Fig. 5: SOX2⁺ adrenomedullary stem cells promote proliferation of chromaffin cells through secretion of paracrine WNT ligands.

A Featureplots for expression of WNT targets Lef1, Axin2 and Lgr5 in the mouse postnatal adrenal medulla dataset, Key of clusters, grouping by lineage. Colour scale represents Log-normalised expression level, with 0 (grey) representing no expression, and 2.5 (red) for Lef1 and Axin2, or 3 (red) for Lgr5 representing highest expression. B Immunofluorescence using antibodies against TH (chromaffin cells) and GFP (cells that have responded to WNT) on mouse TCF/Lef:H2B-EGFP adrenal medulla at P21. Nuclei are counterstained with Hoechst, scale bars 50 μm. n = 1 female, 1 male, 1 technical replicate. C Dot plots of all Wnt genes expressed in the mouse adrenal medulla dataset (left) and in the isolated SOX2-EGFP⁺ cell dataset (right), subdivided by cell clusters. Colour scale represents average expression level, as a sliding scale of expression magnitude from blue (lowest expression) to red (highest expression). Dot size reflects percentage of cells with gene expression. D RNAscope mRNA in situ hybridisation using probes against Sox2 (red) and Wnt6 (blue), showing co-expression. Image in right is magnified region (i). Nuclei counterstained with hematoxylin. Scale bars 20 μm. n = 2 females, 2 males, 2 technical replicates. E Featureplots for Wls in the mouse adrenal medulla dataset (left) and in the isolated SOX2-EGFP⁺ cell dataset. Colour scale represents Log-normalised expression level, with 0 (grey) representing no expression, and 3 (red) in ‘Adrenal Medulla’ or 1.6 (red) in ‘SOX2-EGFP⁺’ representing highest expression. F Representative immunofluorescence using antibodies against Ki-67 marking cycling cells in Sox2^+/+; Wls^fl/fl (control, top) and Sox2^CreERT2/+;Wls^fl/fl (mutant, bottom) samples following tamoxifen induction at P13/13/15 and analysis at P21 (n = 8 controls, 6 mutants). Nuclei counterstained with Hoechst, scale bars 50 μm. Graph showing percentage of Ki-67 positive cells across replicates, revealing a statistically significant reduction in cycling cells in the mutant. Two-sided unpaired t-test, P-value = 0.0083. Whiskers min to max, the box extends from the 25th to the 75th percentile, line is median. Source data are provided as a Source Data file. G Immunofluorescence staining using antibodies against TH (chromaffin cells, green) and Ki-67 (cycling cells, magenta) in Sox2^+/+;Wls^fl/fl (control) and Sox2^CreERT2/+;Wls^fl/fl (mutant) samples following tamoxifen induction at P13/14/15 and analysis at P21 (n = 3 controls, 3 mutants). Scale bars 50μm. Bar graph showing percentage of Ki-67 positive cells, split into TH positive cells and non-TH positive cells. Two-sided unpaired t-test, P-value = 0.0320. Source data are provided as a Source Data file.