Fig. 3. Histopathological changes of Msx2 knockout mice in different hair cycle stages. Left column, normal cycling showed shortened anagen. Differences in skin and hair follicle morphology between wild-type and Msx2 knockout mice start to show at postnatal day 3 (P3). There is a lack of hair cortex differentiation at P5 (A,B). Anagen in Msx2 knockout mutant is shorter and hair follicles at P10 have already entered catagen (C,D). Catagen progression is also delayed compared with wild type. At P21, wild-type follicles have entered telogen, whereas Msx2 knockout mutant follicles are still in catagen (E,F). At P24, control skin has entered anagen, while Msx2 knockout mutants are still in telogen (G,H). Msx2 knockout mutant skin eventually re-entered anagen at P31. Examination of Tgfa expression supported the histological observation (E,F, insets). No Tgfa expression was detected in wild type hair follicles at P21 whereas strong Tgfa expression was still present in Msx2 knockout mutant hair follicles (arrow, in inset of F). Scale bars: 500 µm. Quantification (below) was carried out by counting about 50 hair follicles (at designated days after birth) and converting them into percentage of hair follicles in different hair cycle stages. Orange is anagen, green is catagen and blue is telogen. Error bar represents one standard deviation. (Right column) Regeneration after plucking showed defect in re-entry into anagen. One-month-old mice were stripped with hot wax and followed at 6, 10, 14 and 20 day post-stripping. Note that normal hair follicles re-enter anagen at day 6 (C,E), while those of Msx2 knockout mutants remain in telogen (D,F), and do not enter anagen until 20 days after stripping (H). At this time, control skin has re-entered telogen (G). Quantification is achieved as described in the development column. Scale bar: 500 µm. Arrowheads in E,F indicate dermal papilla stained with alkaline phosphatase.

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