Abstract
Purpose :
The absence of limbal epithelial stem cells (LESCs) due to corneal developmental abnormalities often result in the spread of conjunctival cells into the corneal epithelium. This corneal conjunctivalization can leave individuals with a thin and opaque cornea, which may cause them to be functionally blind. Using the Wnt1Cre recombinase system, conditional deletion of transcription factor activating protein 2 beta (AP-2β) from cranial neural crest cells (NCC) results in a condition with phenotypic similarities to the conjunctivalization seen in humans. This investigation intends to gain further insight into the effects of conditional deletion of AP-2β in the NCC on limbal epithelial stem cells and ultimately corneal cell fate determination.
Methods :
The AP-2β NCC KO model was generated using male mice heterozygous for Tfap2b, Tfap2b+/-, and the wnt1-cre transgene, Wnt1Cre+/-. Male mice were bred with female mice possessing a homozygous floxed Tfap2b gene, Tfap2blox/lox. The control group was age-matched wildtype littermates. BrdU (5-Bromo-2-Deoxyuridine) pulse-chase experiments (100µg/g body weight) were conducted in the control and the mutant mice at postnatal day (P) 4 and P27 for a five-day pulse and ten-day chase. Eyes were enucleated from euthanized mice, following the chase period, and processed and embedded in paraffin. Immunohistochemistry (IHC) was conducted using primary antibodies specific to BrdU, as well as Keratin 15 (K15), a marker of the conjunctival epithelium, in addition to the limbal epithelial stem cell marker, ABCG2.
Results :
At both timepoints, the expression of the conjunctival epithelial marker, K15, showed expansion across the cornea in the AP-2β NCC KO mice, when compared to controls. Additionally, there was a significant reduction in BrdU label-retaining cells (LRCs) in the mutant model as compared to controls at P18. Further, there was also a significant reduction in LRCs between the mutant and control groups at P42. The pattern of ABCG2 expression was also reduced in the limbal region of the mutant mice.
Conclusions :
The reduction in BrdU LRCs observed in the AP-2β NCC KO mice, along with the expansion of the conjunctival epithelial marker, K15, and an alteration in the expression of the stem cell marker, ABCG2, suggest that the deletion of AP-2β in the NCC disrupts maintenance of the limbus and LESCs.
This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.