Fig. 3: Rax⁺ tanycytes participate in injury-induced regeneration.

a A schematic diagram of mechanical injury in ME using acupuncture needle. The coronal atlas figure was adapted from “The Mouse Brain in Stereotaxic Coordinates”⁵⁷. b Representative images of smFISH targeting Scn7a mRNA show colocalization between EdU and Scn7a after mechanical injury. Numbers refer to subregions shown in the magnified images. Scale bars: 50 µm (left) and 10 µm (right). c Sample confocal images stained for Sox2, Olig2, EdU, and DAPI in ME of control and injured mice. Three daily EdU injections were applied to label mitotic cells before tissue collection. Scale bar: 50 µm. d Quantification of the mitotic cell number of tanycytes (Sox2⁺Olig2⁻ or Scn7a⁺), OPCs (Sox2⁺Olig2⁺ or PDGFRα⁺), and astrocytes (S100β⁺) in control and injured mice. Boxes represent the interquartile range (IQR), whiskers extend to ±1.5 IQR and bold black lines indicate the median values (n = 19, 10, 16, 12, 19, 10, 13, 15, 21, and 9 sections from at least three mice from left to right boxes). Statistical significance was analyzed by unpaired two-tailed Student’s t test. **P < 0.01; ***P < 0.001. e Representative confocal images showing the triple labeling of tdTomato, EdU and diverse cell markers in the injured ME, suggesting that mechanical injury induces the self-renewal of tanycytes (tdTomato⁺EdU⁺Scn7a⁺) and their differentiation into OPCs (tdTomato⁺EdU⁺PDGFRα⁺) and astrocytes (tdTomato⁺EdU⁺S100β⁺). White boxes show the magnified view of cells pointed by the arrowheads. The adult tanycytes in Rax-CreER^T2::Ai14 mice were labeled by a single injection of tamoxifen, followed by mechanical injury at 7 dpi. The mice received three daily EdU injection at 1 day after injury and were then sacrificed for smFISH analysis or immunostaining. Scale bars: 20 µm. f Mitotic activity detected by EdU labeling in iDTR mice treated with diphtheria toxin (DT) and Rax-CreER^T2::iDTR (abbreviated as Rax::iDTR) mice receiving vehicle or DT injection. Scale bar: 50 µm. g Immunostaining images showing the mitotic activation of Sox2⁺Olig2^- tanycytes after genetic ablation-mediated injury. Arrowheads indicate EdU⁺Sox2⁺Olig2⁻ tanycytes. Scale bar: 10 µm. h Quantification of the number of Sox2⁺Olig2^- tanycytes undergoing cell division after targeted neural injury. Boxes represent IQR, whiskers extend to ±1.5 IQR, and significance was analyzed by one-way ANOVA with Sidak’s multiple comparison test (n = 22, 12, and 18 sections from at least three mice from left to right boxes). ***P < 0.001. i Quantitative analyses showing the expansion of tanycytes, oligodendrocyte lineage cells, and astrocytes after partial tanycyte ablation. Boxes represent IQR, whiskers extend to ±1.5 IQR, dots represent outliers, and significance was analyzed by one-way ANOVA with Sidak’s multiple comparison test (n = 22, 12, 18, 22, 12, 18, 22, 12, 18, 22, 12, 18, 22, 12, 18, 13, 10, 12, 20, 10, 14, 22, 12, and 19 sections from at least three mice from bottom to top boxes). ***P < 0.001; N.S. not significant. Source data are provided as a Source Data file. The precise P values are summarized in Supplementary Data 3.