Timed Induction of Gli1 (P3 To P30) Was Expressed In Mouse Trabecular Meshwork

Abstract

PurposeThe objective of this project is to determine if Gli1 positive cells contribute to the anterior eye structures including the trabecular meshwork and Schlemm’s canal using an inducible Gli1‐CreERT2; tdTomatoflox (Gli1‐tdTomato) mouse model. Gli1 is downstream of the Sonic hedgehog (Shh) signaling pathway. This pathway is mediated by the Gli transcription factors (Gli1, Gli2, and Gli3) that deferentially activate and repress the expression of specific eye development genes. In addition, Pitx2, FOXC1, and FOXC2 are critical transcription factors for anterior eye development. A second objective is to establish if FOXC1, FOXC2, and Pitx2 are co‐expressed in the Gli1 positive cells.MethodsGli1‐tdTomato expressing mice were induced at different stages with tamoxifen. Eyes were examined that were induced on various embryonic and post‐natal days, P3, P7, and P30 and sacrificed several days to weeks later. Mouse tissues were fixed and prepared for frozen or paraffin sectioning. Tissue antigen retrieval was done using citric acid buffer. Indirect immunohistochemistry for FOXC1, FOXC2, and Pitx2 was completed. Some samples were counterstained with phalloidin. Sections were analyzed with confocal microscopy for spatial distribution of the Gli1+ labeled cells and FOXC1, FOXC2, and Pitx2.ResultsIn Gli1‐tdTomato mice induced on P3, P7, and P30, the periocular mesoderm, trabecular meshwork including Schlemm’s canal, eyelids and optic nerve were positive for Gli1 several days to 3 weeks later. In contrast, the cornea, lens and sclera did not have positive cells for Gli1. Expression levels for FOXC1, FOXC2, and Pitx2 were generally higher in mice harvested at younger ages. After postnatal day 30, the expression decreased with little to no expression of FOXC1 and FOXC2 while Pitx2 expression remained relatively constant in anterior angle tissues.ConclusionThis inducible Gli1‐tdTomato mouse model may be an excellent system for determining the role of Gli1 positive cells in trabecular meshwork and Schlemm’s canal development. The elegance of this model is it can be activated at specific developmental stages and Gli1+ cell derivatives can be tracked and other detection probes (phalloidin, nuclear stains, Immunohistochemistry etc), can be incorporated to determine cell characteristics or over‐all tissue structure. Our results provide evidence that Gli1+ cells are contributing to the development of the anterior angle tissues including trabecular meshwork and Schlemm’s canal.Support or Funding InformationBiomedical Sciences Department Seed Grant (K. Svoboda), NIH K08DE025090 and R21 DE027928 (H. Zhao).