spacer gif spacer gif spacer gif spacer gif spacer gif
QUICK SEARCH:   [advanced]


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

doi: 10.1242/10.1242/dev.00462

This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Email this article to a friend
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrow reprints & permissions
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Frye, M.
Right arrow Articles by Watt, F. M.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Frye, M.
Right arrow Articles by Watt, F. M.
Social Bookmarking
Add to CiteULike   Add to Complore   Add to Connotea   Add to Del.icio.us   Add to Digg   Add to Reddit   Add to Technorati   Add to Twitter  
What's this?

Evidence that Myc activation depletes the epidermal stem cell compartment by modulating adhesive interactions with the local microenvironment

Michaela Frye1, Clare Gardner2, Elizabeth R. Li1, Isabel Arnold1,* and Fiona M. Watt1,{dagger}

1 Keratinocyte Laboratory, Cancer Research UK, 44 Lincoln's Inn Fields, London WC2A 3PX, UK
2 Pfizer Global Research and Development, Sandwich CT13 9NJ, UK
* Present address: Institute for Genetics, University of Cologne, Cologne D-50674, Germany



View larger version (65K):

[in a new window]
  		Fig. 1. Generation of microarray data. (A) Schematic representation of the K14MycER expression cassette (top) and immunofluorescence staining (bottom) of wild-type and transgenic epidermis with an antibody to the murine estrogen receptor (HL7), showing expression of transgene in basal layer of epidermis and outer root sheath of hair follicles after 4 days treatment with OHT. No signal is observed in skin of wild-type mice. (B) Model (top) for effects of Myc on the epidermis: red arrows indicate that differentiation into interfollicular epidermis and sebocytes is stimulated. Red cross indicates that stem cell renewal is inhibited. Histology (bottom) of skin 4 days after daily treatment with OHT. Note thickening of interfollicular epidermis (IFE) and enlarged sebaceous glands (SG) in transgenic relative to wild-type control skin. (C) List of the 6 treatment groups subjected to microarray analysis. Skin was harvested after 1 day or 4 days of daily OHT treatment or was untreated (0d). HF, hair follicle. Scale bars: 10 µm.

 


View larger version (32K):

[in a new window]
  		Fig. 2. (A) Pie chart of the 137 consistently upregulated genes (Table 1) grouped according to their functional roles. (B) Pie chart of the 81 consistently downregulated genes (Table 3) grouped according to their functional roles.

 


View larger version (83K):

[in a new window]
  		Fig. 3. (A) RNA expression profile of upregulated genes in skin of untreated (0d) and treated (1d and 4d) transgenic and wild-type mice. (B,C) Immunolabeling of 4 days OHT-treated skin with antibodies to the proteins indicated. Scale bars: 10 µm (B), 5 µm (C). (D) In situ hybridisation of BSSP using radiolabelled RNA antisense probes (BSSP-as), comparing wild-type (wt) and transgenic (tg) skin untreated (0d) or harvested 1 day or 4 days (1d, 4d) after OHT treatment. A ß-actin probe served as positive control and a BSSP sense probe (BSSP-s) as negative control. The arrowheads indicate the expression of BSSP mRNA in sebaceous glands in untreated transgenic mice (tg-0d) and in the interfollicular epidermis of transgenic mice treated with OHT for 4 days (tg-4d). Scale bar: 10 µm

 


View larger version (78K):

[in a new window]
  		Fig. 4. (A) RNA expression profile of downregulated ECM genes in skin of untreated (0d) and treated (1d and 4d) transgenic and wild-type mice. (B) Immunolabelling of 4 days treated skin with antibodies to the proteins indicated. (C) Western blotting with anti-fibronectin antibody (upper panel) of wild-type (wt) and transgenic (tg) mice skin untreated (0d) or treated with OHT for 1 day or 4 days (1d, 4d). Actin was used as a loading control (lower panel). (D) Relative PCR determination of fibronectin mRNA levels in skin of wild-type (wt) and transgenic (tg) animals treated with OHT for 4 days (4d) (P=0.055). (E) Western blotting with anti-fibronectin antibody (upper panel) and anti-actin antibody as control (lower panel). Primary human keratinocytes overexpressing Myc (Kq-MycER) are compared to controls (Kq-106ER, Kq-pBP). (F) Immunolabelling of fibronectin and laminin at the wound margins of skin from wild-type and transgenic mice that had been wounded and then treated with OHT for 4 days. Skin was harvested after the number of days shown. Arrowheads indicate the expression of fibronectin and laminin at the basement membrane zone. Scale bar: 10 µm.

 


View larger version (102K):

[in a new window]
  		Fig. 5. Myc activation delays epidermal wound healing in vivo and in vitro. (A) Three days after wounding wild-type and transgenic mice received daily OHT treatment for 4 days. Wounds of wild-type mice had completely re-epithelialised, but wounds of transgenic mice remained open. Arrowheads indicate wound edges. Ki67 labelling of wound margins is shown in right-hand panels. Scale bar: 100 µm. (B) Wounds were made in sheets of cultured human keratinocytes (0h) and monitored after 24 or 48 hours. Myc over-expressing keratinocytes (Kq-MycER) were compared to controls (Kq-pBP). Cells were stained with phalloidin. Scale bar: 10 µm.

 


View larger version (52K):

[in a new window]
  		Fig. 6. Effect of Myc activation on keratinocyte motility and spreading. Myc over-expressing keratinocytes (Kq-MycER) were compared with keratinocytes expressing the empty retroviral vector (Kq-pBP) or the mutated Myc construct (Kq-106ER). (A) Movements of individual cells plated on collagen are shown. Green spots: start coordinates; red spots: end coordinates. (B) Average speeds of Kq-MycER (n=24), Kq-106ER (n=24) and Kq-pBP (n=23) on collagen. (C) Cell spreading in serum-free medium (FAD-). Cells were stained with phalloidin. Scale bar: 10 µm. (D) Median of cells spreading in serum-free medium (FAD) or medium supplemented with EGF, IGF or cytochalasin D (cyto. D).

 


View larger version (99K):

[in a new window]
  		Fig. 7. Myc activation did not disrupt cell-cell adhesion or focal adhesions, but did decrease hemidesmosome formation. (A) Cultured human keratinocytes (Kq-MycER, Kq-106ER, Kq-pBP) were stained with antibodies to the proteins shown. Nuclei are stained for Myc using 9E10 antibody. (B) Flow cytometry of primary keratinocytes isolated from transgenic or wild-type mice 2 days after OHT treatment using antibodies against the ß1 and {alpha}6 integrin subunits and CD98. (C) Real-time PCR determination of {alpha}6 integrin subunit mRNA levels in transgenic (tg) mice untreated (0d) or treated with OHT for 4 days (4d) (P<0.01).

 


View larger version (179K):

[in a new window]
  		Fig. 8. Decrease in {alpha}6 integrin levels and hemidesmosomes by activation of Myc. (A,B) Immunolabelling of the epidermis with antibodies to {alpha}6 of (A) wild-type mice or (B) transgenic mice after 4 days treatment with OHT. (C-F) Electron microscopy of the basement membrane zone of skin from transgenic mice (D,F) and wild-type mice (C,E) treated for 9 days with OHT. Arrows in C, D and E indicate hemidesmosomes. Arrows in F indicate absence of hemidesmosomes at the basement membrane. Scale bars: 5 µm (B), 200 nm (D), 1 µm (F). BM, basement membrane; IFE, interfollicular epidermis; HF, hair follicle.

 


View larger version (80K):

[in a new window]
  		Fig. 9. (A) RNA expression profile of downregulated cytoskeleton components and cytoskeleton regulatory factors in untreated (0d) and treated (1d and 4d) wild-type and transgenic mice. (B) Northern blot for MyBP-H of RNA from skin of wild-type (wt) and transgenic (tg) mice, untreated (0d) or treated for 1 day (1d) or 4 days (4d) with OHT. (C) Immunolabelling with antibodies to adducin and myosin II of skin from wild-type and transgenic animals after 4 days OHT treatment. Scale bar: 10 µm. (D) Cultured human keratinocytes (Kq-MycER and Kq-pBP) were stained with antibodies to actin (red; left panels) and myosin II (green; right panels). Cells shown are at the periphery of clones or at the edges of wounds made in confluent sheets. Scale bar: 5 µm. Nuclei are stained for Myc using 9E10 antibody. (E-G) Downregulation of adducin upon activation of Myc visualised by immunofluorescence (E), real-time PCR (F), and Western blotting (G). (E,G) Adducin protein expression in Myc expressing human keratinocytes (Kq-MycER) is decreased compared to controls (Kq-pBP). Scale bar: 10 µm. (F) Relative RNA expression is reduced in skin of transgenic (tg) animals treated with OHT for 4 days (4d) compared to 4 days treated wild-type (wt) and untreated (0d) transgenic mice (P<0.05).

 


View larger version (23K):

[in a new window]
  		Fig. 10. Model for mechanism by which Myc stimulates exit from the stem cell compartment and differentiation into IFE and sebocytes. (A) Location of stem cell populations, showing that stem cells for IFE and sebocytes are in close proximity to their differentiated daughters, whereas the progeny of bulge stem cells migrate to the dermal papilla before differentiating along the hair lineages. (B) Comparison of the role of Myc activation in wild-type and K14MycER epidermis.

 

Add to CiteULike CiteULike   Add to Complore Complore   Add to Connotea Connotea   Add to Del.icio.us Del.icio.us   Add to Digg Digg   Add to Reddit Reddit   Add to Technorati Technorati   Add to Twitter Twitter    What's this?



© The Company of Biologists Ltd 2003