Fig. 3: Fate specification is highly intermingled in planarian stem cells.

a (Top) eye-specialized neoblasts interspersed among non-self neoblasts, which frequently adopt epidermal (middle) or intestinal (bottom) fates. SpNB, specialized neoblast. No ID NB, neoblasts not labeled with assessed FSTFs. Scale bars, (top) 30 µm, (middle and bottom) 10 µm. Representative images from n = 3 animals. b Muscle and protonephridia fates in prepharyngeal regions can be intermingled with neoblasts choosing epidermal fate. Scale bars, 20 µm. Representative images from n = 5 animals. c Directly neighboring neoblasts can make different fate decisions. Scale bars, 5 µm. Representative images from n = 10 animals per comparison. Genes for tissue-specific FSTF pools: epidermis (soxP-3), intestine (hnf-4, gata4/5/6-1), protonephridia (POU2/3, six-1/2-2), muscle (myoD, snail), CNS (pax6A), eye (ovo), pharynx (foxA); see also Supplementary Table 3. d Neighborhood composition analysis of specialized neoblast classes. Stacked bar plots represent percentage of nearest neighbors for sampled cells of a given specialized neoblast query class that are of labeled identities. Ridgeline plots represent percentage of cells in the Voronoi tessellation neighborhoods of sampled cells of a given specialized neoblast query class that are of labeled identities. Top left, middle left, and top right are neoblasts from the pre-pharyngeal region of animals. All other plots are neoblasts from animal tail regions. e Randomized nearest neighbor identity composition for a given query class (n = 1000 simulations) compared to observed nearest neighbor (NN) percentage (red bar). All plots are neoblasts from tail regions. f Cartoon depicting the specification region of eye-specialized neoblasts in homeostasis. Circles represent individual mapped eye-specialized neoblasts and colors represent biological replicates. Data collected from 3 independent animals. g Cartoon depicting the specification region of pharyngeal specialized neoblasts. h Distance measurements of eye-specialized neoblasts to eyes, intestine, CNS, or epidermis. N = 61 from 4 independent animals. i Distance measurements of pharyngeal specialized neoblasts to the pharynx, intestine, CNS, or epidermis. N = 249 from 3 independent animals. j Voronoi neighborhood identity composition of eye-specialized neoblasts. n = 41 pooled from 4 independent animals. Individual neighborhood ridgeline plots available in Supplementary Fig. 24c. k Ridgeline plots of Voronoi neighborhood identity composition for pharyngeal specialized neoblasts. N values for d, e, and k provided in Supplementary Table 4. Two-way ANOVA with Dunnett’s test in h and i, ****p < 0.0001. The central line represents the mean and error bars represent standard deviation in h and i. Mus muscle, Epi epidermal, CNS central nervous system, Int intestine, Neph protonephridia, Phr pharyngeal. Source data are provided as a Source Data file.