Abstract
Purpose :
Schwann cells ensheath and provide trophic support to axons in homeostasis, and they also promote axonal regeneration after an injury. The proteolipid protein 1 (PLP1) is a major component of myelin in the brain, but it is also found in Schwann cells of the peripheral nervous system (PNS). As of now, there are no transgenic reporter mice to study Schwann cells in the cornea. Here, we have investigated the Plp1gene promoter-enhanced green fluorescent protein (EGFP) reporter mice for transgene expression in corneal Schwann cells (cSCs).
Methods :
Both female and male Plp1-egfp mice of different ages (2, 3, 4, and 6.5 months) were used. EGFP expression in the cornea was assessed either immediately after tissue isolation or by corneal whole-mount immunohistochemistry. In some experiments, to reduce background staining, the epithelium was removed by scraping. Briefly, corneas were fixed, blocked/permeabilized, and then stained with antibodies for: L1CAM, bIII-tubulin, and PLP1. Whole mount corneas were analyzed byepifluorescence and confocal microscopy.
Results :
A strong fluorescence was detected along the elongated cell processes of cSCs in transgenic Plp1-egfp mouse corneas at all ages examined. The high degree of transgene expression allowed for clear visualization of EGFP+cSCs even in deep layers of the stroma of freshly isolated corneas examined by epifluorescence microscopy. Fixation, and processing for immunohistochemistry did not affect the intensity of the EGFP signal. Three-dimensional reconstruction of confocalz-stacks showed EGFP+cells enveloping the dense network of bIIItubulin+stromal nerve fibers, from the thick limbal/peripheral nerve fibers to the thinner branches running radially towards the central cornea. EGFP+cells were also co-stained with antibodies against PLP1 and L1CAM. The transcripts of these two SC markers were also found in the cSC cluster identified by single cell RNA-seq analysis of rabbit corneas, which were independently validated using antibodies (R. Mohan et al.,ARVO abstract submitted).
Conclusions :
The Plp1 gene promoter has high specificity for cSCs, as EGFP reporter expression only occurred in these resident glial cells of the cornea. Furthermore, since EGFP fluorescence can be detected in the corneas of live mice, we propose the Plp1-egfp transgenic mouse line as an attractive model suitable for studying cSCs in homeostasis and in injury paradigms.
This is a 2020 ARVO Annual Meeting abstract.