STAT5 Activation in the Dermal Papilla Is Important for Hair Follicle Growth Phase Induction  Julien M.D. Legrand, Edwige Roy, Jonathan J. Ellis, Mathias Francois, Andrew J. Brooks, Kiarash Khosrotehrani  Journal of Investigative Dermatology  Volume 136, Issue 9, Pages 1781-1791 (September 2016) DOI: 10.1016/j.jid.2016.04.014 Copyright © 2016 The Authors Terms and Conditions

Figure 1 Expression and activation of signal transducer and activator of transcription (STAT5) during the first hair follicle cycle. (a) Immunofluorescence detection of total STAT5 (red, top panels) and phospho-STAT5 (P-STAT5) (green, bottom panels) in the dermal papilla (DP) during hair follicle embryonic and postnatal development. P-STAT5 and total STAT5 are detected from P19 and immunofluorescence becomes most prominent at P24. Dotted lines demarcate the DP. Scale bars = 20 μm. (b) P-STAT5 immunofluorescence showing detection only in the DP and not elsewhere in the hair follicle. Is: isthmus; Bu: bulge; SG: sebaceous gland; HG: hair germ. (c) Quantification of P-STAT5-positive DP during hair follicle development. The time points are representative of embryonic hair follicle development (E14.5, E16.5, E18.5), the initial postnatal growth phase (P1, P4), and the late catagen/telogen (P19) and telogen/anagen transition (P24) stages. Mean + SD, n = 3. Journal of Investigative Dermatology 2016 136, 1781-1791DOI: (10.1016/j.jid.2016.04.014) Copyright © 2016 The Authors Terms and Conditions

Figure 2 Expression and activation of signal transducer and activator of transcription 5 (STAT5) during the adult hair follicle cycle. (a) Immunofluorescence of total STAT5 (red, top rows) and phospho-STAT5 (P-STAT5) (green, bottom rows) in the dermal papilla during adult hair follicle cycling. Both total and P-STAT5 are detected from late catagen through to early anagen. Dotted lines demarcate the dermal papilla. Scale bar = 20 μm. (b) Quantification of P-STAT5-positive dermal papilla during each phase of the adult hair follicle cycle. Mean + SD, n = 3. Journal of Investigative Dermatology 2016 136, 1781-1791DOI: (10.1016/j.jid.2016.04.014) Copyright © 2016 The Authors Terms and Conditions

Figure 3 In vivo signal transducer and activator of transcription 5 (STAT5) inhibition in the dermal papilla delays onset of the second postnatal anagen phase. (a) PCR showing the presence of a floxed STAT5 allele (top) and/or deleted STAT5 allele (bottom) for control (Cre− STAT5lox/lox) and STAT5 conditional knockout (cKO) (Cre+ STAT5lox/lox) group samples. (b) Phospho-STAT5 staining of control or STAT5 cKO skin samples. Dotted lines denote dermal papilla (DP). (c) Phospho-STAT5 staining (right) persists in Sox2+ DP (left) of STAT5 cKO skin samples (serial sections). (d) Proportions of hair follicle DP with P-STAT5 in control and STAT5 cKO according to hair follicle type (guard versus all others). (e) Proportions of hair follicle types in control (n = 4) versus STAT5 cKO (n = 4) groups. (f) Bioluminescence of control group (n = 9) versus STAT5 cKO group (n = 6) reporting canonical Wnt signaling changes in back skin over time. (g) Percentage of back skin in anagen for control (n = 9) versus STAT5 cKO (n = 6) groups over time. (h) Bioluminescence at P52 for control (left) versus STAT5 cKO (right) mice. (i) Histology of skin samples taken from control (top) and STAT5 cKO mice at P54. All scale bars = 50 μm. *P < 0.05; **P < 0.01; ***P < 0.001. Values displayed are mean ± SD). Journal of Investigative Dermatology 2016 136, 1781-1791DOI: (10.1016/j.jid.2016.04.014) Copyright © 2016 The Authors Terms and Conditions

Figure 4 Signal transducer and activator of transcription (STAT5−/−) skin-derived precursor (SKP) gene expression analyses show downregulation of Wnt and FGF signaling mediators and upregulation of inhibitory factors. (a) Heat map of gene expression levels comparing STAT5−/− and control SKPs. Hierarchical clustering analysis shows clear clustering according to genotype. (b) Microarray gene expression analyses showed downregulation of Wnt and fibroblast growth factor (FGF) signaling mediators, and upregulation of Wnt and Notch signaling inhibitors in STAT5−/− versus control SKPs. Downregulation of genes implicated in hair follicle morphogenesis and anagen induction was also observed, including follistatin, Tnc, and MMP9. (c) Reverse transcriptase-PCR (RT-PCR) validation of gene expression differences identified by microarray comparing STAT5−/− and control SKPs. cDNA was obtained from STAT5−/− (n = 5) and control (n = 4) SKPs. ∗P < 0.05. (d) Immunofluorescence of MMP9 (left panels) and tenascin C (TnC, right panels) comparing control (top panels) and STAT5−/− (bottom panels) SKPs (scale bar = 50 μm). (e) Wnt6 immunofluorescence on skin sections comparing control and STAT5 conditional knockout (cKO) skin. Reduced staining of Wnt6 is observed in the dermal papilla (DP) of STAT5 cKO hair follicles. Dotted lines demarcate DP. (f) Tumor necrosis factor-α (TNF-α) immunofluorescence on skin sections comparing control and STAT5 cKO skin. Reduced staining is observed in the DP of STAT5 cKO hair follicles. Scale bars = 50 μm. Journal of Investigative Dermatology 2016 136, 1781-1791DOI: (10.1016/j.jid.2016.04.014) Copyright © 2016 The Authors Terms and Conditions