Fig 2: TRPM4 expression in GrCs.a Fluorescence image of a slice cut from the cerebellar vermis (220 μm thick) treated with polyclonal antibodies directed against TRPM4 (magenta) and PAX6 (green) and stained with Hoechst to visualize cell nuclei (blue). TRPM4 expression is visible as a thin-outline around GrCs membrane, while PAX6 and Hoechst fluorescence are confined to the nuclear region (dotted arrow). Note the typical rounded shape of the GrCs and the nucleus occuping most of the cytoplasm. Alternatively, either PAX or Hoechst signals dominate nuclear fluorescence (arrows show two examplar cells) (x40 objective magnification, scale bar 5 μm). b A GrC was selected from a (white circle) and shown at x2 magnification (scale bar 10 μm). The upper panels show the acquisition channels separately (TRPM4, PAX6, and Hoechst, respectively), while in the bottom panel the three channels are merged to highlight the coexpression of TRPM4, PAX6, and Hoechst. It should be noted that TRPM4 staining surrounds the nucleus and that some dots can also be observed inside the cell.

Fig 3: (A) WB results about BDNF, AKT1 and PAX6. The data are expressed as the mean ± SD, *indicates that there is a statistical difference between the two groups (t-test, *P < 0.05, **P < 0.01). (B) The activation of astrocytes in the CA1 of hippocampus. A Fluorescence detection of GFAP (green), Nuclear was labelled with DAPI (shown in blue). Quantification of fluorescence intensity of Astrocyte in two groups (t-test, **P < 0.01). The scale bar is 50 μm.

Fig 4: Stabilizing β-CATENIN selectively in the specified ChPe using FoxJ1Cre causes loss of ChPe fate but not transformation to a neuronal fate.a A cartoon illustrating the hem (cyan asterisks), choroid plaque (white asterisks), and choroid plexus at E10.5 and E12.5. b Foxj1Cre::Ai9 marks differentiated choroid plexus at E12.5 but not the choroid plaque at E10.5 or the hem at either stage. Lmx1aCre::Ai9 marks both hem and choroid plexus/plaque starting from E10.5. Boxed regions (b) are shown at high magnification in the adjacent panels. Representative images are shown of sections taken from N = 3 brains (biologically independent replicates) examined over 2 independent experiments. c–e In E16.5 Foxj1Cre::β-cat GOF embryos, LEF1 levels increase (color key: dark blue indicates grey value =0, white indicates grey value =255), while bonafide ChPe markers AQP1(Boxed regions (c) are shown as high magnification insets), TTR, and OTX2 decrease compared with controls, as seen using (c) immunofluorescence and (d) quantification of the number cells positive for each ChPe marker as a fraction of the Ai9-positive cells. A scatter plot shows that in the E16.5 control ChPe, 100% of 500 Ai9+ cells are also OTX2 + , 98.25% of 942 Ai9+ cells are also AQP1 + , and 98.47% of 1168 Ai9+ cells are also TTR + . In Foxj1Cre::β-cat GOF ChP, 91.6% of 731 Ai9+ cells are also OTX2 + , 33.3% of 781 Ai9+ cells are also AQP1 + , and 47% of 1097 Ai9+ cells are also TTR + . (c, d) N = 5 (OTX2) and N = 6 (AQP1 and TTR) brains (biologically independent replicates) for each genotype examined over 3 independent experiments. Bars represent mean ± SEM; p = 0.088919 (OTX2), p = 0.000129 (AQP1), p = 0.000783 (TTR). (e) A violin plot reveals that the nuclear intensity of OTX2 labeling decreases significantly in Foxj1Cre::β-cat GOF ChPe cells compared with controls. n = 536 nuclei from control and 590 nuclei from Foxj1Cre::β-cat GOF ChPe; N = 5 brains (biologically independent replicates) examined over 3 independent experiments; solid black line represents median and dotted lines represents quartiles; statistical test: two-tailed unpaired multiple Student’s t test with unequal variance; p < 0.0001; *p < 0.05; **p < 0.01; ***p < 0.001; ns if p value > 0.05. f In control and Foxj1Cre::β-cat GOF brains, PROX1 immunostaining is seen in the dentate gyrus, PAX6 and TBR2 appear in the telencephalic ventricular and sub-ventricular zones, respectively, and βIII-TUBULIN is present in postmitotic neurons in the dorsal telencephalon. None of these markers appear in the ChPe of either genotype (solid lines), except for a few βIII-TUBULIN positive neurons that normally populate the ChPe. Boxed regions (f) are shown at high magnification in the adjacent panels. Representative images are shown of sections taken from N = 3 brains (biologically independent replicates) examined over 2 independent experiments. Scale bars: 100 μm (all panels in b, c, and f); 10 μm (all panel in c inset). Source data are provided as a Source Data file.

Fig 5: Characterization of primary cultured AS and MSCs. (A) Immunostaining analysis showed that nearly 95% of cells in the primary AS culture were immuno-positive for astrocytic markers GFAP and S-100β, only a small percentage of cells expressed makers for neural progenitor Nestin and PAX6. (B) Statistical analysis showed that the overwhelming majority of cells in the primary AS culture expressed astrocytic markers (n = 3 independent experiments). (C) MSCs were stained with surface markers CD90, CD105, CD45, and CD34 and subjected to flow cytometry analysis. More than 99% of cells are immunoreactive to mesenchymal stem cell markers CD90 and CD105 and negative for hematopoietic markers CD45 and CD34. (D) The osteogenic differentiation of MSCs was evaluated by Alizarin red staining. Note that the black arrows indicate the calcified tubercles. (E) The lipogenic differentiation of MSCs was detected by oil red staining, and the white arrows indicate lipid droplets. Scale bars: 20 µm in (A), 50 µm in (C,D). Numerical data represent mean ± SEM.