Regeneration in the absence of canonical neoblasts in an early branching flatworm

Abstract

The remarkable regenerative abilities of flatworms are closely linked to neoblasts - adult pluripotent stem cells that are the only division-competent cell type outside of the reproductive system. Although the presence of neoblast-like cells and whole-body regeneration in other animals has led to the idea that these features may represent the ancestral metazoan state, the evolutionary origin of both remains unclear. Here we show that the catenulid Stenostomum brevipharyngium, a member of the earliest-branching flatworm lineage, lacks conventional neoblasts despite being capable of whole-body regeneration and asexual reproduction. Using a combination of single-nuclei transcriptomics, in situ gene expression analysis, and functional experiments, we find that cell divisions are not restricted to a single cell type and are associated with multiple fully differentiated somatic tissues. Furthermore, the cohort of germline multipotency genes, which are considered canonical neoblast markers, are not expressed in dividing cells, but in the germline instead, and we experimentally show that they are neither necessary for proliferation nor regeneration. Overall, our results challenge the notion that canonical neoblasts are necessary for flatworm regeneration and open up the possibility that neoblast-like cells may have evolved convergently in different animals, independent of their regenerative capacity.

Fig. 1. Regeneration and asexual reproduction in Stenostomum brevipharyngium.

a phylogentic distribution of regenerative abilities, asexual reproduction, and adult pluripotent stem cells in flatworms; Catenulida, which includes S. brevipharyngium, are marked in red. b asexual reproduction by paratomy; br, brain; in, intestine; lnc, longitudinal nerve cord; ph, pharynx; pn, protonephridium; rm, rostral muscles; sp, sensory pits; white bars indicate two zooids. c, anterior regeneration; red dotted lines indicate amputation planes. d–f effects of irradiation on paratomy and survival (d), tissue homeostasis (e), and regeneration (f). Morphological structures (c, e and f) are labeled with arrowheads as follows: dark blue, rostral nerves; green, brain neuropile; red, sensory pits; cyan, rostral muscles; magenta, pharynx; yellow, protonephridium. Asterisks in (d) indicate significant differences from controls (at p-value < 0.05) as inferred with the two-sided Mann–Whitney U test (the exact p-values are provided in Supplementary Data 7). Red bars in (e) indicate the disorganization of posterior tissues. Scale bars on all panels represent 20 μm. Source data for panel d are provided as a Source Data file.

Fig. 2. Effects of irradiation on gene expression and cell division in Stenostomum brevipharyngium.

a Volcano plots showing statistical significance (two-sided Wald test) and magnitude of change for differentially expressed genes (DEG) in response to different irradiation doses. b heat map of the DEG that show statistically significant changes (adjusted p-value < 0.05, two-sided Wald test adjusted with the procedure of Benjamini and Hochberg) at all investigated conditions. c heat map and line plots showing expression level changes of germline multipotency program (GMP) components and cell-division genes in response to irradiation, gray boxes indicate insignificant changes (adjusted p-value > 0.05, two-sided Wald test adjusted with the procedure of Benjamini and Hochberg). d anatomical distribution of EdU⁺ cells. e effects of irradiation on mitotic activity as inferred from EdU and H3P stainings. Center lines show the medians, box limits indicate the 25th and 75th percentiles, and whiskers extend to minimum and maximum values. Bars with asterisks indicate significant differences (at p-value < 0.05) as inferred with the two-sided Mann–Whitney U test (For EdU staining: control vs 300 Gy, p-value = 0.000075984; control vs 5 dpi, p-value = 0.0000071517; control vs 12 dpi, p-value = 0.00006619; for H3P staining: control vs 0 dpi, p-value = 0.00014; control vs 3 dpi, p-value = 0.00000965). Source data are provided as a Source Data file. f prominence of the nucleolus in division-competent cells as visualized with antibody staining against the nucleolar protein fibrillarin. g–i distribution of mitotically active cells (red arrowheads) in the head, epidermis, and protonephridium. MIP stands for maximum intensity projection.

Fig. 3. Single-cell atlas of Stenostomum brevipharyngium.

a a two-dimensional uniform manifold approximation and projection (UMAP) showing cell clusters of S. brevipharyngium. b expression patterns of cluster-specific molecular markers (red), overlayed with nuclear Hoechst staining (white). Scale bars represent 20 μm. The markers used for visualizing anatomical cell type distributions are specified in Supplementary Data 3. The newly identified gene C4M1 (go_Sbre_v1_33587_1_1) was used as a marker for cluster 4. c irradiation sensitivity of particular cell clusters. d–g dotplots showing cell type-specific expression of the components of the germline multipotency program (d, f) and cell division genes (e, g) in Schmidtea mediterranea (d, e) and S. brevipharyngium (f, g).

Fig. 4. Gene expression and division competence in stem cells, somatic tissues, and germline of Stenostomum brevipharyngium.

a–d expression of the stem cell markers derived from single-cell experiments combined with 2 h EdU incorporation. e–g co-expression of epidermal and stem cell markers in dividing cells of the epidermis. h, i EdU⁺ cells (arrowheads) in the protonephridium express the protonephridial marker (h), but not the stem cell marker (i). j mutually exclusive expression of gut and stem cell markers. k–m expression of the germline multipotency program (GMP) components and stem cell markers, the presumptive gonadal anlagen on the dorsal side of the animal express GMP components, piwiA and vasa (arrowheads, k), but not the stem cell marker (l). n cells expressing stem cell markers (arrowheads) but not GMP components are present in the blastema of head-regenerating worms. o elevated expression of GMP components and stem cell markers in gonadal anlagen of lethally irradiated worms. Cell nuclei are counterstained with Hoechst (white). Scale bars on all panels represent 20 μm, if not indicated otherwise.

Fig. 5. Functional requirements of GMP and stem cell marker genes in asexual reproduction and regeneration of Stenostomum brevipharyngium.

a, b validation of the efficiency of the dsRNA-mediated knockdown of piwiA (a) and piwiB (b), cell nuclei are counterstained with Hoechst (white), scale bars represent 20 μm. c–e effects of gene knockdowns on mitotic activity (c), anterior regeneration (d), survival and asexual reproduction (e). Bars with asterisks indicate significant differences (at p-value < 0.05) as inferred with the two-sided Mann–Whitney U test (piwiA RNAi vs eGFP, p-value = 0.00094; piwiA RNAi vs untreated p-value = 0.00094; piwiB RNAi vs eGFP, p-value = 0.01078; piwiB RNAi vs untreated p-value = 0.01078). Asterisks indicate a significant difference from the eGFP RNAi control group (at p-value < 0.05) as inferred with the two-sided Fisher’s exact test (d) and two-sided Mann–Whitney U test (e); the exact p-values for both comparisons are provided in Supplementary Data 10, 11. In the boxplots, center lines represent the median, box limits indicate the 25th and 75th percentiles, and whiskers extend to minimum and maximum values. Source data for (a–e) are provided as a Source Data file.

Fig. 6. Divergence of flatworm stem cell systems in the context of metazoan stem cells and regeneration.

a, b Idealized cross-sections through the trunks of Stenostomum brevipharyngium (a) and Schmidtea mediterranea (b) compare their respective stem cell systems. Arrows indicate the dorso-ventral axis of the animals. S. brevipharyngium relies on a complex stem cell system with multiple division-competent cell types, while in S. mediterranea, all somatic cell types are derived from neoblasts (green) as only division-competent cell type that is molecularly defined by germline multipotency program (GMP) expression. c distribution of the GMP-positive stem cells and regenerative abilities across metazoan phylogeny. The flatworm clade is indicated in green.