Progressive alopecia reveals decreasing stem cell activation probability during aging of mice with epidermal deletion of DNA methyltransferase 1

Abstract

To examine the roles of epigenetic modulation on hair follicle regeneration, we generated mice with a K14-Cre-mediated loss of DNA methyltransferase 1 (DNMT1). The mutant shows an uneven epidermal thickness and alterations in hair follicle size. When formed, hair follicle architecture and differentiation appear normal. Hair subtypes exist but hair fibers are shorter and thinner. Hair numbers appear normal at birth but gradually decrease to <50% of control in 1-year-old mice. Sections of old mutant skin show follicles in prolonged telogen with hyperplastic sebaceous glands. Anagen follicles in mutants exhibit decreased proliferation and increased apoptosis in matrix transient-amplifying cells. Although K15-positive stem cells in the mutant bulge are comparable in number to the control, their ability to proliferate and become activated to form a hair germ is reduced. As mice age, residual DNMT activity declines further, and the probability of successful anagen reentry decreases, leading to progressive alopecia. Paradoxically, there is increased proliferation in the epidermis, which also shows aberrant differentiation. These results highlight the importance of DNA methylation in maintaining stem cell homeostasis during the development and regeneration of ectodermal organs.

Figure 1. Expression of DNMT1 in Developing and Cycling Hair Follicles

(A) Sections of mouse dorsal skin at E14, 16, and postnatal day 0, 4, 9 were subjected to immunohistochemistry. DNMT1 is mainly expressed in the basal layer of the epithelium and hair matrix. Some DNMT1 was seen in the dermis. (B) In the adult, DNMT1 was expressed in ORS and matrix region of anagen follicles, and in hair germ region in telogen. (C) Schematic highlighting the expression. DNMT1 is low in the differentiating suprabasal layer, and hair shaft. E, epidermis; D, dermis; dp, dermal papilla; Mx, matrix. Scale bar: 50 μm.

Figure 2. Genotyping and Conditional Deletion Efficiency of K14-Cre DNMT1^fl/fl Mice

(A) K14-Cre DNMT1^fl/fl DNA schematic (Jackson-Grusby et al., 2001). (B) PCR amplification of LoxP and Cre. (C) PCR amplification of floxed vs Cre mediated excision of DNMT1. High DNMT1 excision levels are associated with a phenotype. Muscle is a negative control. (D) Western blot quantification of DNMT1. DNMT1 protein levels inversely correlated with a phenotype. (E) IAP, 5-methyl cytosine and DNMT1 expression were detected by IHC. IAP level was elevated while 5-methyl cytosine and DNMT1 were decreased in mutant mice compared with WT. Scale bar: 10 μm.

Figure 3. K14-Cre DNMT1^fl/fl Mice Shows Reduced Hair Size in All Hair Types, and Reduction of Hair Density in Older Mice

(A) 1-year-old control and mutant. Size bar: 0.5 cm. (A’) Enlargement. Mutant hair is thinner in size and reduced in number. Size bar: 1 mm. (B) Hair numbers gradually decrease from 2-months to 1 year. Scale bar: 50 μm. (B’) Density of hair fibers in mutant and WT mice in young and old mice. Shown as mean ±SD. (C) Comparison of guard, awl, auchene and zigzag hairs. Shapes are generally fine, but mutant hairs are shorter. Size bar: 3 mm (D) Both hair length and width are reduced. Medulla and width of different hair types. Mutant 2 is more affected than mutant 1. Scale bar: 0.2 mm. Bottom: Quantitative comparison of hair lengths by types (mean ±SD).

Figure 4. Molecular Characterization of K14-Cre DNMT1^fl/fl Mouse Skin

(A) Mutant shows unevenness in the thickness of epidermis and the size of hair follicles. Some follicles show larger diameter follicles and hair canals, but others are smaller. Otherwise the morphology of hair follicles appears normal. IHC of 3 month-old mutants show wide and patchy expression of involucrin. (B) IHC show s mutants have normal expression of p63, K15, AE13, and AE15, but reduced CD200 and increased of P16 expression. Scale bar: 50 μm. (C) 1-year-old mutant skin is mainly composed of telogen follicles. (D) First row, skin from old mutants shows most follicles in telogen, and many without club hairs. K14 IHC appear normal. Second row, uneven thickness of epidermis is obvious. Third row, enlarged sebaceous glands. Scale bar: 0.4 mm. (E) Anagen duration is about the same in 1-year old mutants, , but telogen duration increases significantly. Old mutants showed decreased hair follicle density. Bar diagram is shown as mean ±SD.Two-tailed unpaired Student’s t tests were used (*, p < 0.05).

Figure 5. K14-Cre DNMT1 ^fl/fl Hair Follicles Show Reduced Proliferation and Increased Apoptosis in Hair Follicles

(A) One hour BrdU labeling of 3-month-old mice. In the epidermis, BrdU labeling is increased, but reduced in the hair matrix and ORS. Size bar: 20 μm. (A’). Numbers were quantified (A’, mean ± SD). (B) Cells were labeled with CldU (red) for 11.5 hr, then followed by IdU (green) for 30 mins. CldU cells moved above Auber’s line in WT, but not in mutant cells. Size bar: 50 μm. (B’) Upper: Numbers of CldU or IdU positive cells along the proximal-distal axis of hair follicle were quantified. (C) Double staining with K15 (green) and Ki67 (red) in telogen follicles and long term label retention of CldU. LRCs number was lower in mutants. Scale bar: 10 m. (C’) Quantification. Student’s t-test (*, P<0.05). (D) TUNEL positive cells (green fluorescent or brown IHC) in the matrix, bulge and ORS of mutants. DAPI stains nucleus (blue). (D’) Average number of TUNEL positive cells per follicle, bulge or ORS (mean ±SD). (E) γH2AX, indicators of DNA damage, is increased in hair bulbs (brown in IHC). (E’) Numbers of γH₂AX-positive cells in follicle matrix were quantified (mean ± SD). Size bar: 20 μm.

Figure 6. K14-Cre DNMT1 ^fl/fl Hair Follicles Show delay regeneration after plucking and Summary Diagram

A) Hairs in a 1 cm square are striped with wax. At day 11 after waxing, new appear in WT but not in mutants. At day 21, both hairs have entered telogen. Upper half of the plucked region was shaved to see if they are still in anagen (above green arrows). Scale bar: 2 mm. B) Schematic summary showing the roles of DNMT1 in skin morphogenesis. DNMT1 is involved in regulating epidermal progenitors and also hair follicle homeostasis. In epidermis, mutant epidermis becomes thicker, In hair follicles, DNMT1 is involved in regulating the proliferation and apoptosis of bulge LRC stem cells, ORS, and matrix TA cells. When DNMT 1 is reduced, the probability of successful stem cell activation progressively decreases, leading to disrupted homeostasis in epidermis, hair follicle cycling, and response to plucking. Reduction of hair fibers and follicles lead to the progressive alopecia phenotype.