Annelid adult cell type diversity and their pluripotent cellular origins.

Many annelids can regenerate missing body parts or reproduce asexually, generating all cell types in adult stages. However, the putative adult stem cell populations involved in these processes, and the diversity of cell types generated by them, are still unknown. To address this, we recover 75,218 single cell transcriptomes of the highly regenerative and asexually-reproducing annelid Pristina leidyi. Our results uncover a rich cell type diversity including annelid specific types as well as novel types. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. We find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors, in piwi+ cells. Finally, lineage reconstruction analyses reveal computational differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids by single cell transcriptomics and suggest that a piwi+ cell population with a pluripotent stem cell signature is associated with adult cell type differentiation.

ACME Dissociation-Fixation, Flow Cytometry, and Cell Sorting of Freshwater Planarian Cells.

Planarian cell dissociation methods using enzymatic approaches are well established and have been widely used in the field. However, their use in transcriptomics and especially single-cell transcriptomics raises concerns as cells are dissociated alive, and this induces cellular stress responses. Here we describe a protocol for planarian cell dissociation using ACME, a dissociation-fixation approach based on acetic acid and methanol. ACME-dissociated cells are fixed, can be cryopreserved, and are amenable to modern methods of single-cell transcriptomics.

Annelid adult cell type diversity and their pluripotent cellular origins.

Annelids are a broadly distributed, highly diverse, economically and environmentally important group of animals. Most species can regenerate missing body parts, and many are able to reproduce asexually. Therefore, many annelids can generate all adult cell types in adult stages. However, the putative adult stem cell populations involved in these processes, as well as the diversity of adult cell types generated by them, are still unknown. Here, we recover 75,218 single cell transcriptomes of Pristina leidyi, a highly regenerative and asexually-reproducing freshwater annelid. We characterise all major annelid adult cell types, and validate many of our observations by HCR in situ hybridisation. Our results uncover complex patterns of regionally expressed genes in the annelid gut, as well as neuronal, muscle and epidermal specific genes. We also characterise annelid-specific cell types such as the chaetal sacs and globin+ cells, and novel cell types of enigmatic affinity, including a vigilin+ cell type, a lumbrokinase+ cell type, and a diverse set of metabolic cells. Moreover, we characterise transcription factors and gene networks that are expressed specifically in these populations. Finally, we uncover a broadly abundant cluster of putative stem cells with a pluripotent signature. This population expresses well-known stem cell markers such as vasa, piwi and nanos homologues, but also shows heterogeneous expression of differentiated cell markers and their transcription factors. In these piwi+ cells, we also find conserved expression of pluripotency regulators, including multiple chromatin remodelling and epigenetic factors. Finally, lineage reconstruction analyses reveal the existence of differentiation trajectories from piwi+ cells to diverse adult types. Our data reveal the cell type diversity of adult annelids for the first time and serve as a resource for studying annelid cell types and their evolution. On the other hand, our characterisation of a piwi+ cell population with a pluripotent stem cell signature will serve as a platform for the study of annelid stem cells and their role in regeneration.

Smed-egfr-4 is required for planarian eye regeneration.

Planarians are remarkable organisms that can regenerate their entire body from a tiny portion thereof. This capability is made possible by the persistence throughout the lifespan of these animals of a population of pluripotent stem cells known as neoblasts. Planarian neoblasts include both pluripotent stem cells and specialized lineage-committed progenitors that give rise to all mature cell types during regeneration and homeostatic cell turnover. However, little is known about the mechanisms that regulate neoblast differentiation. A recent study demonstrated that Smed-egfr-1, a homologue of the epidermal growth factor receptor (EGFR) family, is required for final differentiation, but not specification, of gut progenitor cells into mature cells. Given the expression by planarians of several EGFR homologues, it has been proposed that these homologues may have diverged functionally to regulate the differentiation of distinct cell types in these animals. In this study, we investigated the role of Smed-egfr-4 in eye regeneration. Compared with controls, animals in which this gene was silenced by RNA interference (RNAi) regenerated smaller eyes. Moreover, the numbers of both mature eye cell types, photoreceptor neurons and cells of the pigment cup, were significantly reduced in Smed-egfr-4(RNAi) animals. By contrast, these animals exhibited an increase in the numbers of eye progenitor cells expressing the specific markers Smed-ovo and Smed-sp6-9. These results suggest that Smed-egfr-4 is required not for the specification of eye progenitor cells but for their final differentiation, and support the view that in planarians the EGFR pathway might play a general role in regulating the differentiation of lineage-committed progenitors.