Defining compartmentalized stem cell populations with distinct cell division dynamics in the ocular surface epithelium

ABSTRACT Adult tissues contain label-retaining cells (LRCs), which are relatively slow-cycling and considered to represent a property of tissue stem cells (SCs). In the ocular surface epithelium, LRCs are present in the limbus and conjunctival fornix; however, the character of these LRCs remains unclear, owing to lack of appropriate molecular markers. Using three CreER transgenic mouse lines, we demonstrate that the ocular surface epithelium accommodates spatially distinct populations with different cell division dynamics. In the limbus, long-lived Slc1a3CreER-labeled SCs either migrate centripetally toward the central cornea or slowly expand their clones laterally within the limbal region. In the central cornea, non-LRCs labeled with Dlx1CreER and K14CreER behave as short-lived progenitor cells. The conjunctival epithelium in the bulbar, fornix and palpebral compartment is regenerated by regionally unique SC populations. Severe damage to the cornea leads to the cancellation of SC compartments and conjunctivalization, whereas milder limbal injury induces a rapid increase of laterally expanding clones in the limbus. Taken together, our work defines compartmentalized multiple SC/progenitor populations of the mouse eye in homeostasis and their behavioral changes in response to injury.


Figure S1 .
Figure S1.Tamoxifen-inducible Cre for cell fate tracking.(A-D) Immunostaining of wild-type mouse eye on section (A, C) or whole-mounts (B, D).K12 and K19 (magenta) were used as markers of the corneal and conjunctival epithelium, respectively.Hoechst nuclear staining in green.Scale bars: 500 μm.(E) Schematic representation of the tamoxifen-inducible CreER system.(F, G) Scheme for long-term lineage tracing.Schedule of tamoxifen injection (magenta arrowheads) and tissue collection (white arrowheads) is shown.(H-J) K14 CreER , Dlx1 CreER and Slc1a3 CreER without tamoxifen injection.The white line outlines the whole-mount epithelial sheets.Magenta, tdTomato.Green, K12 (corneal marker).Scale bars: 500 μm.(K) Diagram shows the measurement of the length between the peripheral edge of the tdTomato+ clone and the corneal/conjunctival boundary.

Figure S3 .
Figure S3.Lineage tracing of Slc1a3 CreER in the cornea.(A-H) Confocal imaging of representative clones at 2-week-, 1-month-, 3-month-, and 1-year-chases.The areas surrounded by the yellow dashed line in Fig. 2E-H are shown.Images are shown as a maximum-intensity projection (A-D) or confocal sections of the xy, yz, and xz planes of basal clones (E-H; BL, basal layer).The yellow dashed line represents the corneal/conjunctival boundary (A-D).Magenta, tdTomato.Green, Hoechst.Scale bars: 20 μm.(I-N) Mouse eyes were analyzed by whole-mount immunostaining at 1-year-chase.The yellow dashed line indicates the corneal/conjunctival boundary.The white dashed line represents the tdTomato+ cell-enriched area in the fornix conjunctiva.Central corneal areas are shown with higher magnification (J, M) and as a side view of Z-stack confocal images (K, N; BL, basal layer).White arrowheads indicate tdTomato+ radial stripes extended from the limbus.Yellow arrowheads indicate tdTomato+ clones expanded laterally within the limbal region.Magenta, tdTomato.Green, K12 (J, corneal marker).Green, K19 (M, conjunctival marker).Green, Hoechst (I, K, L, N).Scale bars: 500 μm (I, L), 200 μm (J, M), 20 μm (K, N). (O) Immunostaining of wild-type mouse eye on whole-mounts.Magenta, CD31 (blood vessels).Green, K12 (corneal marker).Scale bars: 200 μm.(P) Diagram shows the measurement of the length between the peripheral edge of lateral vessel/capillary vessels and the corneal/conjunctival boundary.(Q) The positions of blood vessels are measured from the boundary.N = 3 mice.Data are shown as means ± SD.

Figure S4 .
Figure S4.Lineage tracing of K14 CreER in the cornea.(A-H) Confocal imaging of representative clones at 2-week-, 1-month-, 3-month-, and 1-year-chases.The areas surrounded by the yellow dashed line in Fig. 3E-H are shown.Images are shown as a maximum-intensity projection (A-D) or confocal sections of the xy, yz, and xz planes of basal clones (E-H; BL, basal layer).Magenta, tdTomato.Green, Hoechst.Scale bars: 20 μm.

Figure S5 .
Figure S5.Lineage tracing of Dlx1 CreER in the cornea.(A-H) Mouse eyes were analyzed by whole-mount immunostaining at 2-week-, 1-month-, 3-month-, and 1-year-chases.Central corneal areas are shown as a maximum-intensity projection (E-H, top).The areas surrounded by white dashed lines are shown as a side view of Z-stack confocal images (E-H, bottom; BL, basal layer).The yellow dashed line indicates the corneal/conjunctival boundary (A-D).Arrowheads indicate tdTomato+ cells (A-H).Magenta, tdTomato.Green, Hoechst.Scale bars: 500 μm (A-D), 200 μm (E-H, top), 20 μm (E-H, bottom).(I) The number of tdTomato+ clones per half whole-mount sample is quantified at indicated time points.One-way ANOVA followed by Bonferroni test.*; P < 0.05.ns: not significant.(J) Box plot showing the distance of tdTomato+ clones from the corneal/conjunctival boundary at indicated time points of chase.N = 3 mice at 2-week-chase, N = 3 mice at 4-week-chase, N = 7 mice at 3-month-chase, and N = 6 mice at 1-year-chase.All tdTomato+ clones in whole-mount samples from a half eye were measured and used for quantification.Data are shown as means ± standard deviation (SD).One-way ANOVA followed by Bonferroni test.ns: not significant.

Figure S6 .
Figure S6.Lineage tracing of Slc1a3 CreER and K14 CreER in the conjunctiva.(A-H) Confocal imaging of representative clones of Slc1a3 CreER in the fornix conjunctiva at 2-week-, 1-month-, 3month-, and 1-year-chases.The areas surrounded by the yellow dashed line in Fig. 4A-D are shown.Images are shown as a maximum-intensity projection (A-D) or confocal sections of the xy, yz, and xz planes of basal clones (E-H; BL, basal layer).Magenta, tdTomato.Green, Hoechst.Scale bars: 20 μm.(I-X) Confocal imaging of representative clones of K14 CreER in the bulbar and palpebral conjunctiva at 2-week-, 1-month-, 3-month-, and 1-year-chases.The areas surrounded by the yellow dashed line in main Fig. 4I-P are shown.Images are shown as a maximum-intensity projection (I-L, Q-T) or confocal sections of xy, yz, and xz planes of basal clones (M-P, U-X; BL, basal layer).Magenta, tdTomato.Green, Hoechst.Scale bars: 20 μm.