Tracing the Fate of Limbal Epithelial Progenitor Cells in the Murine Cornea

Abstract

Stem cell (SC) division, deployment, and differentiation are processes that contribute to corneal epithelial renewal. Until now studying the destiny of these cells in a living mammal has not been possible. However, the advent of inducible multicolor genetic tagging and powerful imaging technologies has rendered this achievable in the translucent and readily accessible murine cornea. K14CreERT2‐Confetti mice that harbor two copies of the Brainbow 2.1 cassette, yielding up to 10 colors from the stochastic recombination of fluorescent proteins, were used to monitor K‐14+ progenitor cell dynamics within the corneal epithelium in live animals. Multicolored columns of cells emerged from the basal limbal epithelium as they expanded and migrated linearly at a rate of 10.8 µm/day toward the central cornea. Moreover, the permanent expression of fluorophores, passed on from progenitor to progeny, assisted in discriminating individual clones as spectrally distinct streaks containing more than 1,000 cells within the illuminated area. The centripetal clonal expansion is suggestive that a single progenitor cell is responsible for maintaining a narrow corridor of corneal epithelial cells. Our data are in agreement with the limbus as the repository for SC as opposed to SC being distributed throughout the central cornea. This is the first report describing stem/progenitor cell fate determination in the murine cornea using multicolor genetic tracing. This model represents a powerful new resource to monitor SC kinetics and fate choice under homeostatic conditions, and may assist in assessing clonal evolution during corneal development, aging, wound‐healing, disease, and following transplantation. Stem Cells 2015;33:157–169

Evaluating alternative stem cell hypotheses for adult corneal epithelial maintenance

“…For mouse corneas, the rate of centripetal movement has been estimated as 11-26 µm/d using three different approaches involving direct observations of labelled cells. Corneal epithelial cells labelled with India ink moved 17 µm/d over 7 d [62] , patches of brightly fluorescent cells moved 26 µm/d over 7 wk in mosaic GFP transgenic mice [63] and fluorescent clones of cells extended 11 µm/d over 12 wk in K14-CreERT2;R26R-confetti transgenic mice (from 9 to 21 wk after tamoxifen-activation of the reporter transgene) [64] . Furthermore, the evidence from the mosaic GFP transgenic mice [63] and tamoxifen-activated reporter transgenic mice [64] demonstrated that the same clonal lineage of cells moved across the full radius from the periphery to the centre.…”

“…When Majo et al [1] proposed the CESC cell hypothesis they also proposed that the corneal and conjunctival epithelia continuously expand towards the limbus, which they described as a zone of equilibrium, so any movement in the corneal epithelium was predicted to be centrifugal. This is inconsistent both with the absence of movement in the conjunctiva [62,65] and the convincing, direct evidence that movement of corneal epithelial cells is centripetal not centrifugal [62][63][64] . The evidence for centripetal cell movement in the corneal epithelium is inconsistent with the CESC hypothesis as originally proposed [1] but there is no need to link the stem cell location and movement aspects of the original CESC hypothesis.…”

“…Thus, both the CESC and GBL hypotheses could account for centripetal movement of cells in separately maintained regions on the same radius. However, evidence from transgenic mice shows that a single clone of cells moves across the full radius [63,64] ,…”

“…While this review was in preparation, a similar lineage tracing study was published online, in a preliminary form, and this has already been mentioned in the section on cell movement [64] . This used K14-CreER T2 ; R26R-confetti mice, where the keratin 14 (K14 or Krt14) promoter, rather than a ubiquitous promoter, was used to drive tamoxifen-inducible CreER…”

A Method for Lineage Tracing of Corneal Cells Using Multi-color Fluorescent Reporter Mice

“…Altogether, the Br2.1 cassette allows the random, inducible, and tissue specific expression of one out of four distinct fluorescent genes in each targeted cell. In fact, when a larger number of color combinations is needed, one can generate homozygous mice that contain 2 alleles of Br2.1, thus resulting in the expression of 2 color tags for each cell and in raising the number of possible color combinations to 10 22 . Some of the Br mouse strains enable the expression of a much greater number of possible color combinations since multiple copies of the cassette were integrated into their genome 16 .…”