Sox2 and Pax6 Play Counteracting Roles in Regulating Neurogenesis within the Murine Olfactory Epithelium
Fig 10
CreERT2-mediated excision of Sox2 in HBCs reduces clone size and neuron numbers.
Tamoxifen was administered to unlesioned (A) and lesioned (B) control K5CreERT2; Sox2flox/+; R26fl(stop)-LacZ mice, as well as to unlesioned (C) and lesioned (D) experimental K5CreERT2; Sox2flox/flox; R26fl(stop)-LacZ mice; tissue was analyzed three weeks into epithelial recovery. Sections are X-gal-stained to detect β-galactosidase activity within clones, stained for cytokeratin 14 to mark HBCs, and immunolabeled for Sox2 to confirm gene recombination. (A) In the mice that are heterozygous for the floxed Sox2 allele, the excision of one copy of Sox2 had no discernable effect in the absence of lesion. (A’) The inset shows Sox2 labeling in β-galactosidase-marked CK14 (+) basal cells, as expected (double thin arrows). (B, B’) In the heterozygous animals, excision of one copy of Sox2 had no effect on the composition of the clones that arise following MeBr-lesion from the activated HBCs. X-gal-labeled Sus cells maintain their prominent expression of Sox2 following their regeneration from the HBCs that have undergone genetic recombination, as expected (double thin arrows). (C) In the unlesioned, homozygous floxed Sox2 mice, the absence of Sox2 has no apparent effect. (C’) The inset shows the absence of Sox2 labeling in β-galactosidase-marked CK14 (+) basal cells, as expected (single arrows). (D, D’) However, when the Tamoxifen-treated, homozygous floxed-Sox2 mice were exposed to MeBr, the X-gal-stained clones resulting from lesion-induced activation of the HBCs were smaller and contained fewer neurons. X-gal-labeled Sus cells lack their usual prominent expression of Sox2 following their regeneration from the HBCs that have undergone genetic recombination, as expected (single arrows). (E) Comparing lesioned tissue between floxed Sox2 homozygotes and heterozygotes, the range in the number of cells/clone is depicted in the form of a box-and-whiskers scatter plot, and the median clone size is indicated by the horizontal black line within the box. The median value of clone size between heterozygote vs. homozygote is 10 vs. 4, respectively. The difference between homozygotes and heterozygotes is statistically significant (Mann-Whitney U test, *** p < 0.001). (F) Compositional analysis plotting the relative number of different cell types in each clone, reveals that the percentage of neurons (OSNs) is significantly decreased in the floxed Sox2 homozygotes (white bars) compared to heterozygotes (black bars) (Mann-Whitney U-test, *** p < 0.001), while the percentage of HBCs is increased (Mann-Whitney U-test, *** p < 0.001). Scale bar in A is 25 μm and applies to all panels except A’ and C’. The dashed lines mark the basal lamina.