Anatomical and gene expression resource for Macrostomum lignano

Division Of Basic Sciences - Nci

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Abstract

We are generating a whole-animal single cell (sc)RNA-Sequencing resource for the highly regenerative marine flatworm M. lignano. Vital cells were captured from four developmental stages (1 day, 3 day, 7 day juveniles, sexually mature adults). Juvenile growth, adult tissue homeostasis, and posterior regeneration in M. lignano require somatic stem cells (neoblasts) which have an ill-characterized lineage repertoire encompassing somatic derivatives and germline stem cells. We mapped our sequencing data to the Mlig_RNA_4_5_v5 transcriptome (https://gb.macgenome.org/) using Star Solo to avoid culling multi-mapped reads arising from extensive duplication of the Macrostomum genome, and used Cell Bender to remove empty droplets and low quality cells prior to performing QC, normalization, and clustering using Seurat v5.2.01. We are annotating gene expression clusters using Gene Ontology analysis, as well as bespoke solutions like generating module scores for published cell type-specific marker gene sets and performing cross-species homology-based annotation of M. lignano and S. mediterranea whole-animal data sets using SAMap. To validate assigned annotations and to gain information about clusters with unknown identities, we cloned approximately 300 transcripts that are highly and specifically expressed within clusters, generated riboprobes, and are currently performing whole mount in situ hybridization (WISH) on wildtype adults to determine anatomical site(s) of expression for cluster-enriched genes. Moving forward, we are investigating transcriptional heterogeneity among cycling neoblasts and seek to characterize progenitor populations for major cell types. We will employ trajectory inference algorithms to chart putative differentiation paths, from cycling stem cells to specialized cell types, to construct testable hypotheses about potency regulation and cell-type differentiation programs. This transcriptomic resource will generate needed biomarkers for analysis of neoblast fate decisions and will contribute to comparative evolutionary studies to articulate conserved and novel features of neoblast populations among flatworms and other aquatic invertebrates, as well as commonalities and distinctions between flatworm neoblasts and embryonic or induced pluripotent stem cells in mammalian systems.