Fig. 2

cTEC exhibit a unique morphology that fluctuates during aging and regeneration. Data are from Foxn1[Cre]⁺ Rosa26[Confetti]⁺ thymuses. Blue = Cfp, green = Yfp, red = Rfp. Mouse age was: a–f, 5 weeks, g–i, 12 months, and j is peak regenerated. Comparable panels (e.g., a, i, j) were processed identically. 3D renderings can be found in Supplementary Movies 2–4. a Maximum Z projection of a 40 µm optical stack (the approximate transverse depth of a single cTEC) at 5 weeks. b Zoomed view of the cortex (6 µm Z projection), revealing distinctions between individual cells, in contrast to c, which is conventional cTEC marker expression (Krt8) in the same stack. Scale bar in b also applies to c. d Orthogonal views of a typical cTEC, revealing looping structures forming a complex labyrinth of intracellular voids filled by lymphoid cells (e), as indicated by Thy-1 staining (gray) or DAPI staining (inset, blue). f 2D representation of the interface between two cTEC, showing that most contact occurs via abutment of looping structures, rather than synapses. g Maximum Z projection of a 40 µm-thick optical stack from a 12-month-old mouse. Confetti detection is obscured by autofluorescent pigments associated with aging, but these can be imaged independently (h), and thus subtracted (i; see also Supplementary Fig. 2). Scale bar in g also applies to h and i. Inset in h is at 20× magnification. cTEC in the aged thymus (i) are much less conspicuous than in young (a), reflecting both contraction of total cell dimensions (k–m) and thinning of the remaining projections. j 40 µm Z projection from a 12-month-old mouse on the peak of regeneration (day 20). Confetti intensity is much more robust than in the aged thymus (j), but is not restored to the status of the young (a), with some cells retaining an aged phenotype (atrophic), while others are abnormally large. k–m measurement of relevant physical parameters in cTEC, such as total surface area, feret diameter, or cell volume. Brackets indicate statistical significance (two-tailed t test, p value < 0.05). n = 3 biologically independent tissues each for 5 weeks, 12 months, and regenerated groups. Source data are provided in the accompanying Source Data File