Fig. 1: roboA suppresses formation of EPNs and pharynx muscles in the brain.

a Whole-mount in situ hybridization (ISH) of foxA. foxA is expressed in stem cells and pharynx cells; schematic of stem cell lineage is on the right. Stem cells (purple) are distributed across the body, while only a subset surrounding the pharynx expresses foxA (blue). Scale bar = 500 μm. b, c ISH of pharynx neuron marker npp-1 in (b) regenerating head fragments 14 days post-amputation (dpa) and (c) homeostatic animals 14 days post-feeding (dpf). npp-1 expression is restricted to the pharynx in control animals; ectopic pharynx neurons (EPNs) emerge in the head of roboA(RNAi) animals (red arrowheads). Scale bar = 500 μm; timeline shown below. d Quantification of EPNs in homeostatic animals at 14 (n = 8) and 30 dpf (n = 7); ns = not significant; one-way ANOVA with Tukey test. Statistical comparisons of the control and roboA(RNAi) groups were omitted due to zero variance. e ISH of pharynx-specific neuron markers (pkd2-2 and pkd2-3) and muscle markers (mhc-A and laminin) in the brain regions of homeostatic roboA(RNAi) animals, 14dpf. Scale bar = 100 μm. f Quantification of ectopic pharynx-specific cells in the brain regions of homeostatic roboA(RNAi) and control animals. ctrl: n = 5, roboA(RNAi): n = 8 for pkd2-2, n = 5 for pkd2-3, n = 6 for laminin, n = 5 for vitrin, dd_554 and dd_1320. Statistical comparisons of the control and roboA(RNAi) groups were omitted due to zero variance. Box plots show the median (centre line) and interquartile range (box bounds: 25th–75th percentiles). Whiskers extend to the most extreme values within 1.5× the interquartile range. Source data are provided as a Source Data file. g Confocal images of the brain of a homeostatic roboA(RNAi) animal stained with hybridization chain reaction (HCR) for npp-1 (magenta) and laminin (yellow) with DAPI staining (gray). Insets are zooms of the white box. Dashed lines circle nuclei of cells with ectopic signal. Scale bar = 100 µm; zoomed region = 25 µm. h Scatter plot showing numbers of npp-1⁺ and laminin⁺ cells in roboA(RNAi) homeostatic animals from g. Individual dots represent the cell numbers in the brain. i ISH of brain marker (gluR1) and cholinergic neural marker (chat) in homeostatic animals, 14 dpf (n = 10). Scale bar = 500 μm. j Immunostaining for Arrestin (red) in homeostatic animals, 30 dpf, and body fragments with regenerated heads, 14 dpa. White arrowheads highlight defasciculation. Scale bar = 200 μm. k Quantification of phenotypes from j.