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First published online 3 September 2003
doi: 10.1242/dev.00703

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Manipulation of stem cell proliferation and lineage commitment: visualisation of label-retaining cells in wholemounts of mouse epidermis

Kristin M. Braun1, Catherin Niemann1, Uffe B. Jensen2, John P. Sundberg3, Violeta Silva-Vargas1 and Fiona M. Watt1,*

1 Keratinocyte Laboratory, Cancer Research UK London Research Institute, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
2 Department of Human Genetics, The Bartholin Building, University of Aarhus, DK-8000 Aarhus C, Denmark
3 The Jackson Laboratory, 600 Main Street, Bar Harbor, ME 04609-1500, USA



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  		Fig. 1. Localisation of LRC in different compartments of wild-type mouse epidermis. Epidermal sheets (A-H) or tissue sections (I) were labelled for keratin 14 (red; A-E; H-I) or Ki67 (red; F,G) and BrdU (green) by double-label immunofluorescence. Skin was collected from tail (A-D,F-I) or back (E) of adult wild-type mice. (A) One hour BrdU label. (B-I) Mice received repeated injections of BrdU to generate LRC. Wholemounts prepared following a chase of 2 days (B) or 140 days (C). Asterisk in C indicates site of insertion of dermal papilla. (D,E) Epidermis from 42-day-old mouse (telogen; 31 days post-BrdU injection). (F-I) Epidermis collected following a 70 day chase period. (F) Arrows indicate LRC located in the interfolliclular epidermis. (G) Hair follicle plucked from labelled wholemount. Arrowhead indicates a LRC in the infundibulum. Inset is higher power view of boxed area, showing two cells double-labelled for Ki67 and BrdU. Cells with intense (BrdU heavy) and weak (BrdU light) BrdU labelling are indicated. (H) High power confocal micrograph of sebaceous gland; arrows indicate the position of LRC. (I) Hair follicle in tissue section; arrows indicate keratin 14-positive LRC in the outer root sheath. HF, hair follicle; IFE, interfollicular epidermis; SG, sebaceous gland; BG, bulge. Scale bars: 100 µm.

 


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  		Fig. 2. Localisation of cell lineage markers and putative stem cell markers in tail wholemounts from adult mice. (A-J) Tail epidermal wholemounts from wild-type mice were immunolabelled to detect the proteins indicated or stained with Nile Red (D) to visualise sebocytes. (E) Bracket shows lack of labelling of HF outer root sheath from below the sebaceous gland to the bulb. (J) Brackets show that labelling for keratin 15 is present within the bulge region of hair follicles. Inset is a higher power view of boxed area, showing double-label immunofluorescence for keratin 15 (red) and BrdU (green) in a mouse labelled according to the LRC protocol followed by a chase of 32 days. Staining of sebaceous glands is non-specific because of the use of a mouse primary antibody. (K,L) High-power micrographs of IFE labelled by double-label immunofluorescence for keratin 10 (green) and either ß1 integrin (red; K) or {alpha}6 integrin (red; L). BG, bulge; HF, hair follicle; IFE, interfollicular epidermis; SG, sebaceous gland; CCL, companion cell layer. Scale bars: 100 µm in A-J; 20 µm in K-L.

 


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  		Fig. 3. Examination of tail wholemounts at various stages of the hair growth cycle. Wild-type mice were injected with BrdU according to the LRC protocol and wholemounts were prepared from mice of the following ages: 28 days (early anagen; A,B,E,H,K), 35 days (anagen; C,F,I,L) and 42 days (telogen; D,G,J,M). (A-D) Double labelling for BrdU (green) and Ki67 (red). Arrows indicate representative follicles in early anagen (A), full anagen (C) and telogen (D). Arrowheads in B indicate high-magnification examples of double-labelled cells in early anagen. (E-G) Double labelling for TUNEL positive cells (green) and keratin 14 (red). Arrows indicate TUNEL-positive cells. Arrowheads indicate non-specific staining of hair shafts. Asterisk indicates a follicle in full anagen. (H-J) Immunolabelling for CDP. Arrows indicate representative follicles in early anagen (H), full anagen (I) and telogen (J). (K-M) Immunolocalisation of {alpha}6 integrin. Brackets indicate regions of the hair follicle with strong {alpha}6 integrin labelling during early anagen (K), anagen (L) and telogen (M). Scale bars: 100 µm in A,C-M; 20 µm in B.

 


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  		Fig. 4. Loss of LRC following treatment with TPA. Wild-type mice were injected with BrdU according to the LRC protocol. After a chase period of 10 months, wholemounts of tail epidermis were collected from untreated mice (A) or after thrice-weekly treatment with TPA (B-D,F,H,I) or acetone (E,G,J) for the number of days indicated. Wholemounts were labelled to detect Ki67- (red, AG), BrdU- (green, A-G), keratin 14- (red, H-J) or TUNEL- positive cells (green, H-J). (A-G) Asterisks indicate hair follicles that contain very few LRC. (F,G) Arrowheads in high magnification micrographs indicate double-labelled cells. (H-J) Arrows indicate TUNEL-positive cells. Asterisks indicate non-specific staining of hair shafts. BG, bulge; SG, sebaceous gland. Scale bars: 100 µm in A-E,H-J; 20 µm in F,G.

 


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  		Fig. 5. Consequences of Myc activation in the epidermis of K14MycER transgenic mice. Transgenic mice and nontransgenic (wild-type) littermates were injected with BrdU according to the LRC protocol. After a 70 day chase period, wholemounts of tail epidermis were examined immediately (no OHT) or after daily treatment with 4-hydroxy-tamoxifen (OHT) for the number of days shown. Epidermis was labelled to detect keratin 14 (red) and Nile Red (green) (A,D,G,J,M), keratin 14 (red) and BrdU (green) (B,E,H,K,N), or Ki67 (red) and BrdU (green) (C,F,I,L,O). Arrows (G,J) indicate individual or clusters of sebocytes located in the interfollicular epidermis. Scale bar: 100 µm.

 


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  		Fig. 6. Effects of Myc on interfollicular epidermis of K14MycER mice. (A,B) Wholemounts labelled by double-immunofluorescence for Ki67 (red) and LRC (green). (A) No OHT treatment; (B) 4 days of OHT treatment. (C,D) Serial sections of tail epidermis treated with OHT for 7 days, stained with Haematoxylin and Eosin (C) or Oil Red 0 with Haematoxylin counterstain (D). (E) Wholemount immunolabelled for keratin 14 (red) after 14 days of OHT treatment. K, basal keratinocytes; S, sebocytes. Scale bars: 20 µm.

 


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  		Fig. 7. Effects of {Delta}NLef1 transgene on tail epidermis. Tissue sections (A-D) and wholemounts (E-P) of wild-type (A,C,E,G,I,K,M,O) and K14{Delta}NLef1 transgenic mice (B,D,F,H,J,L,N,P) at 3.5 weeks (A,B.E,F,I,J,M,N) and 3.5 months (C,D,G,H,K,L,O,P) of age. (A-D) Hematoxylin and Eosin staining. Note epithelial cysts (EC) at the base of hair follicles (HF) in transgenic skin. IFE, interfollicular epidermis; SG, sebaceous glands. (E-H) Wholemounts immunolabelled for keratin 14 (red) and LRC (green). Arrows in F indicate bending of base of hair follicles associated with early cyst formation. Arrows in H show loss of LRC in deformed hair follicles of transgenic mice. (I-L) Staining with Nile Red. (I,K) Staining restricted to sebaceous glands (SG). (J,L) Arrows indicate increased sebocyte differentiation along the length of hair follicles. (M-P) Immunolabelling for ß1 integrins (red) and CDP (green). Immunolabelling for CDP is observed in wild-type anagen follicles (arrows in M,O) and some follicles of 3.5-week-old transgenic mice (arrows in N), but is completely lost from the abnormal follicles of older animals (arrowheads in P). Scale bar: 100 µm.

 


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  		Fig. 8. Effects of {Delta}NLef1 transgene on markers of proliferation and differentiation. Wholemounts of tail epidermis from 3.5-month-old wild-type animals (A,C,E,G), 3.5-month-old K14{Delta}NLef1 transgenic mice (B,D,F,H), and 3.5-week-old K14{Delta}NLef1 transgenic mice (I-K) were stained to detect TUNEL-positive cells (green in I,J) or labelled with antibodies against Ki67 (red in A-D,I,J), BrdU (to detect LRC; A,B,K), keratins 10 (green in E,F) and 6 (green in G,H), {alpha}6 (red in E,F) and ß1 integrins (red in G,H). (A,B) Note reduction in number of LRC in deformed hair follicles of transgenic mice (arrowheads in B). In wild-type anagen hair follicles, Ki67 labelling is concentrated at the base of the hair follicles (arrows in A), whereas in aberrant transgenic follicles, Ki67-positive cells are found throughout the deformed hair follicles (arrows in B). (C,D) High power views of interfollicular epidermis. (E,F) In transgenic animals note labelling for keratin 10 extends to the middle and lower part of deformed follicles (arrows in F) and to developing cysts (asterisks in F). (G,H) Keratin 6 labelling is less restricted in transgenic than wild-type follicles (arrows in G,H). (I,J) TUNEL-positive cells are increased in transgenic (I) versus nontransgenic (J) hair follicles. Arrows indicate that TUNEL-positive cells are primarily located in the lower one-third of the hair follicles. Asterisks indicate non-specific staining of hair shafts. (K) High-power confocal micrograph of a hair follicle; arrows indicate examples of double-labelled cells. Scale bar: 100 µm in A,B,E-H,I-J; 20 µm in C,D,K.

 

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© The Company of Biologists Ltd 2003