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doi: 10.1242/10.1242/dev.00453

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Abnormal epidermal differentiation and impaired epithelial-mesenchymal tissue interactions in mice lacking the retinoblastoma relatives p107 and p130

¹ Program on Cell and Molecular Biology and Gene Therapy. CIEMAT, Avenue Complutense 22, E28040 Madrid, Spain
² Instituto de Biomedicina de Valencia. Jaime Roig 11, 46010-Valencia, Spain
³ Department of Animal Pathology, Veterinary School, University of Santiago de Compostela, E27002 Lugo, Spain

View larger version (120K): [in a new window]   Fig. 1. Epidermal abnormalities and altered differentiation in p107/p130 knockout mice. Newborn skin sections of double heterozygous (A,A') and p107/p130-null mice (B,B'). Note the decrease in the number and size of keratohyalin granules of the stratum granulosum in p107/p130-null animals (B') compared with its respective littermate (A'). Expression of the epidermal differentiation markers in double heterozygous (C-E) and p107/p130-null animals (C'-E') showing a similar expression of K10 (C,C') and a decrease in the expression of the terminal differentiation-markers filaggrin (D,D') and loricrin (E,E'). (A,A',B,B') Hematoxylin-Eosin staining. Scale bars: 100 µm.

View larger version (74K): [in a new window]   Fig. 2. Alterations in hair follicles and tooth germs in p107/p130 knockout mice. Hematoxylin/Eosin skin sections of wild type (A-C) and 107/130 null (A'-C') 18.5 dpc (A,A') and 14.5 dpc (B,B') embryos showing the decrease in hair germs (see arrows). Snout of wild-type (C) and 107/130 null (C') 14.5 dpc embryos showed a developmental delay of the whiskers. (D) Quantitative analysis of the number of hair follicles in the skin of 18.5 and 16.5 dpc embryos. (E) Analysis of the developmental stage of hair follicles in 18.5 dpc embryos. Data in D and E come from the quantification of three different sections of at least three different animals and are shown as mean±s.d. The hair follicle stages are as reported (Muller-Rover et al., 2001; Paus et al., 1999). Hematoxylin/Eosin medial sections from the heads of wild type (F,G) and 107/130 null (F',G') 18.5 dpc embryos. A few 107/130-null embryos showed anodontia with the presence of undifferentiated mesenchimal tissue in the site of incisors (compare arrows in F' with those in F). When present, tooth germ in 107/130-null embryos displayed variable degrees of microdontia associated with hypoplasia and disorganization of the odontoblast layer (compare arrows in G' with those in G). pu, dental pulp; od, odontoblast layer; de, dentine; am, ameloblast layer; ep, enamel pulp. Scale bars: 100 µm in A,C,C',G,G'; 50 µm in B,B'; 500 µm in F,F'.

View larger version (134K): [in a new window]   Fig. 3. Normal expression of Edar, Xedar and Troy/Taj and NFB signaling in p107/p130 knockout mice skin. Immunohistochemical detection of Edar (A,A'), Xedar (B,B') and Troy/Taj (C,C') in hair follicles of 18.5 dpc control (A-C) and mutant (A'-C') embryos showing no alterations. Scale bars: 50 µm. (D) EMSA analysis for NFB binding activity of skin protein extracts from mouse of the quoted genotypes, showing no differences between them.

View larger version (96K): [in a new window]   Fig. 4. Normal hair growth in grafts of p107/p130-null epidermis. Growth of hair coat in the grafts from control (A') and p107/p130-doubly deficient mice (A) was evident four weeks after transplantation. Sections of skin grafts from p107/p130-doubly deficient mice (B,C') showed normal anagen hair follicles without alterations in any of the different epithelial layers (B) and hair bulbs (C') undistinguishable from those of control transplants (C). Scale bars: 50 µm.

View larger version (130K): [in a new window]   Fig. 5. Alterations in epidermis, aberrant hair follicle morphogenesis and hair cycling in grafts of p107/p130-null epidermis. Increased number of hair follicles in p107/p130-doubly deficient mice grafts (A,A') compared with controls (B) was evident 4 weeks after transplantation. In addition, a number of abnormalities, including epidermal hyperplasia and parakeratosis (C), dysplastic hairs sharing a unique hair canal (D), misoriented follicles (arrow in E), follicular cysts (arrows in F), and hyperplasic sebaceous glands (G) were commonly detected in p107/p130-doubly deficient grafts. Transplants from p107/p130-deficient skin almost exclusively displayed anagen hair follicles at 2 (H) and 3 (I) weeks after transplantation. Eight weeks after transplantation, control grafts (J) display most of the hair follicles in a resting telogen phase (see inset, right), while in p107/p130-deficient grafts (K), the vast majority of hair follicles persisted in anagen (see inset, right). Scale bars: 500 µm in A,A'; 1 mm in B,H-K; 100 µm in C-G. Brackets in H and J indicate the wound margin.

View larger version (59K): [in a new window]   Fig. 6. Expression of p107 and p130 in anagen hair follicles. Immunohistochemical detection of p107 (A,C) and p130 (B,D) in control anagen hair follicles (A,B) and in hair bulbs from p107/p130-doubly deficient grafts (C,D). Note that in mutant grafts p107 (C) and p130 (D) immunoreactivity is detected in the nuclei of fibroblast forming the dermal papilla. Scale bars: 50 µm.

View larger version (102K): [in a new window]   Fig. 7. Altered differentiation and proliferation of keratinocytes in transplants of p107/p130-deficient skin. K5 was expressed not only at the basal layer but also in scattered suprabasal cells (A). K10 was not expressed in the expected continuous suprabasal pattern (B). K6 was expressed in suprabasal layers of interfollicular epidermis (C). Loricrin expression was dramatically reduced (D). Proliferation was confined to a few keratinocytes in the basal layer of interfollicular epidermis and in the outer root sheath of hair follicles in control grafts (E), whereas the vast majority of these cells are actively proliferating in p107/p130-doubly deficient grafts (F). In addition, proliferating cells were also detected in some cells of the suprabasal layer of interfollicular epidermis in mutant transplants (arrows in F'). Scale bars: 100 µm in D,E; 200 µm in F; 25 µm in F'.

View larger version (103K): [in a new window]   Fig. 8. Decreased Bmp4 signaling in p107/p130 knockout mice epidermis. (A) Northern analysis of different signaling molecules involved in hair development. Total RNA from skin samples of the quoted genotypes were probed for the expression of Axin, ß-catenin, Lef1, Shh, Waf/Cip1 and Bmp4. The membranes were also hybridized with a 7S RNA probe to verify that equal amounts of mRNA were loaded and transferred. Note that a significant decrease in Bmp4 was the only observed alteration in doubly deficient mice samples. (B-H) Immunohistochemical detection of Bmp4 (B,B',F), p75NTR (C,C'), noggin (D,D',G) and Hgf (E,E',H) in hair follicles from control (B-E), p107/p130-deficient skin (B'-E') and in transplants from p107/p130-deficient skin onto NOD/scid mice (F-H). Note the altered expression of Bmp4, noggin and Hgf that are restored in grafts. Scale bars: 25 µm in E'; 500 µm in H.

View larger version (80K): [in a new window]   Fig. 9. Reduced expression of Np63 in p107/p130 knockout mice epidermis is restored in transplants. The expression and localization of Np63 were monitored in epidermis of control (A), doubly deficient mice (B) and skin grafts of doubly deficient mice on NOD/scid mice (C). Note that Np63 expression is severely reduced in p107/p130-null epidermis and that this is restored in grafts. Scale bars: 25 µm.