The distribution and phenotype of Glioma-associated oncogene 1 (Gli1) expressing cells in ocular tissues

Abstract

Purpose : Chronic repetitive injury in the eye can lead to the formation of fibrosis. This can cause alteration to the ocular structure resulting in loss of visual function. At present, there are no medications to prevent or treat this condition. Myofibroblasts are the key drivers of fibrosis. In other organs, recent studies have demonstrated that Glioma-associated oncogene 1 (Gli1) expressing mesenchymal stem cells (MSCs) are precursors to fibrosis-driving myofibroblasts. The purpose of this study is to characterise the distribution and phenotype of Gli1-expressing cells in ocular tissues.

Methods : Mice expressing tdTomato fluorescence in Gli1⁺ cells (Gli1-CreER^T2/tdTomato mice) were generated by crossing Gli1-CreER^T2 animals (Tamoxifen inducible Cre-recombination) with tdTomato Mice (B6.Cg-Gt(ROSA)26Sortm9(CAG-tdTomato)Hze/J). To induce fluorescence, 6–8 week-old Gli1-CreER^T2/tdTomato animals were injected intraperitoneally with Tamoxifen (100mg/kg) once a day for 5 consecutive days. Ten days after the final injection, eyes were enucleated, and processed for ocular flat mounts and cryosection immunofluorescence investigations. Antibodies used included anti-platelet-derived growth factor receptor-β (PDGFRβ), anti-neuron glial antigen 2 (NG2), and anti-β-Tubulin III.

Results : Gli1⁺ cells were readily identifiable in the retina, choroid, ciliary body, and cornea. Flow cytometry indicated that approximately 3-5% and 4-10% of cells in the retina and choroid, respectively, expressed Gli1. In the retina, Gli1⁺ cells were present in the perivascular niche of the superficial, intermediate, and deep vascular plexus. Importantly, a subset of Gli1⁺ cells that were positive for perivascular MSC markers PDGFRβ and NG2 were identified. In the choroid, Gli1⁺ cells were distributed in the stroma and in close association with vasculature. In addition, Gli1+ cells were identified in the stromal layer of the cornea. This included various subsets of cells such as those exhibiting neuronal or stromal morphology. Gli1+ cells that exhibited a neuronal phenotype were also shown to express β-tubulin III.

Conclusions : The cornea, ciliary body, choroid, and retina are rich in Gli1+ cells. A subset of Gli1+ cells in the choroid and retina expressed markers of mesenchymal stem cells. These Gli1+ cells may participate in retinal/choroidal wound healing response and contribute to the development of ocular fibrosis.

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.