Acoel single-cell atlas reveals expression dynamics and heterogeneity of adult pluripotent stem cells

Abstract

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.

PCP components control anterior and posterior regeneration, with a Prickle homolog impacting muscle organization, in the acoelHofstenia miamia

“…S5C). This localization is consistent with our finding that pk-1 RNAi impacts Egr-GRN members and anterior Wnt pathway genes, many of which are expressed in muscle (Hulett et al, 2023b; Ramirez et al, 2020; Raz et al, 2017).…”

“…Double-stranded RNA (dsRNA) was synthesized following a standard protocol previously established in the lab and described in previous work (Gehrke et al, 2019; Hulett et al, 2023b; Srivastava et al, 2014). RNAi injection experiments were performed by introducing dsRNA corresponding to the target gene into the tail area of the worm for 3 consecutive days.…”

“…Digoxigenin and Fluorescein labeled RNA probes for detection of mRNA expression were synthesized as previously described (Srivastava et al, 2014). Fluorescence in situ hybridizations (FISH) were performed following a detailed protocol established for Hofstenia (Srivastava et al, 2014) followed by minor modifications (Hulett et al, 2023b; Ramirez et al, 2020). Specimens were mounted in Vectashield and whole mounts were analyzed and imaged with a Leica SP8 confocal microscope.…”

Chromatin profiling data indicate regulatory mechanisms for differentiation during development in the acoelHofstenia miamia

“…We also noted multiple motifs that corresponded to transcription factors implicated in epidermal differentiation during regeneration in Hofstenia (e.g. TP63 and NFIC) 21 . The high accessibility of these motifs early in development is consistent with the fact that epidermis is one of the first molecularly discernable lineages 14 .…”

“…To gain higher resolution into what was driving this GO term, we looked at the Hofstenia transcripts that contributed to this enrichment (Figure 4E). These genes putatively bound by Runt included several transcription factors that had been previously validated as functional regulators of cell type differentiation during Hofstenia regeneration 13,21 . These included the epidermal TF dlx (Figure 4F), neural TF sox4 (Figure 4G), and endodermal/gut TF foxA (Figure 4H), which all had binding sites for RUNT.…”