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First published online 4 July 2007
doi: 10.1242/dev.02868

Development 134, 2795-2806 (2007)
Published by The Company of Biologists 2007
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Bi-compartmental communication contributes to the opposite proliferative behavior of Notch1-deficient hair follicle and epidermal keratinocytes

Jonghyeob Lee*, Jacob M. Basak, Shadmehr Demehri and Raphael Kopan{dagger}

Department of Molecular Biology and Pharmacology, and Division of Dermatology, Department of Medicine, Washington University School of Medicine, Box 8103, 660 South Euclid Avenue, Saint Louis, MO 63110, USA.


Figure 1
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  		Fig. 1. Notch1 loss leads to reduced cell numbers in hair follicles but to an expansion in the number of epidermal keratinocytes. (A) ß-galactosidase (lacZ, green) immunohistochemistry on Msx2Cre;Notch1flox/flox;Rosa26R/+ skin. Note that ß-galactosidase staining displays the area of Notch1 deletion. (B) Stereoscopic side view of anagen-deleted Notch1 (anN1; left) and embryo-deleted Notch1 (emN1; right) skin at 9 days of age (P9). (C) Stereoscopic view magnified from the images shown in B after dissection. Wild type, left (blue box); anN1, middle (yellow box); emN1, right (red box). Arrows indicate guard hairs. (D) Histological view of the follicles shown in B and C. The different genotypes are designated as in C. Nuclei were stained with DAPI. (E) Average number of hair matrix cells (below the red line in D) per hair follicle. *P<0.00001. Error bars represent s.d. (F,G) Hematoxylin and Eosin staining of P9 (F) and P453 (G) skin. Notice the epidermal hyperplasia in older emN1 skin (G). Scale bars: 50 µm in A; 300 µm in B; 200 µm in C; 20 µm in D; 40 µm in F,G.

 

Figure 2
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  		Fig. 2. Notch1 loss causes decreased mitotic rates and a slight increase in apoptosis, but not acceleration in catagen onset. (A) Hematoxylin and Eosin staining of anagen-deleted Notch1 (anN1), embryo-deleted Notch1 (emN1) and wild-type (WT) skin at 9 days (P9), P13, P17 and P22 old. Note that hair cycling was not altered in Notch1-deficient hair follicles. (B) TUNEL staining of emN1 and anN1 follicles (P9), and of wild-type hair follicles [P9 and P16 (catagen)]. (C) Average number of TUNEL-positive cells per hair follicle in each genotype shown in B. *P<0.05 for both wild type versus emN1 and anN1 versus emN1; **P<0.05 for both wild type versus wild type (catagen) and anN1 versus wild type (catagen); ***P<0.005. (D) Immunofluorescence staining of BrdU (green) and Ki67 (red). (E) Progenitor labeling index of anN1, emN1 and wild-type hair follicles and epidermis. *P<0.01 for anN1 versus emN1 and wild type versus emN1. Error bars represent s.d. Scale bars: 200 µm in A; 20 µm in D.

 

Figure 3
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  		Fig. 3. Reduced mitotic rates in hair follicles correlates with elevated expression of cytostatic and proapoptotic genes. (A) Microdissection of hair bulbs from embryo-deleted Notch1 (emN1) or anagen-deleted Notch1 (anN1)/wild-type (WT) follicles (grouped for this image because they have the same morphology). (B) Quantitative reverse transcriptase (qRT)-PCR of G1 cyclins, cyclin kinase inhibitors (CKIs; Cdkn1a, Cdkn2b and Cdkn1c), gasdermin 1 and Scotin using total RNA isolated from the microdissected samples shown in A. Error bars represent s.e.m. Scale bars: (A, left) 100 µm; (A, right) 200 µm.

 

Figure 4
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  		Fig. 4. p53 family members do not contribute to the Notch1-deficient follicular phenotype. (A) Quantitative reverse transcriptase (qRT)-PCR of p63 and p73. (B) Immunohistochemistry of p73 in hair follicles of anagen-deleted Notch1 (anN1), embryo-deleted Notch1 (em-N1) and wild-type (WT) littermates. (C) qRT-PCR with Notch1, Trp53 (p53) and p53 target genes [Cdkn1a (p21), Igfbp3 and Scotin] using total RNA from the hair bulbs of the genotypes specified (emN1;p53-/-indicates Msx2Cre;Notch1flox/flox;Trp53-/-). Note the sustained increase in Scotin and Igfbp3 expression in the absence of p53. Error bars represent s.e.m. Scale bar: 50 µm.

 

Figure 5
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  		Fig. 5. Altered IGF/IGFBP equilibrium in the hair matrix of Notch1-deficient hair follicles. (A) Quantitative reverse transcriptase (qRT)-PCR of IGF signaling components. Error bars represent s.e.m. (B) Immunohistochemistry (Igfbp2 and Igfbp3) and in situ hybridization (Igf1R and insets for Igfbp3) of IGF signaling components on hair follicles. DAPI was used to label nuclei during antibody staining. Arrows indicate dermal papilla (DP)-specific Igfbp3 immunoreactivity. Note the mRNA and protein expression of Igfbp3 in the DP, in which Notch1 is not expressed. (C) Histological comparisons of the hair follicles from the genotypes specified. Note that the matrix size is restored in embryo-deleted Notch1 (em-N1);Ivl::Igf1 follicles. (D) Average number of hair matrix cells (below the red line in C) per hair follicle. Error bars represent s.d. anN1, anagen-deleted Notch1. Scale bars: 40 µm in B; 200 µm [skin hematoxylin and Eosin (H&E) staining] and 50 µm (follicular H&E and DAPI staining) in C.

 

Figure 6
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  		Fig. 6. Notch1 loss alters the expression of the diffusible ligands Kitl and Tgfß. (A) Quantitative reverse transcriptase (qRT)-PCR of Tgfß signaling components and a downstream target (Col1a2). (B) qRT-PCR of the genes specified using Notch1- and Tgfbr2-deficient hair bulbs. (C) qRT-PCR of Kitl using hair follicle total RNA from the genotypes specified. (D) Immunohistochemical staining of wild-type and Msx2Cre;Notch1flox/flox;Rosa26R/+ mice with Dct (green) and ß-galactosidase (lacZ, red). Nuclei were stained with DAPI. Note that melanocytes (Dct positive) do not show ß-galactosidase staining. (E) The ratio of melanocytes (Dct positive) to the matrix cells in each follicle was obtained from the different genotypes specified. Notice the reduced number of melanocytes in Notch1-deficient hair follicles (*P<0.0001 in both WT versus em-N1 and an-N1 versus em-N1). Error bars represent s.e.m. (A-C) and s.d. (D). Scale bar: 50 µm.

 

Figure 7
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  		Fig. 7. Monitoring the epidermal expression of genes with altered expression in Notch1-deficient hair follicles. (A) Western blot analyses of postnatal day (P)9 epidermal cell lysates from embryo-deleted Msx2-Cre;Notch1flox/flox (N1CKO) mouse (N1EP) and its wild-type littermate (WTEP). (B) Quantification of the western data from A. (C) Quantitative reverse transcriptase (qRT)-PCR of Notch1 and its targets, Hes1, HeyL and Nrarp, using P9 epidermal total RNA. Error bars represent s.e.m. *P=0.05; **P<0.001.

 

Figure 8
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  		Fig. 8. Monitoring epidermal Notch targets for their expression in hair follicles. (A) Quantitative reverse transcriptase (qRT)-PCR for Notch1, Nrarp, Hes1 and Heyl. Notice that Hes1 expression was sustained in embryo-deleted Notch1 (em-N1) hair follicles. Error bars represent s.e.m. (B) mRNA (Notch1, Nrarp and Cdkn1a) and protein (Hes1) expression in skin. Hes1 (red) expression is found in differentiating hair matrix and epidermal suprabasal cells. AE13 (green) is a marker for follicular cortex and cuticle. Nuclei were stained with DAPI. anN1, anagen-deleted Notch1; WT, wild type. Scale bars: 40 µm.

 

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© The Company of Biologists Ltd 2007