(A) Experimental design for testing Lrig1 expression by label-retaining cells (LRCs). BrdU was administered repeatedly to newborn Lrig1Cre-ERT2/+;R26RFP/+ mice (green arrows), followed by an injection-free period. Cre-mediated recombination of RFP was induced in Lrig1-expressing cells 24 hr prior to sacrifice (black arrows). (B–B”) A section of the incisor of an adult Lrig1Cre-ERT2/+; R26RFP/+ mouse stained with DAPI (blue), anti-RFP (red) and anti-BrdU (green), 24 hr after administration of Tamoxifen. Lrig1 is expressed by LRCs in the lower part of the labial cervical loop (laCL, white box in B” but not by LRCs in the upper laCL (red box in B”). Lrig1 expression in the proximal mesenchyme divides the mesenchymal BrdU-LRC population into Lrig1-negative (green, open arrowhead), pulp mesenchyme (pM)-specific and Lrig1-positive (yellow, white arrow), periodontal mesenchyme-specific subdomains. (C–C”) Lrig1-positive cells are found in incisor regions marked by Gli1lacZ-expression. Mesenchymal RFP-positive cells are rare in number when compared to Gli1lacZ-positive cells (arrow in C, C’) and absent from Gli1lacZ-expressing mesenchymal cells distal to the laCL and liCL (open arrowheads). Lrig1-positive cells in the laCL express Gli1lacZ (white box). RFP expression is absent from a population of Gli1lacZ-positive cells in the apical portion of the laCL adjacent to T-A cells (red box). (D) Dosing scheme for in vivo lineage tracing of Lrig1-positive cells. (E–H”) Lineage tracing of Lrig1-expressing cells 24 hr (E, E’, E”), 14 days (F, F’, F”), 1 month (G, G’, G”) and 6 months (H, H’, H”) after Tamoxifen induction. Asterisks indicate the absence of labeled epithelial cells. Green arrowheads denote newly formed labeled progeny in the T-A region or amongst differentiated ameloblasts (Am). Yellow arrows denote increase of labeled cells in the periodontal tissue over time. liCL, lingual cervical loop. Dashed lines delimit epithelium. Scale bars: 100 μm, C as in B; B”, C’, C” as in B’; F, G, H as in E; F’, G’, H’ as in E’; F”, G”, H” as in E”.