DEV031427 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. EGFR and FGFR2(IIIb) expression in developing pelage and vibrissa follicles. (A-L) Mouse skin sections from back skin and whisker pad regions at post-placodal stages of development were labelled with antibodies specific to EGFR (A,B,E-H) or FGFR2(IIIb) (C,D,I-L) and the nuclear label DAPI (blue). Labelling of both receptors was reduced in the early follicular epithelial downgrowths of both pelage (A-D) and vibrissa follicles (E,F,I,J), compared with interfollicular epidermis. However, evidence of increasing intrafollicular expression appeared during later stages of development, more markedly for EGFR (G,H) than for FGFR2(IIIb) (K,L). Dotted lines mark the boundaries between epidermis and dermis. Scale bars: 60 µm.

• Supplemental Figure S2 -

  Fig. S2. The downregulation of placodal FGFR2(IIIb) is unaffected by disruption and delay to hair follicle initiation. The surfaces of skin samples from E14 mice 0 to 24 hours after splitting, recombination and organ culture (A-D). HFs were clearly visible on the surface of recombined skins following 24 hours of culture (black arrows, D). Cryo-embedded samples were sectioned and labelled with antibodies specific to syndecan 1 (red) (E-H), FGFR2(IIIb) (green) (I-L) or laminin (green) (M-P). Localised areas of dermal syndecan 1 became clear from 12 hours of culture (G,H). Epidermis showed corresponding reduced expression of FGFR2(IIIb) from 12 hours (K,L). Laminin expression indicated that recombined skin had begun to re-establish a basement membrane after 6 hours of culture (N). Dotted lines mark the boundaries between epidermis and dermis, nuclear DAPI is blue. Scale bars: 60 µm.

• Supplemental Figure S3 -

  Fig. S3. Expression of EGF- and FGF-related genes during normal skin development. (A) Semi-quantitative RT-PCR shows differential expression of EGF- and FGF-related mRNA transcripts in the epidermal compartment isolated from embryonic back skin of the indicated age. (B) Semi-quantitative RT-PCR shows differential expression of EGF- and FGF-related mRNA transcripts in the dermal compartment isolated from embryonic back skin of the indicated age.

• Supplemental Figure S4 -

  Fig. S4. EGFR and FGFR2(IIIb) signalling has no effect on primary hair follicle morphogenesis post initiation. Skin removed from E14.5 embryos, which had visible placodes on the skin surface (A), and when sectioned and H&E stained (B). (C-N) E14.5 skin was cultured with BSA (250 ng/ml), EGF (50 ng/ml), HBEGF (250 ng/ml) or KGF (250 ng/ml) for 24 hours and 48 hours. After 24 hours of culture, follicles could still be clearly identified on the skin surface of all cultured skin specimens (C-F). Within the EGFR ligand-treated skin, folds and ridges could also be identified (D,E). H&E stained sectioned skin revealed that HF development in EGF-, HBEGF- and KGF-treated skin (H-J) was morphologically indistinguishable from that of the control (G). Scale bars: 0.5 mm. Skin treated with growth factors and labelled with antibodies specific to syndecan 1 (red) and laminin (green) also showed the same expression pattern as the control (K-N). Scale bars: 60 µm.

• Supplemental Figure S5 -

  Fig. S5. Validation of the upregulation of genes known to be involved in the differentiation of interfollicular epidermis. Increased expression of keratin 6, filaggrin and loricrin was verified at the protein level by immunofluorescent labelling on sections from control (A-C) and HBEGF- (D-F) or KGF- (G-I) treated embryonic skin, cultured for 24 hours. Compared with control skin, in all instances treated skins show increased expression of proteins in appropriate regions of the epidermis. Scale bars: 60 µm.

• Supplemental Figure S6 -

  Fig. S6. EGF and KGF ligand treatments elicit their inhibitory effect initially on the epidermis and not the dermis. (A-R) E14.0 mouse back skin (just prior to the initiation of follicle development) was cultured in the presence of BSA (250 ng/ml), HBEGF (250 ng/ml) or KGF (250 ng/ml) for 6, 10 and 24 hours. Skin cultured for 6 hours with control BSA (250 ng/ml), HBEGF (250 ng/ml), or KGF (250 ng/ml) expressed low and diffuse localised dermal syndecan 1, indicating the beginning of dermal condensation development (A,C,E). At the same early timepoint, control epidermis expressed strong differential P-cadherin expression (representing placode formation) (B); no such expression was observed in the epidermis of either ligand-treated skin (D,F). Following 10 and 24 hours, in control skin the expression of dermal syndecan 1 (G,M) and epidermal P-cadherin increased in intensity (H,N). In HBEGF- and KGF-treated skins, weak condensate-like syndecan 1 expression was evident at 10 hours (I,K) but was virtually absent at 24 hours (O,Q). At the same timepoints, some morphological evidence of placodal-like structures were visible, but differential P-cadherin expression was not seen (J,L,P,R). These results suggest that activation of EGFR or FGFR2(IIIb) in skin just prior to the initiation of follicle formation does not immediately inhibit the initiation of a dermal condensation. However, it does appear to inhibit the expression of the molecular signals associated with the epidermal placode formation. These observations suggest that the ligand treatments are eliciting their inhibitory effect initially on the epidermis and not the dermis. P-cadherin and syndecan 1 (red), laminin (green), nuclear DAPI (blue). Scale bars: 60 µm.