Zhu, X.; Xu, M.; Owens, D. M.; Millar, S. E.

Purpose To investigate whether KRT6A or KRT17 label self-renewing stem cells (SCs) in adult Meibomian gland (MG) and palpebral conjunctiva (PC) homeostasis and explore the mechanisms that regulate their expression in these tissues. Methods KRT6A and KRT17 expression in adult mouse MG and PC was examined by single nucleus RNA sequencing and immunofluorescence (IF). Bromodeoxyuridine pulse-chase assays were conducted to assess the turnover rate of PC progenitor cells. Lineage tracing experiments were performed using Krt6a-CreERT2 and Krt17-CreERT2 mice carrying Rosa26RnTnG or Rosa26RmTmG reporter. As Hedgehog (Hh) signaling, the histone deacetylase HDAC3, and the transcription factor KLF4, regulate KRT6A and KRT17 in other contexts, IF was conducted to assess the in vivo effects of overexpression of the Hh pathway activator GLI2{Delta}N, and inducible epithelial deletion of Hdac3 or Klf4, on KRT6A and KRT17 expression in the MG and PC. Results KRT6A and KRT17 are primarily expressed in the MG central duct and ductules. KRT6A also shows robust expression in PC. MG and PC epithelial progenitor cells turn over within 14 days. Lineage tracing indicated that Krt17 labels self-renewing SCs in the MG whereas Krt6a labels SCs in the PC. GLI2{Delta}N overexpression induced ectopic KRT17 expression in MG acini and PC but did not affect KRT6A expression in either MG or PC. Hdac3 deficiency caused expanded expression of KRT6A and KRT17 in MG acini and ectopic KRT17 expression in PC. Klf4 deletion resulted in ectopic KRT17 expression in PC but did not influence KRT6A expression in MG or PC. Conclusions Krt6a- and Krt17-expressing cells contribute to adult PC and MG homeostasis, respectively. KRT17 expression is enhanced by GLI2{Delta}N, and suppressed by HDAC3 and KLF4, whereas KRT6A expression is controlled only by HDAC3.