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First published online 15 February 2006
doi: 10.1242/dev.02278

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NF-B transmits Eda A1/EdaR signalling to activate Shh and cyclin D1 expression, and controls post-initiation hair placode down growth

¹ Max-Delbrück-Center for Molecular Medicine, Robert-Rössle-Strasse 10, 13092 Berlin, Germany.
² Medical Biosciences, School of Life Sciences, University of Bradford, Bradford BD7 1DP, UK.
³ Department of Biochemistry, University of Lausanne, Chemin des Boveresses 155, 1066 Epalinges, Switzerland.
⁴ Department of Dermatology, University Hospital Schleswig Holstein, Campus Lübeck, University of Lübeck, Ratzeburger Allee 160, 23538 Lübeck, Germany.

View larger version (93K): [in a new window]   Fig. 1. Reduced NF-B activity in skin of cIBN and downless mice, but normal perinatal epidermal keratinocyte differentiation in cIBN mice. (A) Total protein extracts of newborn wild-type and cIBN (N) skin were analysed for IBN expression in a western blot using an anti-IB antibody (lower panel). Mouse embryonic fibroblasts isolated from cIBN mice were used as a positive control (N MEF, right lane). ß-Catenin protein levels remained unchanged in all extracts (upper panel). ns, non-specific. (B) EMSA of total skin extracts of newborn wild-type, cIBN (N) and downless (dl) skin. Extracts were treated with specific antibodies against NF-B p65, p50 and RelB as indicated, which inhibited (-p65) or upshifted (-p50) the DNA-binding complex. No effect was seen with RelB. Strong p50 homodimer binding is present in skin extracts. (C) Sagittal cryosections of E17.5 and P0 wild-type and cIBN (N) embryos were incubated with antibodies to different epidermal differentiation markers (AP substrate, in red), as indicated above (loricrin, involucrin, filaggrin, keratin 10). Counterstaining was carried out with Mayer's haemalaun (blue).

View larger version (88K): [in a new window]   Fig. 2. NF-B activity during embryonic vibrissae and hair follicle development analysed in NF-B-driven ß-Gal reporter mice. (A) Whole-mount X-Gal staining of E10-13 (Ig)3xcona-lacZ (Gal) embryos. At E10-11, NF-B activity is observed only in somites and endothelial cells of blood vessels. From E12 onwards, activity is seen in vibrissae follicles and the rim of the eyelids (see also insets E12 and E13). (B) Sagittal sections of X-Gal stained embryonic and newborn skin were performed to analyse the most important stages of murine pelage hair follicle development. At E13.5, arrows indicate endothelial cells of blood vessels. M, matrix; Co, pre-cortex; DC, dermal condensate; DP, dermal papilla. Broken line indicates the boundary between epidermis (Epi) and dermis (Der).

View larger version (70K): [in a new window]   Fig. 3. NF-B regulates down growth, but not initiation of primary guard hair follicle placodes. (A) Upper panel: high-resolution light microscopy of sagittal sections of wild-type, cIBN (N) and downless (dl) mice. Embryos were analysed at E14.5 and E15.5. Developmental stage of placodes is indicated in each panel (0-1/2). Lower panel shows a schematic presentation of the first typical morphological changes observed during hair follicle induction (see also Paus et al., 1999). Brackets indicate placode borders, long arrows indicate apoptosis and short arrow indicate mitosis. Asterisks indicate attempted hair follicle formation; the club indicates loss of structural organisation. (B) In situ hybridization of wild-type and cIBN (N) mice at E14.5 and E15.5 using a cyclin D1 sense and antisense probe. Vibrissae follicles also show cyclinD1 expression in dermal condensate (wild type E14.5). s, sense probe; as, anti-sense probe; vib., vibrissae. Stages of hair follicle development are indicated beneath placodes in wild-type sections. Broken lines indicate the boundary between epidermis and dermis. (C) Analysis of cell proliferation in the epidermis of E14.5 and E15.5 wild-type and IBN (N) embryos. BrdU incorporation into the DNA was detected with an anti-BrdU antibody and subsequent peroxidase reaction (brown nuclei). There are also a few nuclei stained in the interfollicular epidermis and dermis of wild-type and N embryos. Developmental stages are indicated in each panel. P, placode. Brackets indicate placode borders.

View larger version (108K): [in a new window]   Fig. 4. Loss of epidermal structure resulting from reduced cell-cell contacts, significant apoptosis and vacuolization of keratinocytes during early guard hair follicle development in cIBN (IBN) and downless (dl) compared with wild-type mice. High-resolution light microscopy and electron microscopy images of embryos at E14.5 (A) and E15.5 (B). (A, a) Hair follicle placode stage 0/1 in wild-type embryo. Focal reorganization of basal keratinocytes with clustering of mesenchymal cells. (b,c) Cell-cell contact maintained by intercellular junctions, e.g. desmosomes. No vacuoles. (d) Hair follicle placode stage 0/1 in cIBN embryo. Focal reorganization of basal keratinocytes, especially cell parallelization, and an attempt at clustering of mesenchymal cells with mitosis (arrow). (e) Apoptotic cells in epidermis and mesenchyme (black arrows). (e-g) Lack of cohesion of basal cells (white arrows) with associated vacuoles containing lipid-like material. (h,i) Hair follicle placode stage 0/1 in downless embryo. (h) Focal reorganization of basal keratinocytes via cell parallelization and clustering of mesodermal cells. (i) Increased apoptosis in epidermis (black arrows). (j) Lack of cohesion of basal cells (white arrows). (k) Vacuoles containing lipid-like material in epidermis (white arrows). (B, a) Hair follicle placode stage 2 in wild-type embryo. Development of hair peg and significant clustering of mesenchymal cells forming the dermal condensate. (b,c) Epidermal cell-cell contacts maintained by intercellular junctions, including desmosomes. No vacuoles. (d) Hair follicle placode stage 0 in cIBN embryo. No placode development detectable. (d,e,h) Significant apoptosis (black arrows). Cell-cell contacts are maintained by intercellular junctions, including desmosomes, and intercellular spaces are less than at E14.5. Vacuoles containing lipid-like material present. (h,j) Cellular disorganization with poor stratification. (k) Hair follicle placode stage 0 in downless embryo. (k,i,l) No placode development detectable. Cell-cell contacts are maintained as in cIBN and there are fewer intercellular spaces than at E14.5. (m) Vacuoles containing lipid-like material present. P, basal keratinocytes, clustering in hair placodes; M, mesenchymal cells, clustering to form dermal condensate; V, vacuoles; D, dermis; E, epidermis.

View larger version (69K): [in a new window]   Fig. 5. In vivo NF-B activity is downstream of Eda A1/EdaR. (A) Gal NF-B reporter mice ((Ig)3xconalacZ, gal) were mated into cIBN (IBN, N), downless (dl, mutant EdaR) or tabby (ta, mutant Eda A1) mice, and analysed by X-Gal test at the time point of guard hair placode initiation. Upper panels, E14; lower panels, E15. Gal x cIBN mice only revealed some background staining close to the eye and elbow. In Gal x downless and x tabby embryos, X-Gal staining was absent in the placodes, but present in blood vessels (see lower left insets). (B) Technovit sections of X-Gal stained P0 skin of the same matings as indicated in the top left. For X-Gal staining in newborn wild-type Gal skin, see Fig. 2B. (C) In situ hybridization of sagittal paraffin sections of wild-type, cIBN and downless (dl) embryos at indicated time points, using EdaR, mouse IB, Eda A1 and human IB antisense probes. vib., vibrissae. Arrows indicate follicle placodes and (in Eda A1) interfollicular epidermis. 0/1–3, developmental stages of hair placode.

View larger version (47K): [in a new window]   Fig. 6. NF-B is essential and sufficient to induce placode down growth and Shh expression. (A) Embryonic skin explants of E13.5 Gal x tabby matings were incubated for 24 hours with recombinant Fc-Eda A1, Fc-Eda A2, TNF or PMA or left untreated (nt). Upper row, X-Gal staining of skin explants. Lower row, whole-mount in situ hybridization of skins using an Shh antisense probe. Placode down growth was induced more or less strongly by any stimulator of NF-B activity, except for Eda A2. (B) In situ hybridization of sagittal embryonic wild-type and cIBN (N) skin sections at indicated time points (E14.5-P0) using a Shh antisense probe. Broken lines indicate the boundary between epidermis and dermis. Arrows indicate Shh-positive follicles.

View larger version (81K): [in a new window]   Fig. 7. NF-B is needed for secondary zigzag hair development, where it may also be regulated by TROY. (A) Upper panel: cryosections of E17.5 and P0 wild-type and cIBN (N) mice, stained with Haematoxylin/Eosin. Lower panel: hair follicle numbers of E17.5 embryos [wild type, cIBN (N); n=3 each] and newborn (P0; n=9 cIBN, n=6 wild type) were counted per microscopic field (mf). Mean values were calculated and presented in bar graphs, including standard deviations. P values show a significant difference: *P<0.0158 versus wild type. for E17.5, and P<0.0001 versus wild type for P0. At P0, the mean value reveals 50% less hairs in cIBN mice compared with wild type: 38/mf in cIBN versus 75/mf in wild type. (B) The different developmental stages of hair follicle development (stage 1, placode – early bulbous peg stage 5, indicated to the left of the table) in wild type and cIBN (N) mice at E17.5 and P0 are presented in percentage (%) of total number of hairs. (C) In situ hybridization of sagittal skin sections of E14.5, E15.5, E17.5 and P0 embryos using a TROY antisense probe. Broken line indicates the boundary between epidermis (E) and dermis (D). Arrows indicate placodes and (at P0) the matrix of a guard hair follicle.

View larger version (28K): [in a new window]   Fig. 8. Working hypothesis on the role of NF-B in guard, zigzag or awl hair development. The upper panel displays the degree of involvement of NF-B or EdaR/NF-B during the development of the four pelage hair types, proceeding in three different waves. Activation of NF-B is reflected by the X-Gal activity in the NF-B reporter mice (Gal). *The NF-B activating signal for the formation or down growth of zigzag placodes remains unknown. A possible role of EdaR/NF-B in the morphogenesis of guard hairs has also not been identified yet (indicated by `?'). The lower scheme indicates the time point at which Eda A1/EdaR/NF-B contribute to the development of each hair type. Shh and cyclin D1 are downstream of EdaR/NF-B in guard hair follicles. It remains to be determined whether this is also true for zigzag follicles, and whether NF-B directly regulates Shh.

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