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First published online 6 August 2008
doi: 10.1242/dev.022202

Development 135, 3149-3159 (2008)
Published by The Company of Biologists 2008
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Dlx3 is a crucial regulator of hair follicle differentiation and cycling

Joonsung Hwang1, Taraneh Mehrani1, Sarah E. Millar2 and Maria I. Morasso1,*

1 Developmental Skin Biology Unit, NIAMS, National Institutes of Health, Bethesda, MD 20892, USA.
2 Department of Dermatology and Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.


Figure 1
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  		Fig. 1. lacZ knock-in into the Dlx3 locus and Dlx3 expression pattern. (A) Exon-intron organization and enzyme restriction patterns of murine Dlx3 locus (N, NdeI and E, EcoRI), before and after homologous recombination (upper panel). Genotyping by Southern blot (lower panel, left) and PCR (lower panel, right) of wild type (WT) and lacZ knock-in (+/-; Dlx3Kin/+) mice. White and black arrowheads indicate the localization of the oligonucleotides used in the PCR reactions. (B) Whole-mount X-gal staining shows Dlx3/lacZ expression patterns in the Dlx3Kin/+ mice. (i) Clarified head of embryological stage, E15.5 Dlx3Kin/+ embryo by benzyl-benzoate/benzyl alcohol after X-gal staining showing expression in the vibrissae; (ii) cross and (iii) sagittal sections of vibrissae at E16.5. (iv) Dlx3/lacZ expression in hfs in the epidermis of E16.5 head (arrows) and (v) sagittal section through the head, showing expression in the intramembranous bone of the cranium (arrowheads) and the hf (arrows).

 

Figure 2
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  		Fig. 2. Dlx3 expression during hf development. (A) Dlx3 expression was detected using an anti-Dlx3 antibody in the hair matrix cells at postnatal day 1 (P1, early anagen), P9 (full anagen) and P17 (catagen) in the telogen bulge cells at P20 (inset, higher magnification of bulge area in box), P25 (the onset of first postnatal anagen) and P30 (first postnatal anagen). (B) (Top row) Immunofluorescence studies on P1 hf using anti-Dlx3 (green) and anti-β-galactosidase (red) to demonstrate colocalization of Dlx3 and lacZ expression. (Lower rows) Immunofluorescence with anti-keratin antibodies (green): anti-K17 at P9; anti-K35 and anti-K85 at P30. Merged images are shown with DAPI staining in the right column. ORS, outer root sheath; IRS, inner root sheath; CU, cuticle; DP, dermal papilla. Scale bar: 50 µm.

 

Figure 3
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  		Fig. 3. Conditional deletion of Dlx3 in the epidermis using K14cre. (A) Gene targeting to generate the floxed-Dlx3 mice. (Left panel) LoxP sites were inserted into the NotI site (N) between the first and second exons of the Dlx3 gene and immediately downstream of the neomycin gene (Neo). White and black arrowheads indicate the localization of the oligonucleotides used in the PCR reactions. (Right panel) Recombination in the ES cells was assessed by Southern blot analysis; WT, wild type; Dlx3f/+, heterozygote for floxed Dlx3 allele. (B) Gross phenotype of wild type (top) and K14cre;Dlx3f/f (bottom) mice at P9 (left) and 15 weeks (right). (C) Genotype by PCR for wild-type (WT) and K14cre;Dlx3f/f mice showing epidermal specific recombination of the Dlx3 allele in the K14cre;Dlx3f/f skin. (D) The specific deletion of Dlx3 expression was detected in the K14cre;Dlx3Kin/f hf extracts by western blot analysis. Asterisk indicates Dlx3-specific band. (E) The specificity of Cre-mediated recombination was also determined by immunohistochemistry on skin sections from wild-type and K14cre;Dlx3Kin/f littermates. Dlx3 (green) expression is shown in the matrix cells of P1 hf in the wild-type skin; however, only the lacZ (red) expression is detected in the K14cre;Dlx3Kin/f skin. Images are shown with DAPI staining to denote nuclear staining. Scale bar: 50 µm.

 

Figure 4
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  		Fig. 4. Abnormalities in the hair growth and cycling in Cre-mediated conditional Dlx3 knockout mice at late stages of postnatal hf morphogenesis. (A,B) Skin samples were obtained from both wild-type (WT) and mutant (K14Cre;Dlx3Kin/f or K14Cre;Dlx3f/f) littermates at P1, P9, P12, P15, P20 and P26; the deparaffinized sections were stained with Hematoxylin/Eosin (A) and anti-PCNA antibody (B, green). (C) Skin sections of wild-type (WT) and mutant K14Cre;Dlx3f/f at P15 were stained with Hematoxylin/Eosin and antibodies against PCNA, K14 and K1. Scale bars: 50 µm.

 

Figure 5
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  		Fig. 5. Follicular signaling in the regulation of Dlx3 expression. (A) Expression of Lef1, β-catenin and Smad1/5/8 were determined in control and conditional knockout skin to analyze effects of absence of epithelial Dlx3 at P1 and P9 anagen stages. (B) Colocalization of Dlx3/lacZ with Lef1. The skin sections of wild type (Dlx3Kin/+) at P1 were stained with by anti-Lef1, anti-phospho Smad1/5/8 and anti-β-galactosidase antibodies (red, middle); the merged images are shown with DAPI staining. (C) (Top) Lef1 direct binding in vivo to the Dlx3 promoter was demonstrated by ChIP assays using Lef1 antibody. Putative Lef1-binding site in the mouse Dlx3 promoter sequence (-160 to +15 bp) is indicated in red. CCAAT box and TATA box are underlined. The transcriptional start site is indicated by an arrow. (Bottom left) Gel image of ChIP assay. (Bottom right) The specificity of the ChIP assays, determined by both control IgG antibody and a set of PCR primers for Gapdh. Scale bars: 50 µm.

 

Figure 6
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  		Fig. 6. Dlx3 is an indispensable regulator for the expression of follicular-specific structural and transcription factors. (A) Expression of Hoxc13, Gata3, and A13 and A15 hair keratins were significantly reduced in the K14Cre;Dlx3Kin/f follicles at P1 and P9 anagen stages. White arrowheads indicate expression areas of each factor. Scale bar: 50 µm. (B) Luciferase reporter assays of the 1.2 kb K35 promoter and mutants 2, 6 and 7 sites in the absence and presence of Dlx3 co-expression. (C) (Left) ChIP assays were performed in primary and transfected mouse hf cells using anti-Dlx3 and anti-V5 antibodies, respectively. Triangles indicate the putative binding sites in the promoters for hair keratins (K32 and K35) and Hoxc13, which are conserved between human (white) and mouse (black). Each number in the triangles correspond to the sequences analyzed by EMSA (see Fig. S3B in the supplementary material). The PCR amplified areas are indicated by black lines in the mouse promoters for hair keratins (K32 and K35) and Hoxc13. (Middle) Gel images for each ChIP assays; the specificity of the amplified fragments was verified by sequencing. (Right) Specificity of the ChIP assays determined by both control IgG antibody and a set of PCR primers for Gapdh.

 

Figure 7
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  		Fig. 7. Role of Dlx3 during hair cycling. (A) Phospho-Smad1/5/8 and Lef1 expression in K14Cre;Dlx3Kin/f and K14Cre;Dlx3f/f at P18, P20 and P26. White arrowheads indicate specific expression. Scale bar: 50 µm. (B) Schematic diagram of Hf morphogenesis and onset of a new hair cycle. The area of Dlx3 expression is indicated in blue, dermal papilla in yellow and keratin-free germinal matrix compartment in gray. Dlx3 expression is first detected in the hair matrix; by anagen stage, Dlx3 is present in all differentiating hair compartments (matrix, cortex, cuticle and IRS) and later is detected in the telogen bulge. Our results demonstrate that Dlx3 is a target of Wnt, and that colocalization of phospho-Smad1/5/8 and Dlx3 is consistent with a regulatory role for BMP signaling of Dlx3. Our results determine that Dlx3 is a direct transcriptional regulator of Hoxc13, Gata3 and hair keratins, and that Dlx3 expression is necessary for the re-initiation of the hair cycle. HS, hair shaft; S, sebaceous gland; B, bulge; ORS, outer root sheath; IRS, inner root sheath; CU, cuticle; DP, dermal papilla.

 

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© The Company of Biologists Ltd 2008