Sonic hedgehog-dependent activation of Gli2 is essential for embryonic hair follicle development

Abstract

Sonic hedgehog (Shh) signaling plays a critical role in hair follicle development and skin cancer, but how it controls these processes remains unclear. Of the three Gli transcription factors involved in transducing Shh signals in vertebrates, we demonstrate here that Gli2 is the key mediator of Shh responses in skin. Similar to Shh(-/-) mice, Gli2(-/-) mutants exhibit an arrest in hair follicle development with reduced cell proliferation and Shh-responsive gene expression, but grossly normal epidermal differentiation. By transgenic rescue experiments, we show that epidermal Gli2 function alone is sufficient to restore hair follicle development in Gli2(-/-) skin. Furthermore, only a constitutively active form of Gli2, but not wild-type Gli2, can activate Shh-responsive gene expression and promote cell proliferation in Shh(-/-) skin. These observations indicate that Shh-dependent Gli2 activator function in the epidermis is essential for hair follicle development. Our data also reveal that Gli2 mediates the mitogenic effects of Shh by transcriptional activation of cyclin D1 and cyclin D2 in the developing hair follicles. Together, our results suggest that Shh-dependent Gli2 activation plays a critical role in epithelial homeostasis by promoting proliferation through the transcriptional control of cell cycle regulators.

Figure 1

Arrest of Gli2^−/− pelage follicle development phenocopies the skin defects of Shh^−/− mutants. Development of pelage follicles and whiskers in wild-type (A,E,I, M,R), Shh^−/− (B,F,J,N,S), Gli2^−/− (C,G,K, O,T), and Gli3^−/− (D,H,L,P,U) mutants. (A–D) Pelage follicle development in E14.5 skin. Yellow line marks the boundary between epithelium (e) and dermis (d). (E–H) Pelage follicle development in E18.5 skin. Numbers denote stage of hair follicle morphogenesis. (I–L) E18.5 skin sections stained for alkaline phosphatase (blue) and laminin-5 (red). Arrowheads mark the developing dermal papilla, which show high alkaline phosphatase activity. (M–P) Patterning of vibrissae follicles in E14.5 heads as revealed by K17 RNA expression. In M, numbers indicate the normal tract pattern and arrowheads mark the four intervening whiskers. Arrow indicates the whisker formed in Shh^−/− mice (N), and asterisk marks the extra whisker tract in Gli3^−/− mutants (P). (R–U) Histology of vibrissae follicles in E18.5 skin. Arrow indicates the characteristic concentric rings on cross-sections of Shh^−/− vibrissae follicles. Bars, 50 μm.

Figure 2

Gli2^−/− mutant follicles are arrested because of a defect in proliferation. Immunofluorescence of differentiation, and cell proliferation markers of wild-type (A,C,E,G) and Gli2^−/− (B,D,F) skin at E14.5 (A–B) and E18.5 (C–H). (A–D) Staining of Keratin14 (red) and BrdU (green) at E14.5 (A,B) and E18.5 (C,D). (E–F) Staining of Loricrin (red) and PCNA (green) in E18.5 skin. (G–H) Staining of Keratin10 in E18.5 skin. (I) BrdU-positive cells in E18.5 skin. Gli2^−/− hair follicles show a twofold reduction in epithelial proliferation (p < 0.0005). Bars, 50 μm.

Figure 3

Shh responsiveness is reduced in Gli2^−/− skin. Expression of Shh (A,G), Ptc1 (B,H), Ptc2 (C,I), Gli1 (D,J), Gli2 (E,K), and Gli3 (F,L) in E18.5 wild-type (A–F) and Gli2^−/− (G–L) skin. The expression of the Shh-responsive genes, Ptc1 and Gli1, is dramatically reduced in Gli2^−/− mutants (H,J). (M) Shh induces Gli1 and Ptc1 expression in dermal fibroblasts isolated from wild-type skin, as revealed by RT–PCR analysis. This Shh response is lost in Gli2^−/− dermal fibroblasts.

Figure 4

Epithelial Gli2 is sufficient to restore Shh responsiveness, stimulate proliferation, and rescue hair follicle development. (A–G) Sections of E18.5 wild-type (A,E), Gli2^−/− (B,F), K5–Gli2;Gli2^−/− (C,G), and K5–Gli2;Shh^−/− dorsal skin at low (A–D) and high (E–G) magnification. (B) Although most Gli2^−/− follicles are arrested at stages 1–2, some “escape” follicles (arrowhead) can develop further. (H–M) Appearance (H–J) and histology (K–M) of wild-type (H,K), Gli2^−/− (I,L), and K5–Gli2;Gli2^−/− (J,M) skin xenografts after 50 d of incubation. (N–S) Dark-field illuminations of Ptc1 expression in E14.5 follicles in wild-type (N), Gli2^−/− (O), and K5–Gli2;Gli2^−/− (P) skin. Gli1 expression by DIG in situ in wild-type (Q), Gli2^−/− (R), and K5–Gli2;Gli2^−/− (S) skin. (T–V) Staining of BrdU (green) and K14 (red) in E18.5 wild-type (T), Gli2^−/− (U), and K5–Gli2;Gli2^−/− (V) skin. (W) Differences in the progression of hair follicle development at E18.5: wild type (red), Gli2^−/− (green), and K5–Gli2;Gli2^−/− (blue). Bars: E–G,K–M, 50 μm; H–J, 2.5 cm.

Figure 5

Inducible transgenic expression of Gli2 and ΔNGli2 in the developing epithelium. (A) Schematic of the conditional Cre–loxP transgenic approach. The double reporter Z/AP construct (Lobe et al. 1999) contains a strong ubiquitous promoter that drives expression of reporter genes and Gli2. The first reporter, βgeo, which is followed by three polyadenylation signals, is flanked by loxP sites (red arrowheads), which can be excised in the presence of Cre. The second reporter, human placental alkaline phosphatase (hPLAP), and Gli2 are expressed only after Cre excision. This strategy was used to target epidermal expression of full-length Gli2 and N-terminally truncated Gli2, ΔNGli2 (Sasaki et al. 1999). Expression of the Z/AP–ΔNGli2 transgene in E18.5 skin in the absence (B,C,D) and presence (E,F,G) of K5–Cre. (B) In the absence of Cre, lacZ expression was found in all layers of interfollicular epithelium, infundibulum, and outer root sheath (ORS) of developing hair follicles. (C) hPLAP was not expressed in the absence of Cre; however, endogenous alkaline phosphatase activity is detected in dermal papillae. (D) In situ hybridization revealed low levels of endogenous Gli2 expression in the epithelium and dermis. In the presence of Cre, K5–Cre;Z/AP–ΔNGli2 embryonic skin shows efficient excision of the βgeo cassette (E), and induction of hPLAP (F) and Gli2 transgene (G) expression in the epidermis. Bar, 50 μm.

Figure 6

The activator function of Gli2 is required for hair follicle development and is Shh-dependent. Hematoxylin-eosin staining of dorsal skin sections from E18.5 wild-type (A), Gli2^−/− (B), Shh^−/− (C), K5–Cre;Z/AP–Gli2;Gli2^−/− (M), K5–Cre;Z/AP–Gli2; Shh^−/− (N), K5–Cre;Z/AP–Δ NGli2; Gli2^−/− (U), and K5–Cre;Z/AP–ΔNGli2; Shh^−/− (V) mice. In situ hybridization analysis of Shh target gene expression, Ptc1 (D,G,J,O,R,W,Z) and Gli1 (E,H, K,P,S,X,AA). Staining of BrdU (green) and K14 (red) in E18.5 wild-type (F), Gli2^−/− (I), Shh^−/− (L), K5–Cre;Z/AP–Gli2;Gli2^−/− (Q), K5–Cre;Z/AP–Gli2; Shh^−/− (T), K5–Cre;Z/AP–ΔNGli2; Gli2^−/− (Y), and K5–Cre;Z/AP–ΔNGli2; Shh^−/− (BB) skin. Bars, 50μm.

Figure 7

Gli2 activator regulates cyclin D1 and cyclin D2 expression in the developing epithelium. Expression of cyclin D1 (A,B,C,G,H,K,L) and cyclin D2 (D,E,F,I,J,M,N) in E18.5 wild-type (A,D), Gli2^−/− (B,E), Shh^−/− (C,F), K5–Cre;Z/AP–Gli2; Gli2^−/− (G,I), K5–Cre;Z/AP–Gli2;Shh^−/− (H,J), K5–Cre;Z/AP–ΔNGli2;Gli2^−/− (K,M), and K5–Cre;Z/AP–ΔNGli2;Shh^−/− (L,N) mutants. Bar, 50 μm.

Figure 8

Model for Shh regulation of Gli activator and repressor forms in the embryonic hair follicle. In the developing hair follicles, Shh triggers a cascade of events leading to the formation of a potent transcriptional activator Gli2^ACT and inhibiting the formation of Gli repressors (e.g., Gli3). As the balance shifts to predominantly Gli activator forms, the expression of Shh-responsive genes, Ptc1 and Gli1, is induced. Gli1 itself is a potent transcriptional activator that can further potentiate the expression of Shh-responsive genes. Other Shh-responsive genes controlled by Gli activators include cell cycle regulators, such as cyclin D1 and cyclin D2, which are required for the proliferation of epidermal cells in the hair germ. In the absence of Shh, Gli activators will not be formed and Gli repressors will predominate. In our transgenic rescue experiments, we show that both Gli2 and ΔNGli2 can rescue hair follicle development in Gli2^−/− skin, and that wild-type Gli2 requires Shh for its activation, whereas ΔNGli2 can bypass the requirement of Shh. Furthermore, our results suggest a role for Gli repressor in the developing skin, as ΔNGli2 cannot fully restore hair follicle development in Shh^−/− skin, which is expected to possess a high level of Gli repressors (Gli3). (Black boxes) Endogenous Gli; (grey ovals) transgenic Gli.