DEV021295 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. Conditional expression kinetics of the constitutively active β-catenin in the developing skin epidermis. (A) X-gal staining visualizing K5-Cre-mediated expression of β-galactosidase (ROSA-lacZ) at E14.5 in the epidermis. Strong β-gal staining is detected in the dorsolateral skin at E11.0 (data not shown). (B-I) β-catenin protein (β-CAT; red) expression kinetics in the embryonic skin. β-catenin protein accumulates in the prospective hair buds of the control embryos (prominent accumulation shown at E14.5). Upon the K5-Cre-mediated exon3 deletion of β-catenin, it accumulates in the epidermis (initially observed at E11.5 and robust expression is detected at E14.5-E18.5). The increased expression of β-catenin is observed in the supra-basal regions in addition to the basal regions at E16.5 and E18.5 (H,I). (J,K) BAT-lacZ reporter mouse line showing areas with canonical β-catenin signaling activity in the epidermis at E16.5. Scale bars: 100 µm in A,J,K.

• Supplemental Figure S2 -

  Fig. S2. The kinetics of morphological changes of K5-Cre Catnb ^(ex3)fl/+ mutant skin. HE-stained sections of control (A-D) and of K5-Cre Catnb(^ex3)^fl/+ (E-H) skin. The thickened epidermis (arrows in E) starts to form at E11.5. In the mouse embryo, cutaneous hair placodes arise at E14.0. The dermal condensate (arrowheads in F) is formed after E13.5 in the mutant skin. The thickened epidermal region is expanded with its underlying dermal condensate to the entire skin at E16.5-E18.5 in the mutants.

• Supplemental Figure S3 -

  Fig. S3. Abnormal epidermal differentiation of K5-Cre Catnb ^(ex3)fl/+ mutant mice. (A,B) Follicular keratinization with morphological trichilemma-type structures shown by TEM analysis. Scale bar: 5 µm in A,B.

• Supplemental Figure S4 -

  Fig. S4. Altered expression of the hair shaft keratins in K5-Cre Catnb ^(ex3)fl/+ mutant epidermis. (A) Hair shaft keratins detected by the AE13 antibody (red) are expressed at E13.5-E14.5 in the thickened mutant epidermis, which overlap with augmented β-catenin expression (green). (B,C) The region expressing the hair shaft keratins is expanding at E16.5 (red). Subsequently, hair shaft keratins are expressed strongly throughout the epidermis without loricrin expression (green) in K5-Cre Catnb(^ex3)^fl/+ mice at E18.5.

• Supplemental Figure S5 -

  Fig. S5. Induction of Bmp signaling in K5-Cre Catnb ^(ex3)fl/+ skin. (A-H) Visualization of Bmp activity by immunohistochemistry using a pSMAD antibody. The pSMAD level is significantly increased in K5-Cre Catnb(^ex3)^fl/+ mutant epidermis and in the underlying mesenchyme, compared with the control. (E) The pSMAD level is already increased in the mutant epidermis at E11.5 (arrows). (F,G) pSMAD levels are also upregulated in the mutant dermis at E14.5 and E16.5. (H) The widespread pSMAD expression is still visible in the mutant epidermis at E18.5. Dashed lines indicate the dermal-epithelial borders.

• Supplemental Figure S6 -

  Fig. S6. Induced expression of Bmps, Shh and Wnt10b is maintained in K5-Cre Catnb ^(ex3)fl/+BmprIA^fl/fl mutant skin. (A-C) Increased Bmp signaling in the K5-Cre Catnb(^ex3)^fl/+ mutant epidermis is suppressed by the introduction of the compound mutation of K5-Cre Catnb(^ex3)^fl/+BmprIA^fl/fl, whereas it is maintained in the mutant upper dermis (arrowheads in C). (D-O) Compared with controls, the expression of Bmp2, Bmp4, Shh and Wnt10b is already detected in K5-Cre Catnb(^ex3)^fl/+ at E11.5 and is maintained in the K5-Cre Catnb(^ex3)^fl/+BmprIA^fl/fl epidermis. Dashed lines indicate the dermal-epithelial borders.

• Supplemental Figure S7 -

  Fig. S7. The timing of Shh signaling inactivation in K5-Cre Cantb^(ex3)fl/+Shh^fl/− mutant skin. (A,B) Expression of the Shh target gene Ptch1 was dramatically decreased, indicating an effective inhibition of Shh signaling in K5-Cre Cantb(^ex3)^fl/+Shh^fl/− epidermis at E14.5.