Fig. 5: Forced GLI2ΔN expression expands MG stem cell populations and impedes meibocyte differentiation.

a Scheme for bulk RNA-seq of laser-captured MG samples. b GO enrichment analysis of bulk RNA-seq data from GLI2ΔN^Krt5rtTA and littermate control MGs 4 days after induction at 8 weeks of age showing the top 10 enriched pathways. c Volcano plot showing genes upregulated or downregulated at 4 days. Independent biological samples from n = 3 GLI2ΔN^Krt5rtTA mice (2 male and 1 female) and independent biological samples from n = 3 littermate control mice lacking Krt5-rtTA or tetO-GLI2ΔN (2 males and 1 female) were analyzed; differentially expressed genes were defined as padj<0.001 and Log₂FC < -0.5 or Log₂FC > 0.5. FDR calculation was performed by DESeq2 v1.20.0 with the Benjamini-Hochberg procedure. d–g RNAscope showing upregulation of Gli1 and Ccnd1 in GLI2ΔN^Krt5rtTA MGs. h, i IF data showing decreased PPARγ and PLIN2 expression in GLI2ΔN^Krt5rtTA acini. (j-m) RNAscope showing expanded Lrig1 and Lgr6 expression in GLI2ΔN^Krt5rtTA MGs. Independent samples from 3 Gli2ΔN^Krt5-rtTA mice (2 males and 1 female) and 3 littermate controls of genotypes tetO-GLI2ΔN or Krt5-rtTA (2 males and 1 female) were used for RNAscope and IF; all mice were doxycycline-treated for 4 days. (n-s) GLI2ΔN^Krt5-rtTA Rosa26^mTmG mice carrying inducible Cre alleles driven by Lrig1 (n, o), Lgr6 (p, q) or Axin2 (r, s) promoters were tamoxifen-induced at P42 to induce Cre activity and placed on oral doxycycline at P72 to induce GLI2ΔN expression. mGFP expression (red signal) and GLI2 expression (green signal) were analyzed by IF at P74 (n, p, r) or P82 (o, q, s). GLI2ΔN-expressing cells in the acinar basal layer positive for Lrig1 (n), Lgr6 (p), or Axin2 (r) (yellow arrows) give rise to clones that contribute to MG overgrowth (o, q, s, white arrows). n = 3 (1 male and 2 females) samples were analyzed per line per time point. Scale bars: (d–m), 50 μm; (n–s), 25 μm.