Adult pluripotent stem cell (aPSC) populations underlie whole-body regeneration in many distantly-related animal lineages, but how the underlying cellular and molecular mechanisms compare across species is unknown. Here, we apply single-cell RNA sequencing to profile transcriptional cell states of the acoel worm Hofstenia miamia during postembryonic development and regeneration. We identify cell types shared across stages and their associated gene expression dynamics during regeneration. Functional studies confirm that the aPSCs, also known as neoblasts, are the source of differentiated cells and reveal transcription factors needed for differentiation. Subclustering of neoblasts recovers transcriptionally distinct subpopulations, the majority of which are likely specialized to differentiated lineages. One neoblast subset, showing enriched expression of the histone variant H3.3, appears to lack specialization. Altogether, the cell states identified in this study facilitate comparisons to other species and enable future studies of stem cell fate potentials.

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Pluripotent adult stem cell populations underlie whole-body regeneration in many distantly related animal lineages. These collectively pluripotent populations of cells share some features across species, such as the expression of piwi and other germline-related genes. Studies of how these cells operate during regeneration are needed in diverse systems to determine how underlying cellular and molecular mechanisms of renewal and differentiation compare. Here, we sought to characterize stem cells and their dynamics in the acoel Hofstenia miamia, a highly regenerative marine worm with a piwi-expressing stem cell population called neoblasts. Transcriptome profiling at single cell resolution revealed cell types shared across postembryonic stages, including stem cells and differentiated cell types such as neural, epidermal, muscle, and digestive cells. Reconstruction of single-cell differentiation trajectories followed by functional studies confirmed that neoblasts are the source of differentiated cells and identified transcription factors needed for the formation of major cell types. Next, analysis of single-cell transcriptomes from regenerating worms showed that both differentiated cells and stem cells dynamically alter gene expression in response to amputation. Further analysis of the stem cells recovered subpopulations of neoblasts, each with specific transcriptional profiles suggesting that the majority of neoblasts are specialized to differentiated lineages, reflecting putatively lineage-primed progenitors. Notably, neoblast subsets in Hofstenia were identifiable consistently across postembryonic stages and also displayed differential expression dynamics in response to wounding. Altogether, these data suggest that whole-body regeneration is accomplished by the coordination of cells with distinct and dynamic transcriptomic profiles through time. Furthermore, the data generated here will enable the study of how this coordination is achieved, enhancing our understanding of pluripotent stem cells and their evolution across metazoans.

Acoels are marine worms that belong to the phylum Xenacoelomorpha, a deep-diverging bilaterian lineage. This makes acoels an attractive system for studying the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species in which functional genetic studies are possible. We present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large vegetal pole ‘macromeres’ and numerous small animal pole ‘micromeres’. These macromeres become internalized as micromere progeny proliferate and move vegetally. We also noted a second, previously undescribed, cell-internalization event at the animal pole, following which we detected major body axes and tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for mechanistic investigations of acoel development, which will yield insights into the evolution of bilaterian development and regeneration.

Acoels are marine worms that belong to the phylum Xenacoelomorpha. The phylogenetic placement of this group as a deep-diverging lineage makes acoel embryos an attractive system to study the evolution of major bilaterian traits. Thus far, acoel development has not been described in detail at the morphological and transcriptomic levels in a species where functional genetic studies are possible. Here, we present a set of developmental landmarks for embryogenesis in the highly regenerative acoel Hofstenia miamia. We generated a developmental staging atlas from zygote to hatched worm based on gross morphology, with accompanying bulk transcriptome data for each of the stages. Hofstenia embryos undergo a stereotyped cleavage program known as duet cleavage, which results in two large ‘macromeres’ at the vegetal pole and numerous small ‘micromeres’ at the animal pole. The macromeres become internalized as micromere progeny proliferate and move vegetally, enveloping the larger blastomeres. We also noted a second, previously undescribed cell internalization event at the animal pole, following which we detected tissues corresponding to all three germ layers. Our work on Hofstenia embryos provides a resource for future investigations of acoel development, which will yield insights into the evolution of development and regeneration.Summary StatementComprehensive characterization of embryonic development in the acoel worm Hofstenia miamia with accompanying transcriptome data.