Annelid adult cell type diversity and their pluripotent cellular origins

Abstract

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.

Figure 1:. Single cell atlas of adult Pristina leidyi annelids.

A: Experimental workflow. B: UMAP visualisation of the 75,218-cell Pristina leidyi single-cell transcriptomic cell atlas with clusters coloured according to their cell cluster classification. C: UMAP feature plots of markers of the major broad types, including epidermis, gut, muscle, neurons, globin+ cells, polycystin cells, eleocytes, chaetal sacs and lipoxygenase+ cells. D: Lineage reconstruction abstracted graph (PAGA) showing the most probable path connecting the clusters. Each node corresponds to the cell clusters identified with the leiden algorithm. The size of nodes is proportional to the amount of cells in the cluster, and the thickness of the edges is proportional to the connectivity probabilities. E: Cell cluster percentage at the broad cell cluster and the cell cluster levels.

Figure 2:. Pristina leidyi epidermis, muscle, neuron and gut organisation

A: UMAP visualisation highlighting Epidermis (blue), Muscle (red), and Neuron (yellow) clusters. B: Hybridization Chain Reaction (HCR) and UMAP feature plot of expression of epidermis marker PrileiEVm008309t1, showing extensive signal in the epidermal cells across the body, including trunk and tail regions. The bottom left panel is a close up of the top left panel. C: HCR and UMAP feature plot of expression of the neuronal marker PrileiEVm000558t1, showing groups of neuronal cell bodies across the worm’s body, including the head and the stomach region (trunk). The right microscopy panel is a close-up from the middle microscopy panel. D: HCR and UMAP feature plot of expression of the muscle marker PrileiEVm000300t1, showing expression along the worm, including the head and the tail regions shown as examples. The middle microscopy panel is a close-up from the left microscopy panel, evidencing muscle fibres. E: UMAP visualisation highlighting gut and associated clusters, including cell clusters 9, 14, 15, 19, 26, 28, 31 and 39. The colour code matches the colours in the microscopy images. F: General distribution of the marker expression representing each gut region along the worm. For detailed analyses, see Supplementary Figure 7. G: Example HCR sample showing 4 different markers simultaneously, but in distinct regions of the gut (blue, yellow, orange, green). Dashed line indicates the outline of the worm. Circles indicate background signal in the gut. H: UMAP feature plot of expression of diverse gut cluster markers. I: HCR expression of diverse gut cluster markers. In all images anterior is to the left, posterior is to the right. All images are lateral views. Note the strict border between the crop and stomach, where there is no co-expression of the markers. J: UMAP feature plot of expression of anterior and mid intestine marker PrileiEVm010941t1, with expression in cell clusters 9 and 15. K: HCR expression of PrileiEVm010941t1. L: HCR expression of PrileiEVm010941t1 (green) and lumbrokinase+ cell marker PrileiEVm016330t1 (orange), showing non overlapping expression in the same gut region. M: HCR expression of PrileiEVm010941t1 (green) and anterior/mid-intestine marker PrileiEVm008813t1 (pink), showing non overlapping expression in the same gut region. N: HCR expression of PrileiEVm010941t1 and posterior intestine marker PrileiEVm021761t1, showing non overlapping expression in distinct gut regions.In all panels, anterior is left, dorsal is up (unless otherwise noted). Tail in B is ventrolateral. All scale bars are 50 μm unless noted on the figure.

Figure 3:. Annelid specific and novel cell types

A: UMAP visualisation highlighting annelid specific and novel cell types. B: HCR and UMAP feature plot of expression of the ldlrr+ cell marker (cluster 35) PrileiEVm014251t1, showing signal throughout the whole animal body (head, trunk and tail). Detail of the extensions of ldlrr+ cells is shown in the inset of the tail picture. Magenta counterstaining corresponds to eleocytes and nidogen+ cell marker (clusters 7 and 36) PrileiEVm005681t1. C: HCR and UMAP feature plot of lipoxygenase+ cell marker (cluster 17) PrileiEVm000278t1, showing cell expression in the trunk, around the stomach (st), and posterior parts of the animals. D: HCR and UMAP feature plot of globin+ cell marker (clusters 4 and 33) PrileiEVm015446t1, showing expression around the animal’s gut. Magenta staining corresponds to epidermal marker PrileiEVm008309t1. E: HCR and UMAP feature plot of vigilin+ cell markers (cluster 23) PrileiEVm012391t1, in the anterior part of the animal, together with the quantification of cell nuclei area. F: HCR and UMAP feature plots of polycystin cell markers (clusters 10 and 12) PrileiEVm005033t1 and PrileiEVm004079t1, showing the expression of polycystin2+ cells (yellow) segmentally repeated throughout the body wall of the animal and polycystin1+ (magenta) only in the anterior region. G: HCR and UMAP feature plots of chaetal sacs markers (cluster 21 and 22) PrileiEVm000939t1, showing the expression in the fission zone (FZ) and in the tail of the animal. H: HCR and UMAP feature plots of secretory (cluster 34 and 44) and metanephridia (clusters 37) markers PrileiEVm010163t1 and PrileiEVm002621t1. Expression of secretory cells are segmentally repeated mostly ventrally all along the whole body of the animal and metanephridia in some specific segments in midbody and posterior regions. I: HCR and the UMAP feature plot of carbohydrate metabolic cells marker (cluster 29) PrileiEVm001525t1, showing extensive signal in the posterior end of the animal. In all panels, anterior is left, dorsal is up (unless otherwise noted). Head in H is ventrolateral. All scale bars are 50 μm

Figure 4:. The transcriptional landscape of annelid cell type differentiation

A: Expression heatmap of 10,796 genes over 40 WGCNA modules of expression (rows), sorted by cluster expression (columns). B: Summary of Gene Ontology terms associated with example modules. C: Expression heatmap of 958 TFs (rows) sorted by cluster expression (columns). D: UMAP feature plots of TFs associated with individual cell types or broad types. The asterisk points to key differences between TFs. E: Network visualisation of WGCNA modules. In this visualisation, each gene is represented as a dot, coloured according to its cluster of highest expression, and edges represent gene coexpression. F: Module network visualisation summarising coexpression values between different modules, showing associations between different modules. G: Stripplot showing the top central TFs identified in WGCNA modules, and their annotations

Figure 5:. Pluripotent stem cell signature of Pristina piwi+ cells

A: UMAP feature plots of stem cell and proliferation markers, including piwi, nanos, pcna, mcm2, histone h2a, and histone h2b. B: PAGA feature plots of stem cell markers. The graph nodes represent the individual cell clusters and the colour intensity, from dark blue (high) to darkish pink (low), represents the expression of each marker. C: Dot Plot showing the expression of stem cell markers in broad cell types. The colour intensity of the dots represents the mean expression and the size of the dot represents the fraction of cells expressing the marker. Due to the highest expression of histone genes each pair is presented in an individual panel, with its own maximum colour intensity and size. D: PAGA plot coloured according to potency score, ranging from dark blue (low) to yellow (high). E: UMAP visualisation of the 16,247 cells of piwi+ clusters 1, 2 and 8, in their original UMAP embedding (left) and their subclustering UMAP embedding (right) with clusters coloured according to their cell subcluster classification. F: UMAP score plots of markers of piwi+ subclusters 0, 1, 2 and 4, showing differential expression in gut, epidermis, lumbrokinase+, vigilin+ and nidogen+ cell clusters.

Figure 6:. Transcriptomic profile of annelid piwi+ cells

A: UMAP visualisation of scored gene expression of COGs in piwi+ cells. B: Expression heatmap of 200 top TFs expressed in piwi+ cells and their expression in the main broad cell types, showing several clusters of TFs expressed in both piwi+ cells and one or more broad type. C: UMAP feature plots of example TFs coexpressed in piwi+ cells and other broad types. D: Limma differential gene expression analysis of piwi+ cells (clusters 1, 2 and 8) against all other cell types, with annotation lines for the examples indicated in dark green. E: Detail of annotated epigenetic regulators and their expression enriched in piwi+ cells, and example UMAP visualisations of representative epigenetic factors.