DEV017459 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. KRT14-Cre mediates recombination in embryonic surface ectoderm by E11.5. (A-C) X-gal-stained whole mounts of KRT14-Cre ROSA26R (A,C) and control (B) embryos at E11.5 (A) and E12.5 (B, C).

• Supplemental Figure S2 -

  Fig. S2. Formation of Wnt10b-positive placodes and keratinized papules in activated mutant β-catenin embryo footpads. (A,B) Whole-mount in situ hybridization for Wnt10b detects the formation of placodes in developing footpads of KRT14-Cre Ctnnb1^(Ex3)fl/+ (Act β-catenin, B) but not control littermate (A) embryos at E12.0. (C,D) At E15.5, Wnt10b expression is elevated throughout the mutant footpads, raised papules are visible, and digit development fails (D, compare with control in C).

• Supplemental Figure S3 -

  Fig. S3. Failure of acquisition of a permeability barrier in activated mutant β-catenin embryos. (A-F) Dye exclusion assays reveal acquisition of a complete permeability barrier in control embryos by E18.5, and failure to exclude dye at all stages in the mutant.

• Supplemental Figure S4 -

  Fig. S4. Altered expression of P-cadherin and E-cadherin in activated mutant β-catenin embryonic skin. (A-D) Immunohistochemistry (brown) of E15.5 skin sections shows that P-cadherin is expressed broadly in mutant (B) compared with control (A) epidermis (arrows). E-cadherin is downregulated in mutant (D) compared with control (C) epidermal basal cells, similar to its pattern in the hair germ. Black arrows indicate positive signals; red arrow in D indicates absence of E-cadherin expression in differentiating mutant epithelial cells. Purple arrows in C,D indicate downregulation of E-cadherin in control hair germ (C) and mutant basal cells (D). Scale bar in D applies to A-D.

• Supplemental Figure S5 -

  Fig. S5. Induction of a stabilizing β-catenin mutation in adult skin causes formation of ectopic hair follicles. (A) Appearance of Krt5-rtTA tetO-Cre Ctnnb1^(Ex3)fl/+ (right) and control littermate Krt5-rtTA tetO-Cre Ctnnb1^+/+ (left) mice treated with oral doxycycline from postnatal day (P) 50 to P70. (B,C) Dorsal skin from the control (B) and mutant (C) mice pictured in A. Note increased hair growth in the mutant. (D-I) H&E-stained paraffin sections of dorsal skin (D-G) or footpad skin (H,I) from control (D,F,H) and mutant (E,G,I) mice doxycycline treated from P35-P40 (D,E) and P50-P70 (F-I). After 5 days of induction, new hair follicles can be seen budding off from the bulge region of existing follicles in the mutant (E, yellow arrows). After 20 days of induction, control hair follicles are regularly spaced and are in the telogen, resting, stage of the hair follicle growth cycle, whereas mutant skin shows densely packed, irregularly spaced hair follicles in an abnormal growth phase (F,G). Abnormal hair follicles are also induced in mutant footpad skin, which is characterized by a thickened epidermis, and is hairless in controls (H,I). (J,K) Immunofluorescence (red) for the hair follicle outer root sheath companion layer marker KRT6 confirms the identity of hair follicles in induced mutant footpad skin. Scale bars: bar in E applies to D,E; in G, to F,G; in I, to H,I; in K, to J,K. Nuclei in J,K are DAPI counterstained (blue). (L) Efficiency of recombination of the Ctnnb1^(Ex3)fl/+ allele in embryonic and induced adult mutant epithelium. DNA isolated from separated dermis and epidermis was subjected to semi-quantitative PCR using primer pairs that amplify both the KRT14-Cre and tetO-Cre transgenes (left panel), both wild-type Ctnnb1 exon 3 and recombined floxed exon 3 (middle panel), and both unrecombined floxed exon 3 and wild-type exon 3 (right panel), as indicated. Lanes 1-8 represent: (1) E16.5 KRT14-Cre Ctnnb1^(Ex3)fl/+ epidermis; (2) E16.5 control KRT14-Cre Ctnnb1^+/+ epidermis; (3) E16.5 KRT14-Cre Ctnnb1^(Ex3)fl/+ dermis; (4) E16.5 control KRT14-Cre Ctnnb1^+/+ dermis; (5) adult Krt5-rtTA tetO-Cre Ctnnb1^(Ex3)fl/+ epidermis; (6) adult control Krt5-rtTA tetO-Cre Ctnnb1^+/+ epidermis; (7) adult Krt5-rtTA tetO-Cre Ctnnb1^(Ex3)fl/+ dermis; and (8) adult control Krt5-rtTA tetO-Cre Ctnnb1^+/+ dermis. Adult mice were doxycycline treated from P50 and samples were taken at P70. Note that the deleted allele is detected in embryonic mutant epidermis only, and in both adult epidermal and dermal preparations owing to contamination of the adult dermal preparation with hair follicle epithelium. The floxed, recombined allele is detected in adult induced mutant epidermis at slightly higher levels than in embryonic epidermis. The floxed, non-recombined allele is detected at similar intensity in epidermal preparations of both embryonic and adult skin. These data indicate that recombination of exon 3 occurs in induced adult epidermis at a similar or slightly greater efficiency than in embryonic epithelial cells. M, size markers.

• Supplemental Figure S6 -

  Fig. S6. β-catenin-mediated development of de novo hair follicles in adult KRT5-CreERT2 Ctnnb1^(Ex3)fl/+ mice. (A-F) H&E sections of EtOH-treated (A-C) and 4OHT-treated (D-F) skin from KRT5-CreERT2 Ctnnb1^(Ex3)fl/+ mice, after 7 days of treatment starting at P56. 4OHT-treated skin contains de novo hair follicles arising from both interfollicular epidermis and existing hair follicles. (A′-F′) Immunohistochemistry for β-catenin reveals a ‘basket-weave’-like cell membranous localization in control (EtOH-treated) skin (A′-C′), while intense nuclear/cytoplasmic β-catenin localization is seen in 4OHT-treated skin (D′-F′). (G,H) Whole-mount view of hematoxylin/Oil Red O-stained epidermis of tail skin after epidermal-dermal separation. De novo hair follicles arising from existing hair follicles are visible in 4OHT-treated tail skin (yellow arrowheads). (I,J) Whole-mount immunoflourescence reveals expression of the hair matrix marker CDP (I) and the outer root sheath marker KRT17 (J) in existing and de novo hair follicles (white arrowheads).