Smed-pou4-2 regulates mechanosensory neuron regeneration and function in planarians
- Department of Biology, San Diego State University, United States
- Center for Computational Biology and Bioinformatics, University of California, San Diego, United States
Figures
Figure 1 with 1 supplement
Smed-pou4-2 is expressed in the ciliated stripes.
(A) Schematic of dorsal and peripheral sensory cell patterns implicated in mechanosensation. (B) Whole-mount in situ hybridization (WISH) of pou4-2 reveals stereotyped mechanosensory neuron expression in the dorsal head tip, body periphery, dorsal ciliated stripe (dcs), and ventral nerve cords (vnc). The dashed line indicates the cross-section plane shown below. Scale bar = 200 μm. (C) Regeneration time course showing reappearance of pou4-2 expression in the blastema beginning at day 3 post-amputation (asterisk), with re-establishment of dorsal stripe expression by day 7. Blue arrows mark the reappearing dorsal ciliated stripe pattern. Anterior is up. Scale bars = 200 µm; n ≥3.
Figure 1—figure supplement 1
Expression of Smed-pou4-2 during development of S. mediterranea.
BLAST in Planosphere cross-referenced dd_Smed_v6_30562_0_1 to SMED30002016 (E-value=0). RNA sequencing data from Davies et al., 2017 show that Smed-pou4-2 expression peaks during stages 5–6, coinciding with organogenesis and axial patterning. Expression levels are comparable to those in mature asexual worms.
© 2017, Stowers. This figure was reprinted from https://planosphere.stowers.org/feature/Schmitea/mediterranea-sexual/transcript/SMED30002016 with permission from Stowers. It is not covered by the CC-BY 4.0 license and further reproduction of this panel would need permission from the copyright holder.
Figure 2 with 2 supplements
Smed-pou4-2 is positively regulated by soxB1-2.
(A) UMAP of soxB1-2+ neuronal subclusters from scRNA-seq data. (B) pou4-2, synapsin, and synaptogamin are enriched in cluster 8. (C) Heatmap of genes examined in this study demonstrates their differential expression in the pou4-2+ cell cluster. (D) soxB1-2 RNAi reduces pou4-2 expression in the mechanosensory dorsal and peripheral ciliated stripes (dcs and pcs) but not in the ventral nerve cords (vnc) or cephalic ganglia (cg). Scale bars = 200 μm; n ≥3 worms tested, with all samples displaying similar expression patterns.
Figure 2—figure supplement 1
Schematic of RNAi treatment and reciprocal expression analysis.
(A) Timeline of RNAi feeding and fixation for WISH in uninjured and regenerating animals. Planarians were fed twice per week for 4 weeks, amputated pre-pharyngeally 1 day after the final feeding, and fixed 10 days post-amputation for WISH analyses. (B) pou4-2 RNAi leads to reduced 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.
Figure 2—figure supplement 2
Knockdown of atonal genes does not alter pou4-2 expression in regenerating animals.
Scale bars = 200 μm; n ≥3 worms per group (see Supplementary file 6).
Figure 3 with 1 supplement
Identification of genes regulated by Smed-pou4-2 using RNA-seq.
(A) Volcano plot of genes differentially expressed in pou4-2(RNAi) animals compared to controls (FC ≥1.4, p-adj ≤0.1). A subset of genes examined in this study is highlighted on the plot and demonstrates significant downregulation. (B) Co-localization analysis by double-fluorescence in situ hybridization reveals that 74.8% of pou4-2+ cells co-express pkd1L-2, and 28.4% co-express hmcn-1-L. White boxed cells shown in 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 high expression of pou4-2 and low expression of the terminal marker genes. Scale bar = 100 μm. (C) WISH of pkd1L-2 and hmcn-1-L in control and pou4-2(RNAi) 10 day regenerates. Terminal marker expression is strongly reduced in RNAi animals. Numbered red boxes demonstrate a population of scattered hmcn-1-L+ cells that persist following pou4-2 RNAi and are shown in corresponding zoomed-in insets. 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 (magenta arrows). Anterior is to the top. Scale bars = 200 μm; n ≥3 worms tested, with all samples displaying similar expression patterns.
Figure 3—figure supplement 1
Irradiation reveals that pou4−2+cells include progenitors.
(A) Time-course analysis following 100 Gy X-ray exposure shows progressive loss of pou4-2+/terminal marker- presumptive progenitor cells. Double FISH with a combined pkd1L-2/hmcn-1-L riboprobe reveals reduced labeling by 5.5 days post-irradiation (dpi). Red arrows mark pou4-2+ cells lacking terminal marker expression. Scale bars = 200 μm. (B, C) Quantification of pou4-2+/pkd1L-2- hmcn-1-L- cells (B) or pou4-2+/pkd1L-2+ + pou4-2+/hmcn-1-L+ (C) per mm2 dpi. (D) WISH analysis of piwi-1, prog-1, and agat-1 post-irradiation reveals that the pou4-2+/terminal marker- putative progenitors share a spatiotemporal depletion pattern with late progenitor marker agat-1+ cells.
Figure 4
Expression analysis of genes co-expressed in Smed-pou4−2+cells.
(A) WISH images of genes predominantly expressed in mechanosensory neuron regions, dorsal and peripheral ciliated stripes (dcs, pcs). 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 following 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 genes in the stereotypical mechanosensory neuron regions after soxB1-2 and pou4-2 RNAi. The red arrowheads highlight the head tip expression unaffected by soxB1-2 and pou4-2 knockdown in EphR1-labeled cells. 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.
Figure 5
pou4-2 expression is required for mechanosensory neuron regeneration and function.
(A) Acetylated-tubulin staining shows decreased cilia signal in the dorsal ciliated stripe of pou4-2(RNAi) animals. Scale bars = 200 μm, n = 4 worms stained for each of the control and experimental groups. (B) Higher magnification confirms stripe reduction; epidermal and ventral cilia are unaffected. Scale bars = 25 μm. (C) Vibration response assay demonstrating body contractions in wild-type animals following tapping stimulus. (D, E) Quantification of vibration response assay data shows significantly reduced contraction responses in both intact (D) and regenerate (E) pou4-2(RNAi) animals. Data in D and E are represented as mean ± SD; n ≥25 worms for each experimental group. ****p<0.0001, Student’s t-test. (F) Model: pou4-2 acts downstream of soxB1-2 in regulating mechanosensory neuron differentiation.
Author response image 1
In this graph, samples labeled “G” represent four biological controls of gfp(RNAi) control animals, and samples labeled “P” represent four biological controls of pou4-2(RNAi)animals at day 12 in the RNAi protocol.
Additional files
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Supplementary file 1
Gene IDs for transcripts enriched in scRNA-seq soxB1−2+neuronal clusters shown in Figure 2A.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp1-v1.xlsx
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Supplementary file 2
Gene IDs for transcripts shown in Figure 2C heatmap.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp2-v1.xlsx
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Supplementary file 3
Differentially expressed genes in pou4-2 RNAi planarians plotted in Figure 3A.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp3-v1.xlsx
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Supplementary file 4
Gene Ontology analysis of downregulated genes in pou4-2 RNAi planarians.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp4-v1.xlsx
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Supplementary file 5
Primer sequences, eBlock sequences, and accession numbers for genes analyzed in this study.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp5-v1.xlsx
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Supplementary file 6
Replicate numbers of animals used for RNAi experiments reported in this study.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp6-v1.xlsx
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Supplementary file 7
Cell quantification details and total number of cells counted for Figure 3B.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp7-v1.xlsx
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Supplementary file 8
Cell quantification details and total number of cells counted for Figure 3—figure supplement 1A–B.
- https://cdn.elifesciences.org/articles/107718/elife-107718-supp8-v1.xlsx
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MDAR checklist
- https://cdn.elifesciences.org/articles/107718/elife-107718-mdarchecklist1-v1.docx
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Smed-pou4-2 regulates mechanosensory neuron regeneration and function in planarians
eLife 14:RP107718.
https://doi.org/10.7554/eLife.107718.3
