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First published online 11 October 2006
doi: 10.1242/dev.02644

Development 133, 4427-4438 (2006)
Published by The Company of Biologists 2006
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Jagged 1 is a ß-catenin target gene required for ectopic hair follicle formation in adult epidermis

Soline Estrach1,*, Carrie A. Ambler1,*, Cristina Lo Lo Celso1, Katsuto Hozumi2 and Fiona M. Watt1,{dagger}

1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK.
2 Department of Immunology, Tokai University School of Medicine, 143 Shimokasuya, Isehara, Kanagawa 259-1193, Japan.


Figure 1
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  		Fig. 1. Modulation of Notch activity in mouse epidermis. (A) Anti-GFP (green) and anti-Jag1 (red) immunostaining with DAPI (blue) counterstain in a section of back skin anagen hair follicle of a TNR mouse. The bracket represents the pre-cortex, open arrows the outer root sheath, solid arrows the inner root sheath and asterisk the dermal papilla. (B,C) Tail epidermal whole mount of TNR mouse labelled with anti-GFP (green) antibody and DAPI (blue). (B) An optical transverse section at the position of the white line through z-stack images in C. The dashed line in B represents the basal epidermal surface. (D,E) Single plane images of wild-type tail epidermal whole mounts labelled with antibody to activated Notch (NICD) (D) or secondary antibody alone (E) (green) with DAPI (blue) counterstain. (F-H) Whole mounts of Jag1flox/flox (F,G) and K5Cre Jag1flox/flox (H) epidermis immunolabelled with anti-Jag1 antibody. The dashed lines indicate hair follicles and sebaceous glands. (G) Jagged 1 expression in the IFE. (I,J) Sections of Jag1flox/flox (I) and K5Cre Jag1flox/flox(J) hair follicles labelled with antibodies to keratin 14 (green) and cleaved Notch1 (red) and DAPI counterstain (blue). (K) Mouse keratinocytes transiently transfected with Hes1 luciferase reporter (Hes1-luc) alone or in combination with NICD{Delta}OPER transgene and either treated with 4OHT (red bars) or untreated (blue bars). Data are expressed as average light units ±s.d., relative to the Renilla luciferase control. (L-O) Anti-ER immunostaining (red) with DAPI counterstain (green) of back skin from wild-type (L) or K14NICD{Delta}OPER transgenic (M-O) mice treated with acetone (M) or 4OHT (L,N,O) for 1 hour. Scale bars: 50 µm in B,C,I,J,L-N; 100 µm in A-H. (P-R) Gross phenotype of 7.5-week-old K5Cre Jag1flox/flox and Jag1flox/flox littermates (P), and wild-type (Q) and K14NICD{Delta}OPER (R) mice treated with 4OHT for 14 days. Insets in Q and R are higher magnification images of back skin.

 

Figure 2
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  		Fig. 2. Histological analysis of K5Cre Jag1flox/flox, K14CreER Jag1flox/flox and K14NICD{Delta}OPER skin. (A-H) H&E stained sections of back skin of Jag1flox/flox mice aged 5 (A) and 10 (B) weeks, K5Cre Jag1flox/flox mice aged 5 (C), 7.5 (D) and 10 (E) weeks, and 4OHT-treated (21 days) K14NICD{Delta}OPER mice aged 11 (F) and 13 (G,H) weeks. Arrows indicate epidermal cysts, asterisks mark sebaceous glands, and bracket denotes hair follicle doublet. Scale bars: 100 µm in A-E; 200 µm in F-H.

 

Figure 3
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  		Fig. 3. Analysis of differentiation markers in K5Cre Jag1flox/flox, K14CreER Jag1flox/flox and K14NICD{Delta}OPER skin. (A-O) Back skin of Jag1flox/flox (A,H,I,L,M), K5Cre Jag1flox/flox (B,D,J,K,N,O) and 4OHT-treated K14NICD{Delta}OPER (C,E,F,G) mice stained with antibodies to K10 (A-D), CDP (E,H-K), hair keratins (AE13) (F) or trichohyalin (G,L-O). (H,J,L,N) Sections DAPI counterstained (blue). (P-W) Whole mounts of Jag1flox/flox (P-S), K5Cre Jag1flox/flox (T-V) and K14CreER Jag1flox/flox (W) tail epidermis immunolabelled for K14 (P,T,S,W) and CDP (Q,U) or stained with Nile Red (green) and DAPI (blue) (R,V). Dashed lines outline the hair follicles and sebaceous glands. Scale bars: 50 µm in D; 100 µm in A-C,E-W. A, anagen follicles; T, telogen follicles.

 

Figure 4
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  		Fig. 4. Pharmacological inhibition of Notch signalling impairs the ability of ß-catenin to induce new hair follicles. (A) Lysates from total skin of wild-type mice treated with 1 mg DAPT or vehicle alone were analysed by western blotting with antibodies to activated Notch (NICD) and ß-tubulin. (B,C) K14{Delta}N ß-cateninER mouse keratinocytes were cultured with or without 4OHT, DAPT or 4OHT + DAPT for 72 hours, then co-transfected with (B) Renilla luciferase (pRL) and Hes1 luciferase (Hes1-Luc), with or without full-length Notch1 (hNotch1) or (C) with pRL and TOPFLASH or FOPFLASH luciferase reporters. Data are expressed as average light units ±s.d., relative to the Renilla luciferase control. *P<0.011. n, number of replicate samples. (D-G) H&E (D,E) and anti-CDP (F,G) stained sections of back skin from K14{Delta}N ß-cateninER mice treated with 4OHT (D,F) or 4OHT + DAPT (E,G). Arrows indicate ectopic hair follicles. Scale bars: 100 µm.

 

Figure 5
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  		Fig. 5. Deletion of Jag1 blocks the ability of ß-catenin to induce new hair follicles. (A-I) Back skin sections of 4OHT-treated Jag1flox/flox (A,D,E), K14{Delta}N ß-cateninER (B,F,G) and triple transgenic mice (C,H,I). Sections were stained with H&E (A-C) or with antibodies to jagged 1 (D,F,H) and ß-catenin (E,G,I) with DAPI counterstain (blue). E,G,I show nuclear pool of ß-catenin in IFE. Scale bars: 50 µm in E,G,I; 100 µm in A-C,D,F,H. (J-U) Whole mounts of tail epidermis from 4OHT-treated Jag1flox/flox, K14{Delta}N ß-cateninER and triple transgenic mice labelled with antibodies to K14 (J,N,R), CDP (L,P,T), ß1 integrin (red) and Ki67 (green) (M,Q,U), or stained with Nile Red (green) and DAPI (K,O,S). Dashed lines outline hair follicles and sebaceous glands. Scale bar: 100 µm.

 

Figure 6
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  		Fig. 6. Notch activation enhances ß-catenin-induced ectopic hair follicle formation and maturation. (A-L) Back skin of wild-type (A,D,G), K14{Delta}N ß-cateninER (ßcatER) (B,E,H,K) and K14{Delta}N ß-cateninERxK14NICD{Delta}OPER double (ßcatER/NICD{Delta}OPER) (C,F,I,L) transgenic mice treated with 4OHT for 8 days. Sections were stained with H&E (A-C) or with antibodies to CDP (D-F), Ki67 (G-I) or trichohyalin (J-L). Arrows mark new follicles arising from preexisting follicles. (J) Quantitation of number of CDP positive areas (±s.e.m.) per existing follicle in K14{Delta}N ß-cateninER single or K14{Delta}N ß-cateninERxK14NICD{Delta}OPER double transgenics treated with 4OHT for the number of days shown. (M-Q) Epidermis of TNR mice crossed with wild-type (WT;M,O) or K14NICD{Delta}OPER (N,P) or K14{Delta}N ß-cateninER (Q) mice stained with antibodies to GFP (brown). Scale bars: 100 µm.

 

Figure 7
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  		Fig. 7. Jag1 is a direct target gene of ß-catenin. (A-E) Whole mounts of K14{Delta}N ß-cateninER tail epidermis labelled with antibodies to K14 (red) and jagged 1 (green). Mice were untreated (A) or treated for 21 days with 4OHT and either examined immediately (B) or after 21 days without 4OHT treatment (C). Following 21 days without treatment, mice received 4OHT for 7 days (D) or 14 days (E). (F,G) Immunostaining for jagged 1 (red) in back skin sections from K14{Delta}N ß-cateninER mice treated with 4OHT (F) or 4OHT + DAPT (G). Scale bars: 100 µm (A-G). (H-J,L,M) Immunostaining for jagged 1 (green) in back skin sections (H-J) and tail epidermal whole mounts (L,M) of wild-type (H,I,L) and K14{Delta}NLef1 (J,M) mice. H is a control with no primary antibody. DAPI (blue) is shown at lower magnification (F-J). Dashed lines in L,M indicate hair follicles and sebaceous glands. Scale bars: 100 µm in H-J,L,M. (K) Wild-type or K14{Delta}N ß-cateninER mouse keratinocytes were cultured with or without 4OHT for 72 hours, then co-transfected with pRL (Renilla luciferase) and different ratios of empty vector (ev), mNotch1ICD (NICD) and Hes1 luciferase (Hes1-Luc). Data are expressed as average light units ±s.d., relative to the renilla luciferase control. *P<0.01. n, number of replicate samples. (N) Location of putative Tcf/Lef-binding sites in the mouse Jag1 promoter. (O) RT-PCR of Jag1 and ß-actin transcripts from wild-type and K14{Delta}N ß-cateninER mouse keratinocytes treated with acetone or 4OHT in the presence of cycloheximide. Three independent experiments were analysed with consistent replicates and consistent results among experiments. (P) Wild-type and K14{Delta}N ß-cateninER mouse keratinocytes were fixed and subjected to chromatin immunoprecipitation using anti-ß-catenin or control antibodies. Primers surrounding the putative Tcf/Lef-binding sites or an irrelevant region of the Jag1 promoter (831-370) were used for the PCR analysis.

 




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