Affi gel blue beads

Fertilized chicken eggs (Gallus gallus, Rhode Island Red, Petaluma Farms, CA) were incubated at 39° C in humidified conditions and prepared for surgical manipulation. Briefly, 1.5 mL albumin was removed, a hole was cut into the shell directly over the embryo, and the vitelline membrane was removed. Embryos were visualized by staining with neutral red diluted in PBS (Acros Organics, Geel, Belgium) and staged according to Hamburger-Hamilton (HH) criteria.^{4 (link)} Inhibition of neural SHH signaling was achieved by injecting hybridoma cells expressing either immunoneutralizing anti-SHH antibody (5E1) or anti-β-galactosidase antibody control cells (40–1A) into the neural tube at Stage 10 (HH10), as previously described.^{5 (link),60 (link)} SHH-N treatment was performed using affi-Gel Blue beads (50 – 100 mesh, 200 – 250 diameter; Biorad) soaked in recombinant SHH-N protein (400 ng/ml in PBS with 0.1% bovine serum albumin (BSA); Ontogeny) or 0.1% BSA (control) at 37°C for one hour. Forty-eight hours after SHH signal inhibition, SHH-soaked beads were placed between the facial ectoderm and the forebrain neuroectoderm. Embryos were collected at HH22 for genomic analysis or fixed in 4% paraformaldehyde (PFA) for two hours at room temperature or overnight at 4°C and then dehydrated through a graded ethanol series and stored in 100% ethanol at −20°C until analysis.

Transplantation of MECs into cleared fat pads of C57BL/6 mice were as previously described (Watanabe et al., 2014 (link)). Typically, 2000 FACS-sorted basal MECs or 5000 unsorted MECs were used for each transplantation unless otherwise indicated. Lentiviral infection before transplantation and Zeb1 shRNA-expressing lentiviruses were as described previously (Watanabe et al., 2014 (link)). For limiting-dilution transplantation, GFP⁺ cells were FACS-sorted following lentivirus infection and then transplanted into cleared fat pads.
For basal MEC transplantation with Dkk1-coated beads, Affi-Gel blue beads (Bio-Rad Laboratories, 1537301) were washed three times with PBS and incubated with 1 mg/ml recombinant mouse Dkk1 (R&D Systems, 5897-DK-010) in 0.1% BSA for 1 hour before transplantation. Approximately 100 beads were then mixed with 2000 FACS-sorted basal MECs in 10 μL of DMEM/F-12 medium containing 5% FBS, which were injected into cleared fat pads. Beads coated with 0.1% BSA were used as a control.

Foxp3^DTR mice depleted of T_regs according to the early regimen were injected subcutaneously on days −2, −1, 1, and 3 into four adjacent dorsal skin sites with 1 µg control IgG1-Fc or Jag1-Fc fusion protein (both from R&D) conjugated to Affi-gel blue beads (100–200 mesh; Bio-Rad), as previously described (Chen et al., 2012 (link)). Briefly, per mouse: 1 µg of protein (in 10 µl) was soaked with 5 µl Affi-gel blue beads (corresponding to ~2000 beads) for 1 hour at 37° C. Total suspensi ons of 15 µl were then transferred into 29G 3/10 cc insulin syringes (BD) for subcutaneous injection of mice under anesthesia. HFSC Ki67 expression was assessed by flow cytometry on day 4.