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ABSTRACT Over the course of hundreds of millions of years, biomineralization has evolved independently many times across all kingdoms of life. Among animals, the phylum Mollusca displays a remarkable diversity in biomineral structures, particularly the molluscan shell, which varies greatly in shape, size, pigmentation, and patterning. Shell matrix proteins (SMPs) are key components of these shells, and are thought to drive the precipitation of calcium carbonate minerals and influence shell morphology. However, this structure‐function relationship has rarely been studied directly because tools for knocking out genes did not exist in molluscs until recently. In this study, we report the first successful use of CRISPR/Cas9 gene editing to target an SMP in gastropod molluscs. Using the emerging model gastropodCrepidula atrasolea, we generated knockouts of theSMP1gene. Successful gene editing was confirmed by Sanger and MiSeq sequencing, and loss ofSMP1expression was validated through high‐content imaging of crispant embryos. This study establishesC. atrasoleaas a valuable model for investigating the genetic basis of shell formation and provides a framework for applying CRISPR/Cas9 technology in other molluscan species. Our approach will enable future studies to thoroughly test the role of SMPs in shaping the diverse array of molluscan shell structures. 

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 speciesCrepidula fornicataandCrepidula atrasoleabecause they are genetically-enabled mollusc model organisms. First, we established a staging system of shell development inC. atrasoleafor the first time. Next, we leveraged previous findings inC. fornicatacombined with phylogenomic analyses of 95 metazoan species to determine the evolutionary lineage of its adult SMP repertoire. We found that 55% ofC. fornicata’sSMPs 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 ofC. fornicataSMPs that were found conserved inC. atrasoleawere 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 inC. 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 inCrepidulathat enables future comparisons of molluscan biomineralization to reveal mechanisms of shell diversification. 

Abstract ELT-2 is the major transcription factor (TF) required for Caenorhabditis elegans intestinal development. ELT-2 expression initiates in embryos to promote development and then persists after hatching through the larval and adult stages. Though the sites of ELT-2 binding are characterized and the transcriptional changes that result from ELT-2 depletion are known, an intestine-specific transcriptome profile spanning developmental time has been missing. We generated this dataset by performing Fluorescence Activated Cell Sorting on intestine cells at distinct developmental stages. We analyzed this dataset in conjunction with previously conducted ELT-2 studies to evaluate the role of ELT-2 in directing the intestinal gene regulatory network through development. We found that only 33% of intestine-enriched genes in the embryo were direct targets of ELT-2 but that number increased to 75% by the L3 stage. This suggests additional TFs promote intestinal transcription especially in the embryo. Furthermore, only half of ELT-2's direct target genes were dependent on ELT-2 for their proper expression levels, and an equal proportion of those responded to elt-2 depletion with over-expression as with under-expression. That is, ELT-2 can either activate or repress direct target genes. Additionally, we observed that ELT-2 repressed its own promoter, implicating new models for its autoregulation. Together, our results illustrate that ELT-2 impacts roughly 20–50% of intestine-specific genes, that ELT-2 both positively and negatively controls its direct targets, and that the current model of the intestinal regulatory network is incomplete as the factors responsible for directing the expression of many intestinal genes remain unknown.