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.

软体动物门（Mollusca）是一类形态多样性极为丰富的后生动物门类，拥有种类繁多的碳酸钙质外壳。外壳的生物矿化过程涉及壳基质蛋白（shell matrix proteins, SMPs）。尽管学界普遍假设壳基质蛋白的多样性驱动了软体动物外壳的多样性，但目前我们才刚刚开始揭开壳基质蛋白演化历史与生物学特性的面纱。本研究采用两个互补的软体动物模式类群——海洋拖鞋螺（Crepidula fornicata）与暗唇履螺（Crepidula atrasolea），以此解析185个履螺属壳基质蛋白的演化谱系。研究发现，成年C. fornicata的外壳蛋白质组中，95%的蛋白属于保守的后生动物和软体动物同源类群，其中软体动物专属同源类群涵盖了外壳蛋白质组中一半的壳基质蛋白。仅存在少量C. fornicata专属的壳基质蛋白，这与"动物的生物矿化工具库主要由新演化而来的基因主导"这一主流观点相悖。随后，我们选取了覆盖不同演化谱系的一组壳基质蛋白，在C. atrasolea的外壳发育过程中，采用原位杂交链式反应（in situ hybridization chain reaction, HCR）开展时空表达分析。结果显示，本次分析的18个壳基质蛋白中，有12个在外壳组织中表达。这些转录本共呈现5种表达模式，至少界定了外壳发生区域内的3种不同细胞类群。本研究结果是目前为止腹足类壳基质蛋白演化年代与外壳发生区域表达模式领域最为全面的分析。综上，本研究的数据为后续探究软体动物外套膜特化与多样性背后的分子机制及细胞命运决定过程奠定了基础。