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Expression of NLef1 in mouse epidermis results in differentiation of hair follicles into squamous epidermal cysts and formation of skin tumours

¹ Imperial Cancer Research Fund, 44 Lincoln’s Inn Fields, London WC2A 3PX, UK
² Max Delbrück Center for Molecular Medicine, 13125 Berlin, Germany

View larger version (42K): [in a new window]   Fig. 1. Construction, expression and activity of the K14NLef1 transgene. (A,D) Schematic representations of (A) the K14NLef1 transgene construct and (D) the structure of a hair follicle. (B) Immunoblot of epidermis from wild-type (WT) and transgenic (TG) mice using anti-Myc-tag to detect the transgenic protein and anti-actin as a loading control. (C) Semi-quantitative RT-PCR for endogenous Lef1 (WTs + as), transgenic NLef1 (TGs + as) and HPRT of RNA isolated from primary wild-type (wt) or transgenic (tg) keratinocytes. Left-hand lane in each gel shows molecular weight markers and lanes labelled H show controls in which no RNA was added. PCR reactions were analysed after (left to right) 10, 25, 35 or 65 (65 not shown for HPRT) rounds of amplification. (E) Luciferase reporter assay. Keratinocytes were isolated from wild-type (WT) and transgenic (TG) animals and transfected with pTOPFLASH (TOP) or pFOPFLASH (FOP). In some cases, cells were co-transfected with the (ßcat/Lef1 fusion construct (Fusion), N-terminally truncated ß-catenin (T2) or Tcf-3 (TCF). Luciferase activity values are shown in light units and represent the average of triplicate determinations, corrected for transfection efficiency. (F) Immunoblot of total and Triton-soluble protein lysates of primary keratinocytes isolated from wild-type (wt) and K14NLef1 (tg) mice with antibodies to E-cadherin, ß-catenin, plakoglobin and actin (loading control). (G) Flow cytometry of primary keratinocytes from wild-type (wt) and K14NLef1 (tg) mice labelled with anti-ß1 integrin antibody or secondary antibody alone.

View larger version (92K): [in a new window]   Fig. 2. Phenotype of the K14NLef1 mice. (A) 10-month-old K14NLef1 transgenic mouse (line L). (B) Belly of transgenic animal shown in A with small white lumps indicated (arrows). (C-F) Sections of skin from 3.5-month-old wild-type (C,D) and transgenic (E,F) animals. Sections in C,E were stained with Haematoxylin and Eosin. Sections in D,F were labelled with anti-Ki67; arrows, Ki67-positive cells in hair follicles and interfollicular epidermis; arrowheads, Ki67-positive cells in peripheral layer of cyst. (G-I) Expression of the NLef1 transgene (green in G-I) and ß-catenin (green in J,K) in wild-type (G,J) and transgenic (H,I,K) skin. NLef1 was detected with an antibody to the Myc-tag in all cells of the basal layer of the interfollicular epidermis (arrows in H) and the outer root sheath (arrowheads in H,I) of K14NLef1mice (H,I) but not in the skin of wild-type animals (G). Nuclei in G-I were visualised with DAPI (blue). (J,K) ß-catenin antibodies detected the protein at cell-cell borders in hair follicles of wild-type and transgenic animals. In addition, ß-catenin was detected in the nucleus of precortex cells of the hair follicles (arrows in J,K) in wild-type (J) and transgenic mice (K). (L-N) Immunostaining of wild-type skin (L) and skin from K14NLef1 mice (M,N) with anti-ß1 integrin antibody. Positive staining was detected throughout the basal layer of the interfollicular epidermis (arrows in L,M), the outer root sheath of hair follicles (arrowheads in L,M) and the outer layer of epidermal cysts (arrows in N). (O-R) Sections of skin from wild-type (O,Q) and K14NLef1 mice (P,R) 7 days (O,P) and 14 days (Q,R) after wounding. Arrowheads in P show edges of wound where re-epithelialisation has not occurred. IE, interfollicular epidermis; HF, hair follicle. Scale bars: 100 µm (C,E,O-R); 50 µm (D,F,J,K); 25 µm (L-N); 20 µm (G-I).

View larger version (101K): [in a new window]   Fig. 3. Transmission electron microscopy of epidermal cysts and expression of markers of hair and epidermal differentiation in cysts. (A-C) Transmission electron microscopy of interfollicular epidermis of wild-type mouse (A) and cysts from transgenic mouse skin (B,C). Arrowheads indicate basement membrane in A and boundary between epithelial cells and dermis in B,C. c, cornified layers. Asterisks in A and C indicate cells with keratohyalin granules. Scale bar: 2 µm. (D-I) Expression of markers of hair and epidermal differentiation in transgenic skin (D,F-I) and wild-type skin (E). Red fluorescence: DAPI staining of cell nuclei. Green fluorescence: hair keratin Ha5 (D,E; note that staining of cornified layer of interfollicular epidermis is nonspecific); keratin 1 (F); filaggrin (G); keratin 6 (H); and laminin (I). IE, interfollicular epidermis; HF, hair follicle. Arrowhead in E indicates matrix cells. Arrowheads in F,G indicate positive staining in cysts. Scale bars: 50 µm in D,F-I; 100 µm in E.

View larger version (82K): [in a new window]   Fig. 4. Transdifferentiation of hair follicles in K14NLef1 transgenic animals. Hair follicles from K14NLef1 transgenic (A-C,E) and wild-type (D) mice during anagen of the first postnatal hair cycle. (A) Haematoxylin and Eosin staining; arrows indicate residual hair shaft; arrowheads indicate developing cyst. (B) Immunofluorescence staining with antibody to keratin 10 (green) and DAPI counterstain (red). MC, matrix cells; ORS, outer root sheath. Arrows and arrowheads show individual keratin 10-positive cells. (C) In situ hybridisation for Shh; arrows indicate positive signals in hair follicles and arrowheads indicate early cysts that do not express Shh. (D,E) Scanning electron micrographs of plucked hairs. Scale bars: 50 µm in A,B; 100 µm in C, 20 µm in D,E.

View larger version (115K): [in a new window]   Fig. 5. Comparison of the first postnatal hair cycle in transgenic and wild-type mice. (A-F) Haematoxylin and Eosin stained sections of skin from 5- (A,B), 7.5- (C,D) and 9- (E,F) week-old wild-type (A,C,E) and K14NLef1 transgenic animals (B,D,F). Scale bar: 200 µm. (G) Schematic representation of the time of onset (start of bar) and duration (bar length) of first postnatal hair cycle in wild-type (wt) and transgenic (tg) animals. The number of animals examined (n) is indicated. See text for explanation of features marked with arrows and arrowheads.

View larger version (137K): [in a new window]   Fig. 6. Tumours in the skin of adult transgenic mice. Paraffin-embedded sections were stained with Haematoxylin and Eosin (A,B,E,G,H), Oil Red O (C), Ki67 (D) or keratin 10 (F). Tumours shown are sebaceous adenoma (A-D), sebeoma (E,F), squamous papilloma (G) and invasive squamous carcinoma (H). IE, interfollicular epidermis. Scale bars: 100 µm in A,F-H; 50 µm in B-D,F. See text for explanation of features marked with arrows and arrowheads.

View larger version (140K): [in a new window]   Fig. 7. Characterisation of spontaneous tumours of K14NLef1 mice and model of their development. Paraffin-embedded sections of sebaceous adenoma from K14NLef1 mice (A,B,D) and chemically induced squamous carcinoma of wild-type mouse (C) were immunostained with anti-Myc epitope (A), anti-ß-catenin (B), or anti-cyclin D1 (C,D) antibodies. Diffuse staining of stroma and sebocytes in C,D is nonspecific. (E) In situ hybridisation for Ptc in sebaceous adenoma (arrowheads) and overlying interfollicular epidermis (arrow indicates expression in hair follicle). (F) Schematic model of consequences for differentiation and tumorigenesis of activating (green; Gat et al., 1998) or repressing ß-catenin signalling (red) in mouse skin. Scale bars: 50 µm in A,C,D; 25 µm in B; 100 µm in E.