Planarian stem cells specify fate yet retain potency during the cell cycle

Abstract

Planarian whole-body regeneration is enabled by stem cells called neoblasts. At least some neoblasts are individually pluripotent. Neoblasts are also heterogeneous, with subpopulations of specialized neoblasts having different specified fates. Fate specification in neoblasts is regulated by fate-specific transcription factor (FSTF) expression. Here, we find that FSTF expression is common in neoblast S/G2/M cell-cycle phases but less common in G1. We find that specialized neoblasts can divide to produce progeny with asymmetric cell fates, suggesting that they could retain pluripotency. Furthermore, no known neoblast class was present in all neoblast colonies, suggesting that pluripotency is not the exclusive property of any known class. We tested this possibility with single-cell transplantations, which indicate that at least some specialized neoblasts are likely clonogenic. On the basis of these findings, we propose a model for neoblast pluripotency in which neoblasts can undergo specialization during the cell cycle without loss of potency.

Keywords: cell cycle; cell fate; neoblasts; planarians; potency; regeneration; stem cells.

Figure 1.. FSTFs are less frequently expressed in neoblasts in G1 than in S/G2/M in scRNA-seq data.

(A) tSNE plot of smedwi-1⁺ cells from Fincher et al. Color: cell-cycle annotation. (B) tSNE plot of cells annotated as G1/G0 from part A. Color: cluster identity. (C) Cluster 1 cells highly express canonical neoblast markers, whereas cluster 2–7 express candidate post-mitotic markers. Expression is ln(UMI-per-10,000+1). (D) FSTFs and tissue markers strongly mark post-mitotic-cell clusters, but are rarely expressed by G1 neoblasts. (E) Heatmap of the fraction of cells in each cell-cycle stage that express each gene. See Figure S1E and Methods for quantification details. (F) Pairwise Pearson correlation between genes marking major tissue classes in G1 and S/G2/M cells (see Table S1 and Methods). *, p<0.01; **, p<0.00001; ****, p<0.000001, unpaired t-test. Line, median; box, first and third quartile; whisker, min and max. (G) tSNE plot of smedwi-1⁺ cells from 10X sequencing of 2C-gated cells. Color: cluster identity. (H) Cluster 1 cells express neoblast markers; cluster 2–9 cells express post-mitotic markers. Expression is ln(UMI-per-10,000+1). (I) Heatmap of cell clusters from 1G-H, identified with expression of known cell-type-specific genes. Cluster 1 lacks specific expression of any cell-type marker set. *, not a TF. PN, protonephridia (J) UMAP representation of smedwi-1⁺ cells in 10X sequencing of 2C-gated cells. Left: clusters; Right: gene expression. See also Figure S1 and S2

Figure 2.. FSTFs are less frequently expressed in neoblasts in G1 than in S/G2/M in in situ analysis of FACS-isolated neoblasts.

(A-B) FISH on 2C (G1/G0), intermediate (S), or 4C (G2/M) DNA content cells from FACS. Neoblasts, smedwi-1⁺ (blue); post-mitotic cells, P4HB⁺ (yellow) and low/absent smedwi-1. Numerator, double-positives with TF (magenta); denominator, total number of smedwi-1⁺ (blue) or P4HB⁺ (yellow) cells. (C) Quantification of cell FISH data. Heatmap depicts the fraction of cells in each cell-cycle stage that express each gene. (See Methods for quantification details) (D) FISH on 2C (G1/G0) cells from FACS. Animals were pulsed with F-ara-EdU 12 hours prior to sorting. Scale bars, 10 μm. See also Figure S2.

Figure 3.. Asymmetric and symmetric division outcomes and specialized neoblasts.

(A) FISH with BrdU immunofluorescence. BrdU pulse for 24 hours, starting 4 days post-irradiation. Both symmetric and asymmetric divisions can be identified (B) Cell dyads with a smedwi-1-low, FSTF-high cell directly proximal to a smedwi-1-high, FSTF-low cell. (C) Quantification of outcomes in asymmetric dyads. Denominator, total number of asymmetric dyads for that labeling combination. Outcomes observed: FSTF in smedwi-1-low cell only; FSTF in both cells; FSTF in neither cell (D) zfp-1 expression in symmetric divisions. Top row, asymmetric zfp-1 expression; middle row, symmetric zfp-1 expression; bottom row, no zfp-1 expression. Denominator, the total number of symmetric divisions. (E) Quantification of F-ara-EdU⁺ doublets with prog-2 and zfp-1 expression. Denominator, all doublets with one prog-2⁺ and one prog-2⁻ cell (F) F-ara-EdU⁺ doublet contains one prog-2⁺ and one prox1⁺ cell. Box, inset area. Circle, next closest F-ara-EdU⁺ cell. Left, stitched animal tile scan. (G) Quantification of F-ara-EdU⁺ doublets with prog-2 and prox1 expression. Denominator, all doublets in which one cell is prog-2⁺ and the other cell is prog-2⁻ in this condition. Neg, negative for both prog-2 and prox1. Unlabled scale bars, 10 μm. See also Figure S3

Figure 4.. Known neoblast classes display features consistent with both specialization and fate switching.

(A-B) Frequency of epidermal (zfp-1), muscle (foxF-1), intestine (hnf4), pharynx (foxA), and intestine/neuron (prox1) FSTF expression in colonies is similar to a binomial distribution for each gene. Red line, expected colony count in a binomial distribution; blue line, actual observed counts. p values, Fisher’s exact test. (C) (left) Pie chart of enriched genes in tgs⁺ G2/M neoblasts, compared to tgs-1⁻ G2/M neoblasts. Top 50 genes (SCDE, conservative estimate ranking) analyzed. “Many”, enrichment in multiple tissue types. (right) The same comparison for PLOD1⁺ (muscle-specialized) and ACTB⁺ (intestine/phagocyte specialized) G2/M neoblasts (D) (left) tSNE plot of tgs-1 expression in the cell atlas; most tgs-1 expression is in neurons and neoblasts. Expression in ln(UMI-per-10,000+1). (right) Dotplot of tgs-1 expression in non-neoblast clusters in the cell atlas. (E-F) Cell FISH on cells isolated from the G2/M gate. tgs-1 is frequently co-expressed with neural FSTFs but not with epidermal, intestinal, muscle, or phagocyte FSTFs. Scale bars, 10 μm. See also Figure S4

Figure 5.. No known neoblast class is present in all neoblast colonies.

(A) Expression frequency in colonies for neoblast class markers. No marker is expressed in every colony (B) Frequency of tgs-1 expression in early colonies is similar to a binomial distribution (p = 0.53, not significantly different, Fisher’s exact Test). Red line, expected colony count in a binomial distribution; blue line, actual observed counts. Scale bar, 10 μm. See also Figure S5, S6.

Figure 6.. Specialized neoblasts can be clonogenic.

(A) Cells were isolated by neoblast-like morphology and either screened by qRT-PCR (top), or transplanted into lethally irradiated hosts (bottom). Half of transplant recipients were fixed directly after transplantation, and half were fixed nine days later(B) Heatmap depicting qRT-PCR of selected cells. (C) Animals after single-cell transplantation. Single smedwi-1⁺ cells were found in 16.7% of transplanted animals fixed immediately and smedwi-1⁺ colonies were in 10.6% of animals analyzed late (normalized clonogenic rate of 63.5%) (D) Contingency tables analyzing likelihood that unspecialized neoblasts (top) or tgs-1⁺ cells (bottom) alone account for colony formation rate. It is unlikely that unspecialized neoblasts (p_adj = 8.49e-4) or tgs-1⁺ cells (p_adj = 3.64e-6) can alone explain the rate of colony formation. P value calculated by Fisher’s exact test, followed by Bonferroni multiple hypothesis correction. Scale bars, 10 μm. See also Figure S7.

Figure 7.. Single-step model of neoblast specialization and potency.

A specialized neoblast can divide to give rise to cells of different fates. Non-neoblast daughter cells will terminally differentiate, whereas a daughter cell that remains a neoblast can specialize to a different fate associated with cell cycle progression, allowing specialized neoblasts to remain pluripotent.