Lgr5 marks cycling, yet long-lived, hair follicle stem cells.

Transcription

1 Lgr5 marks cycling, yet long-lived, hair follicle stem cells. Viljar Jaks 1,2,*, Nick Barker 3,*, Maria Kasper 1,*, Johan H. van Es 3, Hugo J. Snippert 3, Hans Clevers 3 and Rune Toftgård 1 1 Karolinska Institutet, Department of Biosciences and Nutrition, Novum, SE , Huddinge, Sweden 2 University of Tartu, Institute of Molecular and Cell Biology and Estonian Biocentre, Riia 23, 51010, Tartu, Estonia 3 Hubrecht Institute, Uppsalalaan 8, 3584CT Utrecht, the Netherlands * These authors contributed equally to this work

2 Supplementary Figure 1. The localization pattern and cell cycle distribution of Lgr5+ cells. The expression of Lgr5 can be first detected at E18.5 in the ORS of the growing HFs (a) as detected by X-gal staining of a skin sample obtained from an Lgr5 +/LacZ embryo. In vibrissae Lgr5 expression is limited to the lower part of the ORS (b) and outer cell layer of the vibrissae bulb (c). A similar Lgr5 vibrissae expression pattern is observed in an Lgr5-EGFP-Ires-CreERT2 mouse (d EGFP, e nuclear stain, f overlay). (g) Cell cycle analysis of keratinocytes isolated from telogen skin. Keratinocytes isolated from 8wk-old Lgr5 +/LacZ mice were stained with FDG for β-galactosidase activity, anti CD34-Alexa647 antibody and Vybrant DyeCycle Violet DNA-stain followed by flow cytometric analysis as shown in Fig. 2m. In deep telogen the majority of the cells in the CD34 + as well as in the Lgr5 high cell population reside in the G1 phase.

3 Supplementary Figure 2. Cre recombinase activity in the Lgr5-EGFP-Ires-CreERT2 skin is tightly controlled in spatial and temporal manner. (a) Serial sections of an Lgr5-EGFP-Ires-CreERT2/Rosa26R mouse skin sample at telogen. Mice were injected with 3mg of tamoxifen at P21 and skin samples were harvested 6 days later. Panels 1-6 represent serial sections of the same sample. Note that the labeled (blue) cells reside in the lower bulge and secondary germ. Panel 2 is identical to Fig. 4b. (b-d) Absence of Cre activity in Lgr5-EGFP-IresCreERT2 mice not treated with tamoxifen. X-gal stained dorsal skin samples obtained from a 280-day old Lgr5-EGFP-IresCreERT2/ROSA26-LacZ mouse not treated with tamoxifen revealed no spontaneous activation of the Cre recombinase. (b,c) - longitudinal and cross sections of anagen HFs, (d) - longitudinal section showing telogen HFs. (e) Serial sections of a Lgr5-EGFP-Ires-CreERT2/Rosa26-LacZ mouse skin sample at anagen. Mice were injected with 2mg of tamoxifen at P14 and skin samples were harvested 2 days post injection. Panels 1-3 represent serial sections of the same sample. Note that the labeled (blue) cells reside in the ORS but not in the bulge area at the upper part of the HF close to the sebaceous gland. Panel 1 is identical to Fig. 4p. (f) Serial sections of a dorsal skin sample from the low-dose tamoxifen injected Lgr5-EGFP-Ires-CreERT2/Rosa26-LacZ mice. Mice were injected with 0,5 mg of tamoxifen at P21 and skin samples were harvested 4 days post injection. Panels 1-6 represent serial sections of the same representative sample. Only one single labeled cell in the hair follicle was observed. Panel 3 is identical to Fig. 5a. Forty-four percent of the hair follicles contained labeled cells (n=64).

4 Supplementary Figure 3. Serial sections of a dorsal skin sample from the low-dose tamoxifen injected Lgr5- EGFP-Ires-CreERT2/Rosa26-LacZ mice traced for 6 days. Mice were injected with 0,5 mg of tamoxifen at P21 and skin samples were harvested 6 days post injection. Panels 1-24 represent serial sections of the same skin area. Note that the hair follicles contain either 1 or none labeled cell. Forty percent of the hair follicles contained labeled cell(s) (n=18). Note that the LacZ staining at the openings of the sebaceous glands is unspecific.

5 Supplementary Figure 4. The progeny of Lgr5+ cells can contribute to various compartments of anagen and telogen hair follicle. (a) Serial sections of a dorsal skin sample from the low-dose tamoxifen (0,5 mg) injected Lgr5- EGFP-Ires-CreERT2/Rosa26-LacZ mice traced for 8 days. Panels 1-12 represent serial sections of the same skin area. Labeled cells have formed single clones in the ORS and lower matrix of the 2 labeled anagen hair follicles shown. (b,c) The progeny of Lgr5 + keratinocytes repopulates the MTS24-expressing HF compartment. Samples were obtained from 7wk-old Lgr5-EGFP-Ires-CreERT2/ROSA26-LacZ mice injected with tamoxifen at P21. X-Gal and MTS24 doublepositive areas are indicated with brackets. Sections from skin areas containing catagen (b) and telogen (c) HFs are shown. (d,e) Hair follicles formed after transplantation of Lgr5 + cells contain sebocytes and sebaceous glands. (d) Eosin stained section showing a transplant hair follicle including an incompletely formed sebaceous gland (SG). (e) Haematoxylin-eosin stained transplant HF surrounded by sebocytes.

6 Supplementary Figure 5. Lineage tracing of Lgr5 + cells during telogen. Eight weeks-old Lgr5-EGFP- Ires-CreERT2/ROSA26-LacZ mice were injected with 2x 5 mg of tamoxifen. The initial labeling 6 days after tamoxifen injection was detected mainly in the lower part of the bulge or in the secondary germ area (a,b). Eight days later labeled cells were still predominantly detected in the lower bulge and secondary germ (c-e). A quantitation of representative skin areas of the HF labeling efficiency is given in Supplementary table 2a online.

7 Supplementary Table 1. Quantification of label retaining cells (LRCs) in telogen HFs of the Lgr5 +/LacZ mice and in the lineage traced Lgr5-EGFP-Ires- CreERT2/Rosa26-LacZ mice. Lgr5 +/LacZ mice were injected with BrdU at the indicated time intervals and skin tissue analyzed as shown in Fig. 2n-p. The number of LRCs and X-gal-positive cells in telogen HFs was counted and data collected from 3 independent experiments each including 2-3 mice. For lineage tracing Lgr5-EGFP- Ires-CreERT2/Rosa26-LacZ mice were injected with tamoxifen at P21 and for labeling of LRCs with BrdU at P3-5. Only HFs, which contained LRCs, were analyzed. The average values are given and the standard deviation is shown within brackets. Tamoxifen - - P21 BrdU P10-12 P3-5 P3-5 % of LRC in Lgr5 + cells 5,5 (3.9) 11 (5,8) 12,3 (5,8) % of Lgr5 + cells in LRC population 13,0 (4,9) 21 (1,6) 20,4 (6,7) HFs analyzed

8 Supplementary Table 2. Analysis of hair follicle labeling efficiency in skin samples taken from Lgr5-EGFP-Ires-CreERT2/Rosa26-LacZ mice. 2a: Lgr5-EGFP- Ires-CreERT2/Rosa26-LacZ mice were injected with tamoxifen at the age of 8 weeks. Skin samples were obtained 6 and 14 days post injection. Five serial sections of 5 different skin areas per sample were analyzed. Cells which could be identified in several serial sections were counted as one. Only those hair follicles, which had a visible bulge area and/or secondary germ were evaluated. The microphotographs of representative samples are shown in Supplementary Fig. 5 online. There is a slight increase in the labeling frequency of the hair follicles between days 6 and 14 potentially due to a longer time needed to reach detectable LacZ levels in telogen but no repopulation of compartments other than the lower bulge or secondary germ could be detected. 2b: Lgr5-EGFP-Ires-CreERT2/Rosa26-LacZ mice were injected with a low dose of tamoxifen (0,5 mg) at P21. A similar labeling efficiency is observed at all time points indicating that all labeled HFs were revealed 4 days after tamoxifen injection. The number of labeled cells per hair follicle increases slightly between days 4 and 6 after tamoxifen injection most likely due to cell proliferation occurring at the onset of anagen. In anagen HFs (Table 2b, 12 days post tamoxifen) the labeled cells formed larger clones due to the active proliferation, hence the number of labeled cells was not determined (N/A, not available). Table 2a Table 2b Days post tamoxifen Counted HFs Labeled cells/hf % of HFs % of HFs % of HFs % of HFs % of HFs N/A N/A N/A N/A N/A N/A N/A N/A Total labeled HFs (%)

9 Supplementary Table 3. Primer sequences used for qrt-pcr. Gene/oligo Sequence 5`- 3` Amplicon Length bp marbp f TGCACTCTCGCTTTCTGGAGGGTGT 193 marbp r AATGCAGATGGATCAGCCAGGAAGG mgapdh f GGTGTGAACGGATTTGGCCGTATTG 155 mgapdh r CCGTTGAATTTGCCGTGAGTGGAGT mhprt1 f CAACGGGGGACATAAAAGTTATTGGTGGA 158 mhprt1 r TGCAACCTTAACCATTTTGGGGCTGT mgli1 f CCCATAGGGTCTCGGGGTCTCAAAC 150 mgli1 r GGAGGACCTGCGGCTGACTGTGTAA mgli2 f TGAGGAGAGTGTGGAGGCCAGTAGCA 105 mgli2 r CCGGGGCTGGACTGACAAAGC msmo f GAGGAGCCATATTGCCCCAGGATGT 129 msmo r TCCGGCCCAAACGCTTCTCTAACTC msufu f GGAGCCCTCATCCCTCTCTGCCTAA 140 msufu r TACGGGTGTTCCTCAGTGGCAAAGG mptch1 f TGCTGTGCCTGTGGTCATCCTGATT 121 mptch1 r CAGAGCGAGCATAGCCCTGTGGTTC mlgr5 f CCAATGGAATAAAGACGACGGCAACA 128 mlgr5 r GGGCCTTCAGGTCTTCCTCAAAGTCA mklf5 f GGACTCATACGGGCGAGAAGCCCTAC 98 mklf5 r GTGCTTCCTGTAGTGGCGGGTCAG mtcf4 f CTACGGAGGGATGCTGGGCAATTCT 116 mtcf4 r TGGAGTTGATGTCTGCCGAGGAGTG mtcf3 f CTCAGCAGCAAATCCAAGAGGCAGAG 109 mtcf3 r TGGGAAGACGCAGGGCTATCACAAG mlhx2 f CCTACTACAACGGCGTGGGCACTGT 137 mlhx2 r GTCACGATCCAGGTGTTCAGCATCG msfrp1 f GCAAGCGAGTTTGCACTGAGGATGA 101 msfrp1 r GGCCCCAGCTTCAAGGGTTTCTTCT maxin2 f CAGGAGGATGCTGAAGGCTCAAAGC 127 maxin2 r CTCAAAAACTGCTCCGCAGGCAAAT mcd34 f CCTGATGAACCGTCGCAGTTGGAG 140 mcd34 r GGGTCACATTGGCCTTTCCCTGAGT mkrt15 f TGGACATCAAGACTCGGCTGGAACA 107 mkrt15 r ACCGCTGCTTCCTTCTCTGACACCA msca1 f TGCCCCTACCCTGATGGAGTCTGTG 104 msca1 r GGAGGGCAGATGGGTAAGCAAAGATTG