Fig. 5

Regeneration of the cortex occurs independently of cTEC proliferation. a Wide area view of a single optical plane of thymus from a 5-week-old mouse in which H2b:mCherry (red) was conditionally activated in TEC by Foxn1[Cre], 1 h after a single injection of the thymidine analog 5-ethnyl-2′-deoxyuridine (EdU, green). EdU⁺ nuclei are not obvious in the deeper tissue because display levels are limited by the intense staining of cortical lymphoblasts, but they are nonetheless found throughout the organ. b, c Similar wide area views from 12 month thymuses, or thymuses from 12-month mice at the midpoint of regrowth (day 14 post-castration), respectively. d–f Higher magnification views of the medulla in thymus from 5 weeks, 12 months, or regeneration day 14. Edu⁺ mTEC nuclei are readily detectable (arrows) in all tissues, although the frequency does decrease with age (g), a feature that does not change after regeneration. Data points represent the percentage of labeled nuclei in individual image volumes from three biologically independent thymuses for each sample type (young, aged, regenerated). Red bars indicate the mean of these percentages. Numbers below sample types indicate pooled EdU⁺ nuclei/pooled total nuclei for each sample type. h–j Similar analysis of the cortex in the same order of appearance. A single Edu⁺ cTEC was found out of 1024 nuclei that were formally counted in multiple volumes from at least 3 tissues) (h, k); many more volumes were examined (but not formally counted) without finding additional labeled cells. No labeled nuclei were found in cTEC from 12 months or regenerating thymus showing that tissue regeneration does not correlate with an increase in cTEC proliferation. l Consistent with the near absence EdU labeling in cTEC, genes associated with cell cycle do not fluctuate during castration-induced regeneration; only one of these (cyclin b1) exhibits a 2-fold or greater change (indicated in gray) during induced regrowth. m For comparison, an equal number of dynamically regulated genes found within the cell projection ontologies (see Fig. 1). Source data are provided in the accompanying Source Data File