot rudiment; ft, foot; hv, head vesicle; lk, larval kidney; in, intestine; ir, intestinal rudiment; oc, ocellus; sf, shell field; sfe, shell field edge; st, stomodeum; tn, tentacles; ve, velar lobes. Scale bars each represent 100 μm

Fig. 3

Shell matrix proteins of Crepidula fornicata clustered into orthogroups. Rows represent SMP orthogroups that were generated through ortholog inference techniques, and ordered based on the taxonomic level to which they are found to be lineage-restricted. Columns depict counts of proteins per taxon with their order (tree depicted above columns) based on current understandings of their phylogenetic positions (Kocot et al. 2011; Laumer et al. 2015; Laumer et al. 2018; Cunha and Giribet 2019). Gray boxes indicate presence of at least one protein from a taxon in an orthogroup. White boxes indicate that no protein was found for a respective taxon in an orthogroup. Green boxes indicate the presence of a skeletal matrix protein in the given orthogroup

Fig. 4

Lineage-restricted shell matrix proteins selected for HCR in Crepidula atrasolea. Rows represent SMP orthogroups that were examined by HCR in C. atrasolea. They are ordered based on the taxonomic level to which they are found to be lineage-restricted (Metazoa-restricted, Gastropoda-restricted, and Crepidula-restricted). Columns depict counts of proteins per taxon with their order (tree depicted above columns) based on current understandings of their phylogenetic positions (Kocot et al. 2011; Laumer et al. 2015; Laumer et al. 2018; Cunha and Giribet 2019). Gray and white boxes indicate presence or absence of respective species within SMP orthogroups. Green boxes indicate the presence of a skeletal matrix protein in the given orthogroup

Fig. 5

Shell field expression of Shell Matrix Protein 1 in Crepidula fornicata and Crepidula atrasolea embryos. Using hybridization chain reaction (HCR), mRNA expression was detected for SMP1 in both species (A-D). Wheat germ agglutinin (WGA) was used to mark the larval shell. CfSMP1 is expressed throughout the shell field in late ovoid staged C. fornicata embryos (A), and more intensely around the shell field edge during organogenesis (B). Similar expression patterns are seen for CaSMP1 in late ovoid staged (C) and organogenesis staged C. atrasolea embryos (D). Hoechst is shown in gray, SMP1 in magenta, and WGA in yellow. fr, foot rudiment; ft, sf, shell field; ve, velar lobes; lk, larval kidney. Scale bars represent 100 μm

Fig. 6

Shell field expression of SMPs in Crepidula atrasolea at organogenesis and veliger stages. Using hybridization chain reaction, mRNA expression was detected for Ca211122 (A and B), Ca163409 (C and D), Ca94322 (E and F), CaSMP10 (G and H), CaSMP4 (I and J), and CaSMP12 (K and L). Hoechst is shown in gray and each SMP in cyan. ft, foot; sf, shell field; sfe, shell field edge; tn, tentacles; ve, velar lobes; ls, larval statocyst; lk, larval kidney. Scale bars in each represent 100 μm

Fig. 7

Summary of regionalized SMP expression during larval shell development in Crepidula atrasolea. Summary table showing the lineage-restriction of each SMP, the best blast hit description, and shell field region(s) of expression (A). Circles in (A) indicate presence or absence of expression in the outer edge (outer Sf), inner edge (inner Sf), and/or the broader shell field (broad Sf). Half circles indicate presence of expression in one stage (late organogenesis or veliger) but not both. Cartoons depicting 5 different SMP expression patterns within the shell field of organogenesis and veliger stage C. atrasolea embryos (B). The SMPs are color coded in (A) based on their expression patterns shown in (B). Cartoons showing regionalization of the shell field into 3 potential zones of biomineralization in late organogenesis and veliger stage C. atrasolea embryo (C). These zones are highlighted in different colors: outer shell field edge in dark blue, inner shell field edge in light blue, and broader shell field in dark gray (C)

Fig. 8

Co-expression of CaSMP3 and CaSMP20 mRNA throughout embryonic shell development in Crepidula atrasolea. Right lateral (A), left lateral (B), and posterior views (C) of late ovoid embryos show co-expression of CaSMP3 (magenta) and CaSMP20 (green) in the shell field. These two expression domains continue during early organogenesis (D-F) and mid organogenesis (G-H). In the veliger stages, laterally asymmetric expression is most apparent in the shell field edge (I-J). Hoechst is shown in gray, CaSMP3 in magenta, and CaSMP20 in green. ft, foot; sf, shell field; sfe, shell field edge; tn, tentacles; ve, velar lobes; ls, larval statocyst. Scale bars each represent 100 μm unless otherwise specified