Lrig1 Expression Defines a Distinct Multipotent Stem Cell Population in Mammalian Epidermis

Summary Lrig1 is a marker of human interfollicular epidermal stem cells and helps maintain stem cell quiescence. We show that, in mouse epidermis, Lrig1 defines the hair follicle junctional zone adjacent to the sebaceous glands and infundibulum. Lrig1 is a Myc target gene; loss of Lrig1 increases the proliferative capacity of stem cells in culture and results in epidermal hyperproliferation in vivo. Lrig1-expressing cells can give rise to all of the adult epidermal lineages in skin reconstitution assays. However, during homeostasis and on retinoic acid stimulation, they are bipotent, contributing to the sebaceous gland and interfollicular epidermis. β-catenin activation increases the size of the junctional zone compartment, and loss of Lrig1 causes a selective increase in β-catenin-induced ectopic hair follicle formation in the interfollicular epidermis. Our results suggest that Lrig1-positive cells constitute a previously unidentified reservoir of adult mouse interfollicular epidermal stem cells.


Flow analysis of BrdU labelled cells was performed with the APC-BrdU flow cytometry kit (BD biosciences) in combination with CD34-pacific blue, α6
integrin-FITC and Sca1-PE conjugated antibodies.
Conventional frozen and paraffin sections as well as tail epidermal whole mounts were prepared and immunolabelled as described previously (Braun et al., 2003;Lo Celso et al., 2004).
Tissue grafts were imaged using a Leica M165 FC stereo microscope.
Confocal images were acquired on a Leica TCS SP5 confocal microscope. Zstacks were acquired at 200Hz with an optimal stack distance and 1024x1024 dpi resolution. Z-stack projections were generated using the LAS AF software package (Leica Microsystems).

Image Analysis
Confocal 3D images were imported into Volocity 5.0 high performance 3D imaging software (Perkin Elmer) for accurate measurement of GFP clone volumes in 3-dimensions. Clones were initially identified by thresholding the maximum intensity of the GFP channel, and restricting measurements to areas greater than 80µm 3 (the approximate volume of a single cell) to eliminate background fluorescence. Clones were subsequently categorised according to whether they lay completely in the IFE, whether they lay in the infundibulum, with or without additional IFE contribution (infundibulum clone); contained junctional zone cells, with or without additional contribution to the IFE, SG or infundibulum (junctional zone clone); lay exclusively in the SG (SG clone); or resided in the HF beneath the junctional zone. Four representative wholemount images acquired at 20x magnification were analysed for each of three animals in control and ATRA treated groups. Images of isosurfaced GFP clones were generated using Imaris 6.2 (Bitplane).

RNA Isolation and Quantitative Real-Time PCR
RNA was isolated from whole skin using TriZol (Collins and Watt, 2008), and from primary cells using SV total RNA isolation kit (Promega). RNA from whole skin was treated with RQ1 RNase free DNase (Promega) before reverse transcription using Superscript III (Invitrogen) and random hexamer primers. Specific gene expression assays from Applied Biosystems were used for amplification using an Applied Biosystems 7900HT Real Time PCR System (Applied Biosystems, Foster USA) (Probe IDs available on request).
Samples were normalised to GAPDH using the ΔCt method.

Chromatin Immunoprecipitation
Primary mouse keratinocytes were isolated from dorsal skin of K14MycER mice and littermate controls treated with 4OHT for 4 days or K14DN-β-cateninER mice and littermate controls treated with 4OHT for 10 days.
Samples were pooled from at least 6 mice for 1 ChIP experiment. ChIP experiments for the MycER samples were performed as described (Odom et al., 2007), and for the K14ΔNβ-cateninER samples according to the To activate β-catenin signalling, primary mouse keratinocytes (P1-P4) were cultured to 80% confluence and starved overnight in 0.5% FBS supplemented FAD medium. Cells were treated with purified Wnt3A as described previously (Pálmer et al., 2008) for 6 hour in calcium-free FAD medium containing 0.5% FBS, then lysed. RNA was purified using an SV total RNA isolation kit (Promega).

Western Blotting
Tail skin was snap frozen, transferred to 1 x TBS supplemented with 2 x proteinase and phosphatase inhibitor cocktails (Roche) and homogenised for 30 seconds in a Polytron PT-MR2100 (Kinematic AG, Switzerland). 2 x RIPA buffer was subsequently added and samples were incubated on ice for 20 minutes. Equal amounts of protein from Lrig1 heterozygous and null littermates were analysed by Western blotting using antibodies to cMyc (N262, Santa Cruz) and β-tubulin (Sigma).

Promoter Analysis
The Lrig1 promoter was cloned from 1577bp upstream of exon1 to the start codon and inserted into the XhoI and NcoI sites of pGL3 basic. Keratinocytes from wild type and K14MycER transgenic mice were transiently transfected with the promoter construct as well as pRL-TK renilla. After overnight recovery cells were treated with 0-50 nM 4OHT for 24 hours in calcium-free FAD in the presence of 0.5% fetal calf serum. Luciferase activity was measured using a dual luciferase reporter assay (Promega) on a Glomax (Promega).