Distribution of label-retaining cells in the limbal epithelium of a mouse eye

Abstract

Corneal epithelial stem cells are believed to be localized in the limbus, an annular zone between the cornea and the conjunctiva, but it has not been possible to identify individual stem cells in situ because of the lack of specific molecular markers. Description of stem cell distribution has also been ambiguous because limbal boundaries are ill defined. In this study, we investigated whether distribution of slow cycling, label-retaining cells (LRCs) could be determined precisely against a definable anatomical structure of an eye. We found that a boundary between the cornea and the limbus could be determined reliably by distinct epithelial nuclear staining patterns. Using this boundary line as a fiduciary marker, we determined that LRCs were located exclusively in the basal epithelium at the limbal side of the cornea-limbus boundary line along the entire circumference, within an annular zone of 100-200 mum wide. LRC density was highest in the superior temporal quadrant and lowest in the inferior nasal quadrant. These results show that LRCs are present asymmetrically in a narrow zone within the limbus that can be defined precisely in reference to a newly defined anatomical boundary line between the cornea and the limbus.

Figure 1

Anatomical distinction of the cornea, limbus, and conjunctiva by the epithelial nuclear appearance in a CAG-EGFP mouse eye. An ocular surface flat mount was stained with 4′,6-diamidino-2-phenylindole (DAPI) to show nuclear appearance, and the basal epithelium was imaged. (A) Conjunctiva. (B) Limbus. (C) Limbus and cornea. (D) Cornea. Dashed line in C represents approximate position of a cornea–limbus boundary line, based on the nuclear appearance. Bar = 20 μm.

Figure 2

Ocular surface staining patterns with keratins 12 and 15 in reference to a cornea–limbus boundary line. A flat whole mount tissue was processed for double immunofluorescence with keratin 12 (A,D) and keratin 15 (B,E) and also DAPI nuclear staining (F) to show a cornea–limbus boundary line, which is drawn with dashed white lines (D–G). C and G show merged images of keratins 12 and 15. A–C show an entire superior half of the ocular surface (central cornea at the bottom, a lid margin at the top, with slits to mount the tissue flat) from a 30-week-old mouse eye. D–G show a high-power image of the same tissue at an area of the cornea–limbus border. Original images were all in grayscale but converted to pseudo-color for presentation. For D–G, several high-power microscopic fields were patched together to generate each panel. Bar = 50 μm.

Figure 3

Anatomical distinction of the cornea, limbus, and conjunctiva by the epithelial nuclear appearance in a histone H2B-EGFP mouse eye. The basal epithelium of an ocular surface flat-mount was imaged for intrinsic nuclear green fluorescent protein (GFP) (A,C,E,G) or Hoechst 33258 nuclear staining (B,D,F,H). Representative areas of conjunctiva (A,B), limbus (C,D), cornea–limbus border (E,F), and cornea (G,H) are shown. Dashed lines in E and F represent approximate position of a cornea–limbus boundary line. Bar = 20 μm.

Figure 4

Bromodeoxyuridine (BrdU) staining in basal epithelium of the limbus. A and B show basal epithelium of a P4 mouse cornea labeled with BrdU for 4 days and fixed without a chase, double stained for BrdU (A) and DAPI (B). C shows a merged image of DAPI and BrdU. Arrowheads point to all cells that did not label with BrdU in the field. D–F show basal epithelium of the limbus after 4-day BrdU labeling and 9-week chase, double stained for BrdU (D) and DAPI (E). F is a merged image of DAPI and BrdU. Arrows indicate BrdU-positive cells [label-retaining cells (LRCs)], which could be clearly identified and located to the basal epithelium with a manual inspection under the microscope by changing the focal plane slightly up and down and by switching between the BrdU and DAPI channels. Original images were all in grayscale but converted to pseudo-color for presentation. Bar = 20 μm.

Figure 5

Distribution of LRCs along the entire circumference of the limbus, presented as a superior and an inferior half. Each blue dot in the limbus represents an LRC that was manually identified under the microscope. Background is MECA32 immunofluorescence images that depict limbal capillary vessel arcades. Dashed yellow lines along the limbus indicate a cornea–limbus boundary as determined by the DAPI nuclear appearance of high-power images. This is a left eye and a representative of 12 eyes that showed similar patterns. Bar = 500 μm.