Niche-induced cell death and epithelial phagocytosis regulate hair follicle stem cell pool.

Methods

Mice
K14-H2BGFP17, Lef1-RFP18 and K14-GFPActin19 were obtained from the Fuchs Laboratory. TGFβRIfl/fl mice were obtained from Vesa Kaartinen15. β-catnflox(Ex3)/+ mice were obtained from Makoto Taketo20. Lgr5-CreER (Clevers Lab), Shh-CreER (Tabin Lab), LysM-Cre (Foerster Lab), CX3CR1-GFP (Littman Lab) and Rosa-stop-tdTomato (Zeng Lab) were obtained from Jackson Laboratory (JAX)21–25. The Yale Transgenic Facility generated the K14-H2BmCherry mice. All studies and procedures involving animal subjects were approved by the Institutional Animal Care and Use Committee at Yale School of Medicine and conducted in accordance with the approved animal handling protocol. Lgr5-CreER and Shh-CreER were used to recombine alleles and label cells conditionally within specific hair follicle populations and temporally during the regression phase. Cre-induction for the lineage tracing experiments was induced with a single intraperitoneal injection of Tamoxifen (1μg/g in corn oil) at postnatal day 14. TGFβRIfl/fl recombination was induced with three intraperitoneal injection of Tamoxifen (100μg/g in corn oil) at postnatal day 10, 12 and 14. Intravital microscopy and laser ablation procedures were carried out as described previously4,5. For lineage tracing experiments only cells that were unambiguously separated from other were sampled to ensure the identity of individual lineages. Mice from experimental and control groups were randomly selected for live imaging experiments. No blinding was done. All lineage tracing and ablation experiments were repeated in at least three different mice.

Generation of K14H2BmCherry mice
Transgenic mice expressing H2BmCherry under the control of the Keratin 14 promoter (K14-H2BmCherry) were generated using the following procedure. The H2BmCherry insert (provided by D. Egli) was amplified by PCR from the TopoTA vector (Life Technologies) using primers 5′-CGGCGGATCCATGCCAGAGCCAGC and 3′-CGCTCTAGATTACTTGTACAGCTCGTCC, which introduced cleavage sites for BamHI and XbaI restriction enzymes immediately upstream and downstream, respectively, of the open reading frame. The 1.1Kb PCR product was inserted between the BamHI and XbaI sites in the pG3Z*K14cassette vector (provided by E. Fuchs). The resulting transgene was digested with SacI and SphI, and the 4.3Kb fragment was injected into blastocysts at the Yale Transgenic Facility (T. Nottoli). Chimeric mice were screened initially by PCR and founder mice were selected to establish transgenic mouse lines. These initial lines were subsequently screened by histological analysis, and the line displaying the highest expression levels of the K14H2BmCherry reporter was selected to establish the final colony.

In vivo imaging and laser ablation
Mice between postnatal day 17 and 35 were anesthetized with intraperitoneal injection of 7 μl/g of ketamine/xylazine cocktail mix (15mg/ml and 1mg/ml, respectively in PBS). Anesthesia was maintained throughout the course of the experiment with vaporized isofluorane delivered by a nose cone as previously described10. Image stacks were acquired with a LaVision TriM Scope II (LaVision Biotec, Germany) microscope equipped with a tunable Chameleon Ultra (Coherent, USA) Ti:Sapphire laser. To acquire serial optical sections a laser beam (740nm for Alexafluor 350; 940 nm for H2BGFP; 1040 nm for RFP and tdTomato; 990 nm for simultaneous excitation of GFPActin and H2BmCherry) was focused through a 20X or 40X water immersion lens (N.A. 1.0 and 1.1 respectively; Zeiss, USA) and scanned with a field of view of 0.5 or 0.25 mm2 respectively at 600Hz. Z-stacks were acquired in 1–3 μm steps to image a total depth of 150 μm of tissue. We revisited the same hair follicles in separate experiments as previously described10. For time-lapse recordings, serial optical sections were obtained between 1 to 5 minute intervals, depending on the experimental setup. Laser ablation was carried out with the same optics as used for acquisition. An 800nm laser beam was used to scan the target area (10–50 μm2) and ablation was achieved using 30–50% laser power for ~1sec. Ablation parameters were adjusted according to the depth of the target (50–100 μm).

Image Analysis
Raw image stacks were imported into Fiji (NIH, USA) or Imaris software (Bitplane/Perkin Elmer) for further analysis. Provided images and supplementary videos are typically presented as a maximal projection of 3 to 6 μm optical sections. For visualizing individual labeled cells expressing the tdTomato Cre reporter, the brightness and contrast were adjusted accordingly for the green (GFP) and red (RFP/tdTomato) channels and composite serial image sequences were assembled as previously described. Hair follicle length and labeled cell position values were measured from the top of the stem cell compartment. Apoptotic cell tracking analysis was performed in Imaris software (Bitplane).

Electron Microscopy
Trimmed skin samples were fixed (2% gluteraldehyde and 2%paraformaldehyde in 0.1M sodium cacodylate buffer pH7.4) for 1 hour. The samples were rinsed in sodium cacodylate buffer and were post-fixed in 1% osmium tetroxide for 1 hour. The samples were rinsed and en-bloc stained in aqueous 2% uranyl acetate for an hour further, followed by rinsing, dehydrating in an ethanol series to 100%, and rinsing several times in 100% propylene. Then samples were infiltrated with Embed 812 (Electron Microscopy Sciences) resin and baked overnight at 60° C. Hardened blocks were cut using a Leica UltraCut UC7. 60 nm sections were collected and stained using 2% uranyl acetate and lead citrate for transmission microscopy, and 250 nm thick sections were stained with either Richardson’s stain or 1% Toluidine Blue for light microscopy. For immunolabeled electron microscopy, dissected skin samples were fixed in 4% paraformaldehyde/0.1% gluteraldehyde in phosphate buffer for 30 minutes and then in 4% paraformaldehyde/PB overnight at 4° C. The samples were rinsed in 0.1M HEPES. To quench, aldehydes were placed in 50mM NH4Cl +100mM glycine +2% sucrose for 1 hour then washed in Hepes buffer and placed in 0.1% Tannic Acid/0.1M Hepes for 1 hour, then rinsed in 50mMTris/50mM Maleate and placed 2% UA/50mMTris/50mM Maleate for a 1 hour. After rinsing, they were dehydrated through a graded series 50 % to 95% of ethanol at 4C, then infiltrated with 50:50 ethanol/LR White (EMS) for 1 hour followed by several changes of pure 100% LR White overnight on a rotator at 4°C. Samples were polymerized at 60°C for 18 hours. 50nm resin sections were cut on a Leica UC7 ultra-microtome and collected on nickel formvar/carbon grids, and immunolabeled using a primary chicken anti-GFP (Abcam) diluted to 1:50 for 1 hour, rinsed and placed on protein A gold secondary 1:50 (University of Utrecht). The sections were counterstained with 2% uranyl acetate and lead citrate. Grids were viewed FEI Tencai Biotwin TEM at 80Kv. Images were taken using Morada CCD and iTEM (Olympus) software.

Immunostaining on paraffin sections and whole mount skin
Skin was fixed in 4% PFA for whole mount or in 10% formalin for paraffin embedding and used for histological analysis as previously described26. Immunohistochemistry was performed by incubating sections at 4°C overnight with primary antibodies as follows: mouse anti-β-catenin (1:100, BD #610153; 14/Beta-Catenin), rat anti-CD11b (1:250, eBioscience #14-0112; M1/70), goat anti-P-cadherin (1:100, R&D #AF761), rabbit anti-pSmad2 (Ser465/467) (1:1000, Cell Signaling #3108; 138D4), and rabbit anti-Lef-1 (1:100, Cell Signaling #2286; C18A7). pSmad2 immunostaining required TSA Plus kit (PerkinElmer). For brightfield immunohistochemistry, biotinylated species-specific secondary antibodies, followed by detection using the ABC kit (Vector Labs) and DAB kit (Vector Labs), were used according to the manufacturer’s instructions. M.O.M. kit was used for mouse antibodies (Vector Laboratories). Secondary antibodies conjugated with FITC, RRX and Cy5 (Jackson Immunoresearch Laboratories) were used at a concentration of 1:100 for 1 hour at room temperature. Alexafluor 350 phalloidin (Life Technologies) was used according to the manufacturer’s instructions.

FACS
Back skins of K14-H2BGFP; Lef1-RFP and Lgr5-CreER; TGFβRIfl/fl or TGFβRIfl/+; tdTomato; K14-H2BGFP mice were harvested at P12, P16, or P20 and were placed dermis down on 0.2% collagenase (Sigma) at 37°C for 20 minutes, and then 0.25% trypsin (Gibco) at 37°C for 10 minutes to obtain epithelial cells as previously described27. Cells were stained for 10 minutes with biotinylated rat anti-CD34 (1:50, eBiosciences #14-0341; RAM34), biotinylated rat anti-CD45 (1:50, BD #553077; 30-F11), biotinylated rat anti-CD117 (1:50, BD #553353; 2B8), and goat anti-Integrin alpha 9 (1:50, R&D #AF3827). Cells were washed for 5 minutes and then incubated with streptavidin-Pacific blue (1:200, Invitrogen) and Alexafluor 647 donkey anti-goat IgG (Jackson Immunoresearch Laboratories). Cells were isolated on DAPI exclusion and as follows: DP = RFP+, CD34−, CD45−, CD117−, Integrin-alpha 9+ and enriched ORS = RFP−, GFPHigh using a FACSAria II Cell Sorter (BioScience), as previously described28. Cells were sorted into RNA lysis buffer for RNA isolation (RNease Mini Kit, Qiagen). FACS profiles were analyzed through FlowJo software.

RT-qPCR
cDNA was made using Superscript III First-Strand Synthesis kit (Invitrogen). RT-qPCR was performed in triplicate with SYBER Green I reagents (Invitrogen) using 5.0ng cDNA per reaction on the ViiATM 7 Real-Time PCR system (Invitrogen – Life Technologies). Data were analyzed by ViiATM software, Microsoft Excel and PRISM. Gene-specific primers were designed and are listed in Supplementary Table 1.

Statistical Analysis
Data are expressed as percentages, box and whisker plots (error bars represent max and min), or mean ± SD. An unpaired Student’s t-test was used to analyze data sets with two groups and *p < 0.005 to **** p<0.0001 indicated a significant difference. Statistical calculations were performed using the Prism software package (GraphPad, USA).

Extended Data

Supplementary Material
1