Bulk RNA-sequencing of Meibomian glands from K5-rtTA tetO-GLI2DN mice and littermate controls
The role of Hh signaling in adult Meibomian gland (MG) is unclear. In this study, we overexpressed an activated form of GLI2 (GLI2?N) in basal epithelial cells, including those in the MG, in a doxycycline-dependent manner. Four days post doxycycline treatment, GLI2?N+ MG acinar basal cells expand and replace differentiating PLIN2+ meibocytes. We laser-captured control and GLI2?N+ MG tissues and performed bulk RNA-seq. Our data showed that the most significantly upregulated genes were those involved in regulation of cell proliferation, such as Ki67 and Ccnd1. Expression of Hh target genes Gli1, Ptch1, and Hhip was also significantly elevated in GLI2?N-expressing MGs compared to controls. The most significantly downregulated genes were those associated with control of lipid metabolism such as Pparg, and meibocyte differentiation including Scd3, Scd4, and Plin2. Notably, cell populations expressing MG stem cell markers Lrig1 and Lgr6 were expanded upon GLI2?N expression in MGs. These data suggest that forced GLI2?N expression in MG epithelium promotes proliferation, expands the number of cells expressing Lrig1 and Lgr6, and, either directly or indirectly, suppresses meibocyte differentiation Overall design: Three pairs of control and K5-rtTA tetO-GLI2?N mice were fed with doxycycline water for 4 days. Their eyelids were dissected and frozen sectioned. The MG tissues were laser-captured and collected for RNA extraction. The resulting RNA samples were subjected to bulk RNA-seq.