Bulk RNA-sequencing of Meibomian glands from K5-rtTA tetO-GLI2DN mice and littermate controls. 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 (GLI2ΔN) in basal epithelial cells, including those in the MG, in a doxycycline-dependent manner. Four days post doxycycline treatment, GLI2ΔN+ MG acinar basal cells expand and replace differentiating PLIN2+ meibocytes. We laser-captured control and GLI2ΔN+ 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 GLI2ΔN-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 GLI2ΔN expression in MGs. These data suggest that forced GLI2ΔN 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-GLI2ΔN 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.

成年睑板腺（Meibomian gland, MG）中Hh信号通路（Hh signaling）的作用尚不明确。本研究以强力霉素依赖的方式，在包括睑板腺上皮在内的基底上皮细胞中过表达激活型GLI2（GLI2ΔN）。强力霉素处理4天后，GLI2ΔN阳性的睑板腺腺泡基底细胞发生扩增，并取代了正在分化的PLIN2阳性睑脂细胞。我们通过激光捕获显微切割获取对照组与GLI2ΔN阳性的睑板腺组织，并开展了批量RNA测序（bulk RNA-seq）。测序结果显示，上调最显著的基因多参与细胞增殖调控，例如Ki67与Ccnd1。与对照组相比，表达GLI2ΔN的睑板腺中，Hh靶基因Gli1、Ptch1与Hhip的表达水平也显著升高。而下调最显著的基因则多与脂质代谢调控（如Pparg）及睑脂细胞分化（如Scd3、Scd4与Plin2）相关。值得注意的是，在睑板腺中表达MG干细胞标志物Lrig1与Lgr6的细胞群在GLI2ΔN表达后发生了扩增。上述结果表明，在睑板腺上皮中强制表达GLI2ΔN可促进细胞增殖、扩增表达Lrig1与Lgr6的细胞群，并通过直接或间接途径抑制睑脂细胞分化。实验整体设计：将3对对照组与K5-rtTA tetO-GLI2ΔN转基因小鼠饲喂含强力霉素的饮水4天，随后摘除其眼睑并进行冰冻切片。通过激光捕获显微切割收集睑板腺组织，提取RNA后将所得RNA样本进行批量RNA测序。