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First published online 17 July 2007
doi: 10.1242/jcs.007104

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Miz1 is required for hair follicle structure and hair morphogenesis

¹ Institute for Cell Biology, University of Marburg, Robert-Koch-Str. 6, 35033 Marburg, Germany
² Institut de recherches cliniques de Montreal, 110 Avenue des Pins West, Montreal, QC, H2W 1R7, Canada
³ Institute for Molecular Biology and Tumor Research (IMT), University of Marburg, Emil-Mannkopff-Str.2, 35033 Marburg, Germany
⁴ Institut de recherches cliniques de Montreal, Department de Microbiologie et Immunologie, Université de Montréal, 110 Avenue des Pins West, Montreal, QC, H2W 1R7, Canada

View larger version (22K): [in this window] [in a new window]   Fig. 1. Conditional knockout of Miz1 POZ/BTB domain in keratinocytes. (A) K14cre mediates efficient recombination of the Miz1 lox allele in primary keratinocytes. For genotyping, PCR was performed on genomic DNA from keratinocytes that were isolated from back skin of 1-day-old mice without subsequent culture, using primers indicated in supplementary material Fig. S1. Note that in K14Cre+/Miz1+/lox keratinocytes, the wild-type fragment using primers 1 and 5 is inefficiently amplified because of competition in the PCR by the shorter fragment. The wild-type and loxP allele fragment amplified with primers 1 and 5 (about 1400 bp) differed only in 70 bp and could not be distinguished. (B) Levels of Miz1 mRNA in keratinocytes K14Cre+/Miz1lox/lox and control mice. Miz1 mRNA was almost undetectable in K14Cre+/Miz1lox/lox keratinocytes using primers 3 and 4, which are located in the region coding for the POZ/BTB domain. By contrast, when primers 6 and 7, which are located outside the POZ/BTB domain coding region, were used, a signal was obtained as in control mice, indicating the presence of a truncated Miz1 transcript lacking the POZ/BTB coding region. (C) Western blot analysis, using the anti-Miz1 antibody 10E2, of proteins immunoprecipitated from keratinocyte lysates using the anti-Miz1 antibody H-190. A truncated Miz1 protein could be detected in K14Cre+/Miz1lox/lox and K14Cre+/Miz1+/lox keratinocytes (arrow). Arrowhead indicates Miz1.

View larger version (95K): [in this window] [in a new window]   Fig. 2. Phenotype and histological analysis of K14Cre+/Miz1lox/lox mice. (A) Eight-day-old (P8) K14Cre+/Miz1 lox/lox mice (foreground) exhibit rough fur compared with control littermates. (B,C) Histological analysis of hematoxylin-stained sections revealed an impaired arrangement and wavy structure of hair follicles (C), when compared with sections taken from control mouse skin (B). (D) Sections from control mice (anagen) stained with an antibody against K14 (red). Expression of K14 was observed in the basal layer of the epidermis in the entire hair follicle. This was strong in the upper part of the hair funnel and decreased towards the hair bulb. (E) K14Cre+/Miz1lox/lox mice showing strong K14 expression extended into the lower part of some hair follicles (arrows). (F,G) Boxed areas of E at a higher magnification. (H) In addition, cyst-like hair follicles occurred with hair remnants. (I-K) Ectopic strong expression of K14 in hair follicles of K14Cre+/Miz1lox/lox mice which were delayed in the catagen stage. Note the extreme misorientation of one follicle in K (arrow). Bars, 400 µm (B,C,D,E,I,J); 100 µm (H,K); 50 µm (F,G).

View larger version (93K): [in this window] [in a new window]   Fig. 3. (A,B) Histological analysis of the epidermis in the hair follicle and around the hair funnel orifice. (C) Using an antibody against the proliferation marker Ki67 only basal keratinocytes were stained in control mice. (D) In K14Cre+/Miz1lox/lox mice, suprabasal cells were also positive for Ki67, correlating with an increase in the number of cell layers in the epidermis. (E-H) In these areas some suprabasal cells were not only positive for keratin 1 (E,F) but also for keratin 14 (H; green), which was restricted to basal keratinocytes in control mice (G, green). Furthermore, K14Cre+/Miz1lox/lox mice had a thicker cornified layer positive for filaggrin (G,H; red). Note that the suprabasal cells of the epithelium from cyst-like structures (asterisks) also express K1, which is usually found in the interfollicular rather than in the intrafollicular epidermis (compare with Fig. 2H). Bar, 100 µm (A,B,E-H); 20 µm (C,D).

View larger version (83K): [in this window] [in a new window]   Fig. 4. Hair cycle alteration in K14Cre+/Miz1lox/lox mice. (A-H) Representative sections of comparable areas of back skin from P19 (A,B), P23 (C,D), P26 (E,F) and 1-year-old (G,H) control (A,C,E,G) and K14Cre+/Miz1lox/lox mice (B,D,F,H). Note the sharp boundary between dermis and subcutis (arrow). Such sections were used to count the number of all hair follicle and those which are extending into the subcutis, representing hair follicle either in catagen or anagen, depending on the chosen time point and the hair follicle morphology. (I) Quantitative analysis of the percentage of subcutaneous follicles at different ages as indicated. The bars represent the medians. The differences of the medians for P18/19 and for one year were statistically significant (for both P<0.0001). (J) Proliferation of keratinocytes measured by the BrdU-labeling index. Mice received BrdU 1.5 hours before being sacrificed and sections were stained with an anti-BrdU antibody to count the percentage of S-phase cells, either in the interfollicular skin or in the hair follicle. The twofold difference found in the hair follicle epithelium was statistically significant (P<0.0001). The bars represent the medians. n, number of animals analyzed.

View larger version (79K): [in this window] [in a new window]   Fig. 5. Histological analysis of skin from 1-year-old control (A,E) and K14Cre+/Miz1lox/lox mice (B,C,F-H). Hematoxylin and eosin staining (A-C) revealed aberrant hair follicle morphology with thickened epithelium and distorted hair remnants in the follicle (B,C). In addition, pigmentary incontinence was observed in K14Cre+/Miz1lox/lox (F,H) but not in control (E) mice with Fontana-Masson staining. (G) Melanin deposits in the upper third of the hair follicle epithelium. (D) Hair remnants and ectopic melanin deposition appear as black spots in stereomicroscopic analysis of back skin (arrows). (I-K) K14Cre+/Miz1lox/lox mice showed a decrease in hair density (K) while specifically zigzag hairs were reduced (I; P<0.0001; bars represent mean values). In the upper panel in K, the light area is due to the unpigmented tips of the zigzag hairs which are missing in K14Cre+/Miz1lox/lox mice (lower panel). The pigmentation of the hairs was irregular in K14Cre+/Miz1lox/lox mice with coarse melanin inclusions, and the bends of zigzag hairs were diminished (J; arrow). Bars, 100 µm (A-C,E-H); 200 µm (D); 50 µm (J); 5 mm (K).