Fig. 7: Aged MGs exhibit fewer cells with Hh activity and elevated association of GLI2 with acetyl-lysine.

a Violin plots derived from snRNA-seq data show relatively reduced percentages of cells expressing Gli2 and Ptch1 in the ductule and acinar basal layer, respectively, in aged compared to young MGs. b, c Ptch1 is expressed in acinar basal cells (b, white arrow), some meibocytes (b, yellow arrow), and surrounding stromal cells (b, blue arrow) at 8 weeks; fewer Ptch1-expressing cells are present in aged MG (c). (d, e) Fewer Gli2-expressing cells are present in aged MG particularly in the ductule (d, e, white arrows). f, g RNAscope using a pan-Hh probe which detects both Ihh and Shh mRNAs reveals similar expression of mRNA for Hh ligands in the acini of young (f) and aged (g) MGs. h, i PLA for GLI2 and acetyl-lysine shows that close association (<40 nm) of GLI2 with acetyl-lysine is elevated in subsets of cells within aged (i) compared with young (h) MG acini (i, white arrows) and stroma (i, blue arrows). 3 male C57BL/6 J mice were analyzed at each age in (b-i); representative data are shown. j, k Association of GLI2 and acetyl-lysine is elevated in the acinar basal layer (k, white arrows) and meibocytes (k, yellow arrows) but not stromal cells (j, k, blue arrows) in Krt5-rtTA tetO-Cre Hdac1^fl/fl Hdac2^fl/fl (Hdac1/2^DcKO) mice doxycycline induced from P48 and analyzed at P58 compared to control mice of genotype Hdac1^fl/fl Hdac2^fl/fl and lacking Krt5-rtTA or tetO-Cre. (j). l, m Ptch1 expression in control acinar basal cells (l, white arrows) and differentiating acinar cells (l, yellow arrow) is reduced in HDAC1/2-deficient MG acini (m); stromal Ptch1 expression is similar in mutants and controls (l, m, blue arrows). 3 male mice of each genotype were analyzed in (j-m); representative data are shown. n Violin plots of snRNA-seq data indicate that there is a relatively lower percentage of Hdac1-expressing acinar basal cells (cluster #16) but a higher percentage of Hdac2-expressing acinar basal cells and ductular cells (cluster #17) in aged compared to young MG. o–r IF for HDAC1 (o, p) and HDAC2 (q, r) shows similar expression in young (o, q) and aged (p, r) MGs. 3 male C57BL/6 J mice of each age were analyzed in (o–r); representative data are shown. White dashed lines in (b–m; o–r) outline MG acini and/or ducts. Scale bars: 25 μm. See also Supplementary Fig. S6.