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Requirement of NF-B/Rel for the development of hair follicles and other epidermal appendices

¹ Max-Delbrück-Center of Molecular Medicine, Robert-Rössle Straße 10, 13092 Berlin, Germany
² Max-Planck-Institute for Infection Biology, Schumannstraße 21/22, 10117 Berlin, Germany

View larger version (55K): [in a new window]   Fig. 1. Generation of mice with systemic NF-B suppression. (A) Integration of IBN (Krappmann et al., 1996) into the ß-catenin locus by homologous recombination. IBN lacks the phosphorylation and ubiquitination sites, which are required for signal-induced degradation (Krappmann et al., 1996). The murine ß-catenin locus (exons, white boxes) and targeting vectors are shown. IBN and loxPIBN cDNAs were inserted in frame to the start codon in the second exon of the ß-catenin gene and replaced exons 3 to 6, resulting in a ß-catenin null allele (Huelsken et al., 2000). In cloxPIBN, a stop codon inserted in frame is flanked by loxP sites (black arrowheads) (Zhang et al., 1996). (B) Western blot of cloxPIBN (D5-C7) and cIBN (D7-H9) ES cell clones using an antibody directed against the IB C-terminus (C-21). ns, non-specific. IBN protein is detected only in the ES cells carrying the IBN transgene. (C) EMSA of cIBN ES clones (G2 and G3), and cloxPIBN clones (B7 and A3) before Cre-mediated recombination as controls. ES clones were treated with PMA and specific complexes inhibited with anti-p65 antibody, as indicated. NF-B activity can no longer be induced by PMA in cIBN ES cells clones. (D) Embryonic fibroblasts (MEFs) of wild-type (WT) and cIBN littermates (IBN) were stimulated with IL1ß and TNF for the times indicated and extracts were assayed by EMSA. In cIBN fibroblasts, DNA-binding activity of NF-B p50/p65 complexes is severely blocked after stimulation with TNF and completely impaired after IL1ß stimulation. (E) The same extracts were analyzed in Western blots for IB and ß-catenin proteins, as indicated. As expected, endogenous IB is degraded after stimulation leading to the observed variations of the protein in wild-type and cIBN fibroblasts. De novo synthesis of IB protein, which depends on active nuclear NF-B complexes, is delayed in cIBN fibroblasts, as NF-B translocation is strongly suppressed. (F) A wild-type littermate compared with a cIBN mouse (right). (G) Increased apoptosis in the embryonic liver of cIBN mice. Cryosections of embryonic livers of wild-type and cIBN embryos at E12.5, E14.5 and at birth, P0, as indicated, were analyzed by in situ TUNEL assay.

View larger version (97K): [in a new window]   Fig. 2. Hair growth defects and lack of exocrine glands in cIBN mice. (A) Only an intermediate type of hair is seen in cIBN mice: monotrich/awl. In wild-type animals, all four types are found: Au, auchenes; Aw, awl; M, monotrich; Z, zigzag. Pictures were taken on a dark field using a Leica DC100 camera. (B) Strongly reduced numbers of hair follicles in newborn cIBN pups compared with wild-type littermates, shown in plastic sections of back skin (upper panels). No hair follicle anlagen are seen in the tail of newborn cIBN pups (middle panel, right), although they are readily detected in wild-type littermates (black arrows, left). Lower panels, dorsal skin sections of adult (P30) wild-type and cIBN littermates. Owing to the decreased number of hair follicles in the adult cIBN animal, the skin appears thinner. (C) Development of sweat glands in the foot pads is severely impaired. Compared with wild-type littermates, only very small residual glands can be detected in adult cIBN mice (black arrows). (D) Meibomian glands in the eyelid of a control littermate (left panel, right arrow) are absent in a 1-month-old cIBN mouse (right panel). The lid epidermis in wild-type animals (upper left panel, left arrow) is much thinner than in cIBN mice (right panels). The lower two panels show a magnified view of the part of the lid displaying the mucous membrane and the Meibomian glands (left panel, arrow), which are absent in cIBN mice (right). Connective tissue and the thickened epidermis of the lid margin (right panel, arrows) and a hair follicle (arrowhead) are indicated.

View larger version (76K): [in a new window]   Fig. 3. Whole-mount X-Gal staining of epidermal appendices in adult (Ig)3conalacZ mice. (A,B) Pelage (body) hair follicles. Hair (white arrow head) and follicle (black arrow head) are indicated. (A) A discrete area of the follicle is stained blue (arrow). (B) ß-galactosidase activity is also seen in the matrix of pelage hair follicles (arrow). (C) Vibrissal (whisker) follicle. X-Gal staining is found in the matrix (lower arrow) and hair shaft or inner root sheath of the vibrissal follicle (upper arrow). (D) X-Gal staining is also picked up in the bulge of this whisker follicle (arrow). Here, only a few blue cells are detected in the matrix. (E) Tail skin. Hair follicles of the tail stain blue (arrow). (F) X-Gal staining is found in the sweat glands of foot pads (arrow).

View larger version (84K): [in a new window]   Fig. 4. Edar (dl) expression and hair placode formation in E15.5 embryos of cIBN and wild-type mice. Increased apoptosis in hair follicles of cIBN embryos at day E17.5. (A) In situ hybridization (ISH) was used to assess Edar (dl) expression at E15.5. Panels dl-w.t.1 and dl-N1 show skin of whole mount embryos. dl-w.t.2 and dl-N2 show plastic sections of the same. At E15.5, Edar (dl) expression (arrow in dl-w.t.2) and concomitant hair placode formation is observed in torso skin of only wild-type mice. (B) Plastic sections of vibrissal follicles at E15.5, after ISH using a probe for Edar (dl). In follicles of vibrissae, Edar expression is observed in wild-type and cIBN mice (arrows). (C) Increased apoptosis is seen in hair follicles of pelage hair (right panel N1) and vibrissae (right panel N2) in cIBN mice at E17.5. Left panels show nuclear DAPI stain, right panels show the in situ TUNEL assay. White arrows in wild-type point to two pelage hair follicles.

View larger version (44K): [in a new window]   Fig. 5. Chronic otitis media, impaired macrophage activity and loss of peripheral lymph nodes in cIBN mice. (A) Plastic sections of the middle ear of a wild-type littermate and a cIBN mouse showing the middle ear cavity and the surrounding epithelium (arrowheads). Note the large amounts of pus and the swollen irregular mucous membrane in the middle ear of the cIBN mouse (upper right panel). Magnified view of the mucous membrane surrounding the middle ear cavity (lower panels), showing large amounts of granulocytes and monocytes in the cavity, and infiltrations of granulocytes and monocytes in the mucous membrane of the cIBN mouse (arrowheads). Arrowhead in wild-type (w.t.) points to a healthy mucous membrane. (B) cIBN mice are more susceptible to Leishmania major infection. cIBN mice (n=5; black square) and control littermates (n=3; white circle) were infected with L. major V121 promastigotes in the hind footpad. Data are expressed as mean increase in thickness compared with uninfected contralateral feet. (C) The higher susceptibility of cIBN mice to L. major correlates with strongly reduced iNOS induction in bone marrow-derived macrophages after stimulation with TNF. The data are from three independent experiments. cIBN mice (black square); wild-type littermates (white circle). Note that the error bars for cIBN mice are smaller than the symbols and are not visible. (D) IFN production of spleen cells after restimulation in vitro with Leishmania antigens (lysate; see Material and Methods). For IFN production analysis after restimulation with L. major antigens (lysate) spleen cells were isolated from cIBN or control mice infected with L.major for 8 weeks. Spleen cells were restimulated for 48 hours in the presence or absence of L.major antigens or were stimulated by Concanavalin A (ConA). IFN contents in culture were determined in a bioassay using WEHI-279 cells as described (Morris et al., 1992). Note the tenfold amount of IFN (U/ml) in IBN mice (black bars) when compared with control littermates (white bars). No difference is seen when the mitogen ConA or medium is added to the spleen cells.

View larger version (76K): [in a new window]   Fig. 6. (A) Absence of inguinal lymph nodes in cIBN mice. Mice were injected intraperitoneally with 1% Chicago Sky Blue in 1xPBS. Two weeks after injection, the skin in the groin of cIBN (N) and control mice (wild type) was dissected to visualize the inguinal lymph nodes. (B) Peyer’s patches of cIBN mice are absent or reduced in size and numbers. (Top) Peyer’s patches (arrowheads); Scale bar, 200µm. (Bottom) Plastic sections of PPs.

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