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First published online 14 May 2008
doi: 10.1242/dev.017459

Development 135, 2161-2172 (2008)
Published by The Company of Biologists 2008
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Activation of β-catenin signaling programs embryonic epidermis to hair follicle fate

Yuhang Zhang1, Thomas Andl1, Steven H. Yang2, Monica Teta1, Fei Liu1, John T. Seykora3, John W. Tobias4, Stefano Piccolo5, Ruth Schmidt-Ullrich6, Andras Nagy7, Makoto M. Taketo8, Andrzej A. Dlugosz2 and Sarah E. Millar1,*

1 Departments of Dermatology and Cell and Developmental Biology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
2 Department of Dermatology, University of Michigan, Ann Arbor, MI 48109, USA.
3 Department of Dermatology, University of Pennsylvania School of Medicine, Philadelphia, PA 19104, USA.
4 Bioinformatics Core, University of Pennsylvania, Philadelphia, PA 19104, USA.
5 Department of Histology, Microbiology and Medical Biotechnologies, Section of Histology and Embryology, University of Padua, 35121 Padua, Italy.
6 Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
7 Samuel Lunenfeld Research Institute, Mount Sinai Hospital, Toronto, Ontario, M5G 1X5, Canada.
8 Department of Pharmacology, Graduate School of Medicine, Kyoto University, Yoshida-Konoé-cho, Sakyo, Kyoto 606-8501, Japan.


Figure 1
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  		Fig. 1. Abnormal skin development and Wnt/β-catenin pathway activation in stabilized β-catenin mutant embryos. (A-H) Control littermate and KRT14-Cre Ctnnb1(Ex3)fl/+ (Act β-catenin) embryos at the stages indicated. Arrows indicate keratinized plaques in mutant embryos. (I,J) Higher magnification views of dorsal skin from G and H, respectively. Pigment (arrows) accumulates in mutant skin. (K,L) Immunohistochemistry for β-catenin at E15.5 showing elevated β-catenin levels in control placodes (K), and cytoplasmic and nuclear β-catenin throughout mutant ectoderm (L, arrows). (M-P) X-gal-stained control BATgal (M,O) and KRT14-Cre Ctnnb1(Ex3)fl/+ BATgal (Act β-catenin BATgal) (N,P) littermates at E13.5 (M,N) and E16.5 (O,P), showing increased BATgal expression and its irregular pattern in the mutant. Scale bar in L applies to K and L.

 

Figure 2
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  		Fig. 2. Premature and expanded placode development in β-catenin mutant embryos. (A-D) Premature placode development at E12.5 indicated by Hematoxylin and Eosin (H&E) staining (A,B, arrows) and Shh mRNA expression (C,D; purple brown) in KRT14-Cre Ctnnb1(Ex3)fl/+ (Act β-catenin) embryos compared with controls. (E-H) Placodes are enlarged in the mutant at E14.5 (E,F) and immunofluorescence for PGP9.5 reveals precocious development of enlarged nerve fibers (G,H, arrows). (I-P) Whole-mount in situ hybridization for Wnt10b (I-N) or Edar (O,P) reveals accelerated formation of enlarged placodes in the mutants. (Q-T) Whole-mount X-gal staining of control {kappa}Gal (Q,S) and KRT14-Cre Ctnnb1(Ex3)fl/+ {kappa}Gal (R,T) embryos reveals an irregular pattern of NF{kappa}B activity (arrows) in the mutants. Scale bars: bar in B applies to A,B; in D, to C,D; and in H, to E-H.

 

Figure 3
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  		Fig. 3. Aberrant hair follicle development and global expression of placode and hair shaft markers in activated β-catenin mutant embryos. (A-J) Histological analysis of dorsal skin from control littermate and KRT14-Cre Ctnnb1(Ex3)fl/+ mutant embryos reveals the failure of hair follicle downgrowth in mutants. Mutant skin displays evaginations (B,D,J; arrows) and fails to stratify normally (H; arrow). (G,H) Higher magnification views of E and F, respectively. (K-X) The placode marker Shh (in situ hybridization, brown; K,L) and the dermal condensate marker alkaline phosphatase (AP; enzymatic assay, purple brown; M,N) are expressed broadly in the mutant. Expression of the inner root sheath marker GATA3 (O,P) and the outer root sheath marker SOX9 (Q,R; immunofluorescence, red) is reduced or absent, but hair shaft keratins (AE13 antibody, immunohistochemistry, brown) are ectopically expressed in mutant ectoderm (S,T). Proliferation is decreased (U,V; Ki67 immunofluorescence, red) and the pattern of apoptosis/terminal differentiation is altered (W,X; TUNEL staining, green) in mutant ectoderm. Arrows indicate expression; dashed lines mark dermal-epidermal boundaries. Samples were E17.5 (K-N,Q-X) or newborn (O,P). Scale bars: bar in F applies to A-F; in H, to G,H; in J, to I,J; in X, to K-X.

 

Figure 4
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  		Fig. 4. Mutation of β-catenin after placode initiation blocks hair follicle downgrowth and causes abnormal epidermal stratification and pigmentation. (A-F) H&E-stained sections of E16.5 (A,B) or P1 (C-F) dorsal skin from littermate control and KRT5-rtTA tetO-Cre Ctnnb1(Ex3)fl/+ embryos doxycycline treated from E13.5 (A,B) or E15.5 (C-F). (E,F) Higher magnification views of C,D. Placodes (arrows) are abnormal and expanded at E16.5 (A,B), but hair follicle downgrowth (arrows) fails (C,D). Epidermal stratification is abnormal in the mutants (blue arrows; E,F). (G,H) Precocious pigmentation (arrow) of newborn mutant induced from E15.5. Scale bars: bar in D applies to A-D; in F, to E,F.

 

Figure 5
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  		Fig. 5. Epidermal differentiation fails in activated β-catenin mutants. (A-N) Expression of KRT14 (A,B), DSC1 (I,J) and DSG1 (K,L) is decreased, and expression of KRT10, filaggrin and loricrin (C-H) is absent in stabilized β-catenin mutants, but laminin {alpha}5 expression (M,N) indicates that the basement membrane remains intact. Scale bar in N applies to A-N. Arrows indicate expression. (O-S) TEM of E16.5 control (O,R) and mutant (P,Q,S) dorsal epidermis (O-Q) or epidermal-dermal junction region (R,S) reveals the presence of basal (Ba), spinous (Sp) and granular (Gr) layers in control epidermis (O), and their absence in the mutant (P). Pigment granules are present in mutant ectoderm (red arrows in Q). The basement membrane (BM) is intact in the mutant, but hemidesmosomes (red arrows in R) are reduced in number (S).

 

Figure 6
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  		Fig. 6. Verification of array data and identification of Sp5 as a direct β-catenin target capable of suppressing epidermal differentiation gene expression. (A) qRT-PCR assays for the indicated transcripts in epidermal extracts from control and activated β-catenin mutant E15.5 embryos. Average relative expression levels, measured in triplicate assays of two independent pairs of experimental and control samples, were normalized to levels of actin transcripts. (B,C) Immunohistochemistry for hair keratin KRT82 (brown) demonstrates its upregulation in activated mutant epithelium at E16.5. (D,E) Similar patterns of Topgal (whole-mount X-gal staining, blue) and Sp5 (whole-mount in situ hybridization, purple) expression (arrows) in wild-type primary hair follicle placodes at E16.0. The less-sensitive in situ hybridization assay does not detect Sp5 expression in all secondary hair follicles at this stage. D,E were photographed at the same magnification. (F-I) Expanded Sp5 expression in KRT14-Cre Ctnnb1(Ex3)fl/+ mutant embryos at E14.5 (F,G; yellow arrows) and E15.5 (H,I; yellow arrows), and its ectopic expression in mutant cornea (white arrow). (J) qRT-PCR shows decreased expression of Sp5 in epidermal extracts of KRT5-rtTA tetO-Dkk1 E14.5 embryos doxycycline treated from E0.5 compared with control littermates. (K,L) Absence of placodal Sp5 expression in Dkk1-expressing E15.5 embryos (L) compared with littermate controls (K). (M) ChIP assay for β-catenin binding to a region of the Sp5 promoter containing multiple conserved LEF/TCF-binding sites. Chromatin prepared from E15.5 control and KRT14-Cre Ctnnb1(Ex3)fl/+ epidermis was incubated with anti-β-catenin antibody (β-cat Ab) or control IgG. Semi-quantitative PCR detects β-catenin binding to the Sp5 promoter in control extracts and increased binding in KRT14-Cre Ctnnb1(Ex3)fl/+ extracts. β-catenin does not bind to Gapdh sequences (bottom panel). (N) qRT-PCR detects expression of transfected mouse Sp5, decreased levels of endogenous KRT10 and involucrin, and increased expression of endogenous DLX3 in Sp5-transfected HaCAT cells compared with empty vector-transfected controls. Average relative expression levels measured in triplicate assays of three independent transfection experiments are shown, normalized to levels of actin transcripts.

 

Figure 7
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  		Fig. 7. Model of the effects of activated β-catenin on ectodermal differentiation. (A) Wnt/β-catenin signaling (blue) is weakly activated in subsets of wild-type epithelial cells in response to dermal signals. Positive-feedback signaling enhances pathway activity in placodes (Liu et al., 2008Go; Liu et al., 2007Go), activates secreted factors that promote dermal condensation and attract melanoblasts and nerve fibers, and induces expression of secreted Wnt inhibitors that block pathway activation in adjacent cells by a reaction-diffusion mechanism. Epithelial signaling reappears after birth in hair shaft precursor cells that attract pigment deposition. (B) In the constitutive mutant, elevated epithelial β-catenin overrides the requirement for a dermal message, and stimulates enhanced positive-feedback signaling, dermal condensation, and expression of secreted Wnt inhibitors that reduce Wnt/β-catenin signaling in dermal cells, but not in epithelial cells expressing activated mutant β-catenin. The imbalance between positive and negative signals leads to an expanded adoption of placode fate, broad suppression of epidermal differentiation, enhanced innervation, precocious pigmentation and differentiation towards hair shaft. (C) Induced mutation in embryonic epithelium after the placode stage blocks follicle downgrowth, stimulates hair shaft differentiation, and attracts pigment deposition and innervation. (D) Induced mutation in adult telogen skin induces abnormal growth of existing follicles, and the formation of ectopic follicles from outer root sheath and epidermis. Diagrams are not to scale.

 

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© The Company of Biologists Ltd 2008