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First published online 8 March 2006
doi: 10.1242/dev.02325

Development 133, 1553-1563 (2006)
Published by The Company of Biologists 2006
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p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation

Johanna Laurikkala1,*, Marja L. Mikkola1,*, Martyn James1, Mark Tummers1, Alea A. Mills2 and Irma Thesleff1,{dagger}

1 Institute of Biotechnology, University of Helsinki, 00014 Helsinki, Finland.
2 Cold Spring Harbor Laboratory, Cold Spring Harbor, NY 11724, USA.


Figure 1
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  		Fig. 1. Localisation of p63 mRNA in frontal sections of embryonic mouse heads and in developing skin. (A) Prior to morphological tooth formation (E10), p63 transcripts were seen throughout the simple epithelium. (B-D) During the initiation (E11), placode (E12) and cap (E15) stages, p63 expression continued in all oral and dental epithelia. (E) At the bell stage, p63 expression was particularly intense in the outer enamel epithelium and weaker in the inner enamel epithelium. (F) At E14, transcripts were intense in the epithelium of palatal shelves. (G) p63 transcripts were present in simple surface ectoderm at E11. (H) At E15, p63 expression continued in the basal epithelial cell layer, whereas it was downregulated in the most superficial cells. Dashed line indicates the epidermal surface. (I) At E17, p63 expression continued in the basal epithelial cells and it was intense in the stage 1-4 hair follicles. iee, inner enamel epithelium; ns, nasal septum; oee, outer enamel epithelium; ps, palatal shelf. Scale bar: 200 µm.

 

Figure 2
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  		Fig. 2. Expression of TAp63 and {Delta}Np63 transcripts, and of p63 protein. (A-C) No TAp63 mRNA was seen in surface ectoderm or tooth germ epithelia at the initiation (E11), placode (E12) or cap stages of molar tooth development (E14). (D-G) Intense expression of {Delta}Np63 was seen in the oral and dental epithelium, as well as in hair follicles, in similar patterns to those observed with the p63 probe that detects all isoforms (see also Fig. 1). (H) Expression of the TAp63 isoform was not detected in the epithelia of the newborn (NB) mouse. (I) In P2 incisors, {Delta}Np63 expression was intense in the enamel organ epithelium contacting the ameloblasts, but ameloblasts did not express {Delta}Np63. (J) Immunohistochemical analysis showed p63 protein expression throughout the oral and dental epithelia at E11. (K) Expression was particularly intense in the outer enamel epithelium in the molar at bell stage. (L) In the P1 incisor, p63 protein expression was most intense in the outer enamel epithelium. (M) Western blot analysis of p63 isoforms in E13 and E14 skin samples of p63-/- and wild-type (combined +/- and +/+) embryos. NIH3T3 cells transfected with {Delta}Np63{alpha}, {Delta}Np63ß or {Delta}Np63{gamma} were used as control samples. The major band detected in wild-type tissues corresponds to {Delta}Np63{alpha}; TAp63{alpha} typically migrates substantially more slowly in SDS-PAGE. (N) Quantification of the expression of {Delta}Np63 and TAp63 isoforms in E7 and E8 whole embryos, and in E9 and E13 skin by real-time PCR. Trace amounts of the TAp63 isoform were detected at E13. A, ameloblasts; cl, cervical loop; oee, outer enamel epithelium; n.d., not detectable.

 

Figure 3
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  		Fig. 3. Comparison of molar tooth and hair follicle development between wild-type and p63 mutant embryos. (A,B) At E11, no difference could be detected between wild-type and p63-/- tissues during the initiation of tooth development (arrows). (C-F) At E12 and E13, dental placodes and tooth buds (arrows) had formed in wild-type embryos, whereas in the mutant littermates development had arrested at the stage of thickened dental epithelium (arrows). (G,H) At E16, wild-type molars had advanced to the bell stage, whereas in p63 mutants the epithelial thickening had degenerated. (I) The first wave of hair follicles (guard; arrow) had developed to stage 3-4, the placodes of the second wave awl follicles had appeared in wild-type skin at E16.5 and the epidermis had increased in thickness. (J) p63-/- epidermis lacked stratification and hair follicles. sr, stellate reticulum; dp, dental papilla.

 

Figure 4
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  		Fig. 4. Tooth and hair placodes do not form in p63 null mutants. (A,C) The placodes of incisors and molars are visualised by Pitx2 and Shh expression at E12 in the wild-type mandible. (B,D) In mutant mandibles, Pitx2 and Shh remained continuous in the dental lamina, indicating the absence of placodes. (E,G) ß-catenin and Edar are early markers of hair placodes in wild-type skin at E14. (F,H) In p63 null skin, ß-catenin and Edar expression was absent. i, incisor; m, molar placode.

 

Figure 5
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  		Fig. 5. Expression of Fgfr2b, Fgf8, Bmp7 and ß-catenin in wild-type and p63 mutant embryos. (A-D) Fgfr2b transcripts were seen throughout simple oral epithelium and thickened dental epithelium (E11) and dental placode (E12) in wild-type embryos, but were absent in the p63 mutant littermate. (E,F) Fgfr2b transcripts were absent in skin epithelium of p63 mutants. (G,H) Fgf8 expression was detected in the area of tooth development both in wild-type and in mutant embryos prior to morphological tooth formation (E10). (I,J) Bmp7 transcripts were absent in p63 null oral epithelium. (K,L) At E14, Bmp7 was expressed throughout surface ectoderm in a wild-type embryo, whereas expression was completely absent in a p63 mutant littermate. (M,N) ß-catenin was intensely expressed both in wild-type and mutant oral epithelium. (O,P) ß-catenin was downregulated in skin epithelium in the p63 null embryo. Scale bars: in A, 200 µm for A-L; in M, 500 µm for M,N; in O, 200 µm for O,P.

 

Figure 6
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  		Fig. 6. Expression of NOTCH pathway genes and Edar in wild-type and p63 mutant embryos. (A,C,E,G) Notch1, Notch2 and Notch3 were co-expressed in dental and oral epithelium in wild-type suprabasal cells. (B,D) Notch1 transcripts were absent in p63 null epithelium at E11 and E12. (F,H) Notch2 and Notch3 showed normal expression in the p63 mutants. (I) At E12, Jag1 was intensely expressed in suprabasal cells in the epithelium and in the mesenchyme surrounding the tooth bud in wild-type embryo. (J) The p63 mutant epithelium was devoid of Jag1 transcripts but faint expression was seen in the mesenchyme. (K,L) Jag2 was expressed in the oral epithelium in both wild-type and mutant embryos. (M,N) In E12 skin epithelium, intense Notch1 expression was seen in the wild-type embryo, whereas transcripts were not detected in the mutant littermate. (O,P) Jag1 was not observed in p63-deficient skin epithelium. (Q-T) The intensity of Edar expression was weaker in the oral, dental and skin epithelium of p63 mutants than in wild-type embryos. Scale bars: in A, 200 µm for A,B; in C, 500 µm for C-L; in M, 200 µm for M-T.

 

Figure 7
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  		Fig. 7. Induction of p63 expression by BMP2, BMP7 and FGF10 in dental epithelium in E13 tooth explants, as analysed by whole-mount in situ hybridisation. Proteins were introduced with beads (A-I) or transfected cells (J) on explants and cultured for 24 hours. Note the endogenous expression of p63 in epithelium in all explants. (A,B) BMP2 and BMP7 induced p63 expression. (C) FGF10 induced p63 expression. (D-J) None of the other signals tested (FGF4, FGF8, Activin, EGF, SHH, TGFß1, WNT6) affected p63 expression. (K,L) Expression of p63 does not require FGFR2b. p63 transcripts were observed throughout ectoderm, in a similar pattern, in both wild-type and Fgfr2b null mutant embryos at E11.

 

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© The Company of Biologists Ltd 2006