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


spacer gif
     Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents    

First published online 11 July 2007
doi: 10.1242/dev.005041

Development 134, 2947-2958 (2007)
Published by The Company of Biologists 2007
This Article
Right arrow Summary Freely available
Right arrow Full Text
Right arrow Full Text (PDF)
Right arrow Supplementary Material
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 Fadloun, A.
Right arrow Articles by Davidson, I.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Fadloun, A.
Right arrow Articles by Davidson, I.

The TFIID subunit TAF4 regulates keratinocyte proliferation and has cell-autonomous and non-cell-autonomous tumour suppressor activity in mouse epidermis

Anas Fadloun1, Dominique Kobi1, Jean-Christophe Pointud1,*, Arup Kumar Indra1,{dagger}, Marius Teletin1, Christine Bole-Feysot1, Barbara Testoni2, Roberto Mantovani2, Daniel Metzger1, Gabrielle Mengus1 and Irwin Davidson1,{ddagger}

1 Institut de Génétique et de Biologie Moléculaire et Cellulaire, CNRS/INSERM/ULP, 1 Rue Laurent Fries, 67404 Illkirch Cédex, France.
2 Dipartimento di Scienze Biomolecolari e Biotecnologie, U. di Milano, Milano, Italy.


Figure 1
View larger version (50K):
[in this window]
[in a new window]

  		Fig. 1. Inactivation of TAF4 in foetal epidermis. (A,B) Gross morphology and X-Gal dye diffusion assay of E18.5 mice of the indicated genotypes. (C) Immunostaining of skin biopsies of E18.5 animals of the indicated phenotypes with anti-TAF4 antibody. The epidermis (E), dermis (D) and basal keratinocyte layer (BK) are indicated. Arrows indicate representative nuclei. Left panel, immunostaining; right panel, Hoechst stain. (D) Histological analysis of representative Haematoxylin-Eosin-stained paraffin sections from dorsal or ventral (upper and lower panels, respectively) regions of control and mutant foetuses at E18.5. The epidermis is indicated and the arrows indicate the location of the basal membrane. (E) Transepidermal water loss (TEWL) of three independent E18.5 foetuses of each genotype after 20 or 60 minutes as indicated. (F) Weight loss of three independent E18.5 foetuses of each genotype after the indicated times. (G) Quantitative RT-PCR on RNA from three independent E18.5 foetuses of each genotype. Duplicate reactions were performed for each RNA preparation. In each case, the expression in the Taf4lox/+ animals is set as 1. Scale bars: 30 µm in C; 50 µm in D.

 

Figure 2
View larger version (85K):
[in this window]
[in a new window]

  		Fig. 2. Inactivation of TAF4 in adult epidermis. (A) Ethidium bromide-stained agarose gel of products of a triplex PCR. The locations of the floxed and deleted fragments amplified from the dermis (D) or epidermis (E) 2 weeks after oil or Tam injection are shown. (B) Immunostaining of skin biopsies 2 weeks after oil (above) or Tam (below) injection with anti-TAF4 antibody. Abbreviations as described in Fig. 1. (C) Immunostaining of skin biopsies 2 weeks after oil or Tam injection with anti-Ki-67 antibody. Arrows indicate representative positive staining nuclei. (D) Immunostaining of skin biopsies with anti-keratin 6 antibody. The arrows indicate the interfollicular epidermis. (E) Histological analysis of representative Haematoxylin-Eosin-stained paraffin sections from dorsal or ventral regions of oil- or Tam-injected mice. Hair follicles (HF) are indicated and arrows indicate the location of the basal membrane. Scale bar: 25 µm in D; 50 µm in E.

 

Figure 3
View larger version (93K):
[in this window]
[in a new window]

  		Fig. 3. Hair cycle defects in TAF4 mutant epidermis. (A-D) Animals were injected with oil or Tam as described in Materials and methods and two representative examples photographed at the indicated times post-injection. Reversible hair loss in the same Tam (foreground), but not oil (background) -injected animal is observed. (E,F) Defective depilation-induced anagen. Animals were injected with oil or Tam as indicated and depilated by wax-stripping 6 weeks after injection. Images show mice at the day of depilation and after 10 and 20 days.

 

Figure 4
View larger version (58K):
[in this window]
[in a new window]

  		Fig. 4. Changes in gene expression upon TAF4 inactivation. (A,B) A restricted list of up- (A) or down- (B) regulated genes are shown along with their log2 values classified by their function or intracellular location. (C) RT-PCR analysis of gene expression of the indicated genes in RNAs from skin biopsies from two independent oil- or Tam-injected mice. (D) RT-PCR analysis of gene expression of the indicated genes in RNAs from skin biopsies of two independent E18.5 foetuses. (E) Immunoblot analysis of CDKN1A and cyclin D1 expression in epidermal extracts. 20 µg of total extract was loaded in each lane and the presence of CDKN1A, cyclin D1 and p53 revealed using the appropriate antibodies. In the lower part of the panel, phosphorylated p38 and CREB/ATF1 were revealed in total epidermal extracts prepared and quantified as described in Materials and methods.

 

Figure 5
View larger version (69K):
[in this window]
[in a new window]

  		Fig. 5. TAF4 has tumour suppressor activity. (A) Quantitation of tumours in oil- and Tam-injected mice after DMBA/TPA treatment. N, number of animals per group. (B) Number and size of tumours from oil- or Tam-injected animals after 26 weeks of treatment. (C) Quantitation of tumours in animals treated with DMBA and TPA for 15 weeks prior to oil or Tam injection. (D) Quantitation of tumours in oil- and Tam-injected animals after two sequential DMBA treatments. (E) Representative animals after 20 weeks of DMBA/TPA treatment. Black arrows, epidermal tumours; yellow arrows, melanocytic growths. (F) Representative animals after 20 weeks of DMBA treatment.

 

Figure 6
View larger version (132K):
[in this window]
[in a new window]

  		Fig. 6. Histological analysis of tumours. Twenty-six weeks after DMBA treatment of Tam-injected mice, epidermal and melanocytic tumours were excised, fixed in parrafin, sectioned and stained with Haematoxylin-Eosin for histological analysis. (A) Representative section from a moderately differentiated squamous cell carcinoma. (B) An invasive carcinoma amongst muscle tissue. Note the irregular, elongated cell morphology and the keratin pearl (arrow). (C,D) Large melanocytic tumours invading deeply into the muscle and adipocyte layers of the dermis. (E) Section of a locally invasive squamous cell carcinoma infiltrated by melanocytes. Black arrow, a keratin pearl; yellow arrow, melanocytes. (F) Section of a lymph node from a DMBA-treated Tam-injected animal. Yellow arrows, representative melanocytes. Scale bar: 100 µm in A,B; 240 µm in C; 50 µm in E; 100 µm in F.

 

Figure 7
View larger version (123K):
[in this window]
[in a new window]

  		Fig. 7. Diminished T-RA response in TAF4 mutant epidermis. (A) Haematoxylin-Eosin-stained sections from epidermis of oil- or Tam-injected animal after treament with T-RA or acetone for 5 days. (B) Epidermal sections from mice treated with calcipotriol for 5 days. Scale bar: 50 µm.

 




© The Company of Biologists Ltd 2007