Smed-pou4-2 is expressed in the ciliated stripes.

(A) Planarian cartoon illustrates the dorsal and peripheral cell patterns implicated in detecting mechanosensory stimulation in Schmidtea mediterranea. (B) Whole-mount in situ hybridization to pou4-2 revealed a stereotyped pattern depicted in (A), in the dorsal head tip, body periphery, dorsal ciliated stripe (dcs), and additional expression in the ventral nerve cords (vnc). The dashed line represents the cross-section plane shown in the bottom panel. Anterior is to the top. Scale bar = 200 μm. The cross-section image shows pou4-2 expression in the dorsal ciliated stripe (dcs), dorsal and ventral peripheral stripes (pcs), and ventral nerve cords (vnc). Scale bar = 200 µm. (C) Expression analysis of pou4-2. The worms were amputated pre-pharyngeally, and regenerates were fixed at the designated time points. The asterisk highlights the first reappearance of expression within the blastema. The blue arrows at 4 dpa denote the appearance of the anterior dorsal ciliated stripe pattern. Anterior is to the top; dpa, days post-amputation. Scale bar = 200 μm; n ≥ 3 worms tested, with all samples displaying similar expression patterns.

Smed-pou4-2 is positively regulated by soxB1-2.

(A) UMAP representation of soxB1-2+ neuronal subclusters (denoted by numbers) inferred from a scRNA-seq dataset. (B) pou4-2, synapsin, and synaptogamin are all enriched in the soxB1-2+ cluster 8. (C) Heatmap of genes examined in this study demonstrates their differential expression in pou4-2+ cells. (D) soxB1-2 RNAi caused a reduction in sensory neuron-patterned pou4-2 expression in the dorsal ciliated stripe (dcs) and the peripheral ciliated stripe (pcs) but did not affect ventral nerve cord expression (vnc). Anterior to the top. Scale bars = 200 μm; n ≥ 3 worms tested, with all samples displaying similar expression patterns.

Identification of genes regulated by Smed-pou4-2 using RNA-seq.

Volcano plot of differentially expressed genes after pou4-2 RNAi treatment in intact planarians.

pou4-2 is required for expression of pkd1L-2 and hmcn-1-L.

(A) Double-fluorescence in situ hybridization to visualize expression of pou4-2 with pkdL1-2 or hmcn-1-L in sensory neurons; 74.8% and 28.4% of pou4-2+ cells were pkd1L-2+ and hmcn-1-L+, respectively. (B) Planarians were fed twice per week for 4 weeks, amputated pre-pharyngeally one day after the last feeding, and allowed to regenerate for 10 days before fixation. Whole-mount in situ hybridization showed pkd1L-2 and hmcn-1-L expression were drastically decreased in pou4-2(RNAi) worms. Blue arrows denote expression in the dorsal and peripheral ciliated stripes (dcs and pcs, respectively). Note that some pkd1L-2 and hmcn-1-L expression was detectable in regenerates (red arrows). Regions denoted by a numbered dashed box indicate that hmcn-1-L expression was unaffected by pou4-2 RNAi, which is shown in zoomed-in insets. Anterior is to the top. Scale bars = 200 μm; n ≥ 3 worms tested, with all samples displaying similar expression patterns.

Expression analysis of genes co-expressed in Smed-pou4-2+ cells.

(A) WISH images of genes predominantly expressed in the dorsal and peripheral ciliated stripes. WISH post-RNAi revealed reduced expression of mechanosensory neuron-patterned genes (labeled on the left) after soxB1-2 and pou4-2 RNAi (labeled on the top). loxhd-1 was also expressed in a punctate pattern (black arrowheads) that appeared largely unaffected by pou4-2 RNAi. The RNAi treatments did not affect nsun-7 expression in the photoreceptors (blue asterisks). (B) In situ hybridization images from whole-mount and cross-sections of genes expressed in mechanosensory neurons and other cell types. Note reduced expression of mechanosensory neuron-patterned genes after soxB1-2 and pou4-2 RNAi. The red arrowheads highlight the tip cell expression unaffected by soxB1-2 and pou4-2 knockdown in ephA4. The insets show the corresponding cross-section of the worm. Anterior to the left. Blue arrows mark ciliated stripe cell regions. Dashed boxes denote cross-section regions. Abbreviations: cephalic ganglia (cg), dorsal ciliated stripe (dcs), dorsal and ventral peripheral stripes (pcs), epidermis (ep), ventral nerve cords (vnc). Scale bars = 200 µm for intact animals and 100 µm for cross sections; n ≥ 3 worms tested with all samples displaying similar expression patterns.

pou4-2 expression is required for mechanosensory neuron regeneration and function.

(A) Acetylated-tubulin staining revealed decreased cilia labeling along the dorsal ciliated stripe after pou4-2 RNAi. Anterior is to the top. Scale bars = 200 μm, n = 4 worms stained for each of the control and experimental groups. (B) Higher magnification of acetylated tubulin staining of control and pou4-2 RNAi animals. Scale bars = 25 μm. (C) Significant reductions in vibration sensation and rheosensation were observed in pou4-2 RNAi worms. These reduced behaviors following stimulation were consistent in intact and regenerates. Data in C are represented as mean ± SD, and n > 25 worms for each experimental group. ****p < 0.0001, Student’s t-test.

(A) Schematic of RNAi treatment time course used prior to whole-mount in situ hybridization analysis for uninjured and regenerating animals, respectively. Planarians were fed twice per week for 4 weeks, amputated pre-pharyngeally one day after the last feeding, and allowed to regenerate for 10 days before fixation. (B) pou4-2 RNAi caused a reduction in soxB1-2 expression in the dorsal ciliated stripe (dcs). Anterior is to the top. Scale bars = 200 μm; n ≥ 3 worms tested, with all samples displaying similar expression patterns.

Reciprocal RNAi/WISH analysis of pou4-2 and atonal genes.

(A) showed no changes in expression compared to controls. Scale bars = 200 μm (whole worms) and 100 μm (head region); n ≥ 3 worms tested with all samples displaying similar expression patterns (see Supplementary File S6).

pou4-2 is expressed in irradiation-sensitive cells.

(A) Double fluorescent whole-mount in situ hybridization revealed pou4-2 co-expression with pkd1L-2 or hmcn-1-L. White boxed cells zoomed in within insets show high pou4-2 and terminal marker expression and are displayed at higher magnification. White arrowheads point to examples where terminal marker gene expression is much brighter than pou4-2 expression. White arrows mark pou4-2+ cells with low expression of the terminal marker genes. Scale bar = 100 μm. (B) timecourse X-ray exposure experiment to examine pou4-2 expression in presumptive progenitor cells. Double-FISH detection of pou4-2 with a pkd1L-2 and hmcn-1-L riboprobe mix was severely reduced after 5.5 days post-irradiation (dpi). Red arrows highlight cells expressing pou4-2 only. Scale bars = 200 μm. (B-C) Plots of measurements to track the number of pou4-2+/pkd1L-2- hmcn-1-L- cells (B) or pou4-2+/pkd1L-2+ and pou4-2+/hmcn-1-L+(C) per mm2 over days post-irradiation. (D) Spatiotemporal expression of piwi-1 after 100 Gy irradiation along with early-stage epidermal progenitor prog and late-stage epidermal progenitor agat-1. The WISH analysis revealed that pou4-2 shares the same spatiotemporal expression pattern as agat-1.