Fig 1: Canonical Wnt signaling components are present in the developing human and mouse choroid plexus epithelium.a–g Canonical Wnt signaling pathway molecules FZD1, ß-CATENIN, LEF1, and AXIN2, and also ChPe markers OTX2 and AQP1 are detected at gestational week (GW) 11 (b–e) and GW13 (f, g) in the human choroid plexus epithelium. d, e ß-CATENIN immunohistochemistry intensity measurements across three ChPe cells (identified by colored arrowheads in d corresponding to traces in e). ß-CATENIN intensity is seen in the nucleus, indicative of active canonical Wnt signaling and the boundaries between cells. b–g Representative images of sections taken from 1 fetal brain at each age. h In the E12.5 mouse telencephalon, Wnt3a and Ttr expression identifies the hem the ChPe, respectively. i, j Nuclear localization of ß-CATENIN and its quantitation (Bkgd, background; scatterplot represents mean ± SEM; n = 150 nuclei; N = 3 brains (biologically independent replicates) examined over 3 independent experiments. k Canonical Wnt signaling components Axin2, Fzd1, Fzd2a, and LEF1 are expressed in the E12.5 mouse ChPe. (h, k) Representative images of sections taken from N = 7 brains (biologically independent replicates) examined in 5 independent experiments. Statistical test (j): One Way ANOVA followed by post hoc Dunnett’s multiple comparison test (p < 0.0001), *p < 0.05, **p < 0.01, ***p < 0.001, ns if p value > 0.05. Scale bars: 500 µm (all panels in b and f); 50 µm (all panels in d, i low mag, g, h, and k); 10 µm (d and i high mag). Boxed regions (b, c, d, f, i) are shown at high magnification in the adjacent panels. Further information on replicates and reproducibility for this figure is mentioned in the “Statistics and Reproducibility” section of the Methods. Source data are provided as a Source Data file.
Fig 2: Stabilization of ß-CATENIN leads to activation of canonical Wnt signaling.(a) At E12.5, in situ hybridization for Lmx1a shows endogenous expression; the Ai9 reporter labels the Lmx1a-expressing lineage, including the hem, ChPe, and hem-derived Cajal-Retzius cells. (b, c) At E14.5, nuclear localization of active (non-phosphorylated) ß-CATENIN is increased in the Lmx1aCre::ß-cat GOF ChPe compared with controls (arrowheads) as seen by (b) immunohistochemistry and (c) a violin plot representing quantitation of n = 294 nuclei (control) and n = 301 (GOF); N = 3 brains (biologically independent replicates) for each genotype examined over 2 separate experiments. Boxed regions (b) are shown at high magnification in the adjacent panels. (d) Microdissection of E14.5 Ai9-labeled telencephalic hemispheres to isolate ChP (inset) for RNA extraction. (a, d) Representative images of sections taken from N = 3 brains (biologically independent replicates) examined over 2 independent experiments. (e) Heatmap (showing normalized reads) of Wnt signaling target genes from RNA-seq data from control and Lmx1aCre::ß-Catenin GOF ChP (N = 2 biologically independent replicates). Color key: red (high expression) and blue (low expression) (f–i) In situ hybridization (Axin2), qPCR Axin2 and Lef1, and immunohistochemistry (AXIN2, LEF1, and SOX9) reveal upregulation of AXIN2 and LEF1 and downregulation of SOX9 in the Lmx1aCre::ß-cat GOF ChPe compared with controls. (f, h) Representative images of sections taken from N = 3 brains (biologically independent replicates) examined over 2 independent experiments. Bar graphs (g) represent mean ± SEM, N = 3 brains (biologically independent replicates) for each genotype examined over 2 independent experiments. (i) Violin plots quantifying the nuclear intensity of LEF1 and SOX9, for LEF1: n = 505 nuclei for control and 500 nuclei for Lmx1aCre::ß-cat GOF ChPe, N = 5 brains (biologically independent replicates) for each genotype examined over 3 independent experiments; color key: dark blue (grey value =0) and white (grey value =255). For SOX9, n = 457 nuclei for control and 444 nuclei for Lmx1aCre::ß-cat GOF ChPe; N = 4 brains (biologically independent replicates) examined over 2 independent experiments. For violin plots (c, i) solid black line represents median and dotted lines represent quartiles). Statistical tests: Two-tailed unpaired Student’s t test with unequal variance (c, g), p < 0.0001 (c), p = 0.0005 (g, Lef1) and p = 0.0003 (g, Axin2); two-tailed unpaired multiple Student’s t test with unequal variance (i), p < 0.000001 (i, LEF1 and SOX9). *p < 0.05, **p < 0.01, ***p < 0.001, ns if p value > 0.05. Scale bars: 10 µm (all panels in a, b, f and h); 100 µm (all panels in d). Source data are provided as a Source Data file.
Supplier Page from Abcam for Anti-Axin 2 antibody