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First published online 15 September 2004
doi: 10.1242/dev.01377

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Ectodysplasin A1 promotes placodal cell fate during early morphogenesis of ectodermal appendages

Tuija Mustonen^1,*, Maritta Ilmonen^1,*, Marja Pummila¹, Aapo T. Kangas¹, Johanna Laurikkala¹, Risto Jaatinen¹, Johanna Pispa¹, Olivier Gaide^2,, Pascal Schneider², Irma Thesleff¹ and Marja L. Mikkola^1,

¹ Developmental Biology Program, Institute of Biotechnology, PO Box 56 (Viikinkaari 9), University of Helsinki, 00014, Finland
² Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland

View larger version (109K): [in a new window]   Fig. 1. Eda-A1 overexpression leads to increased size and irregular shape of the early hair placodes. Placodes were larger in K14-Eda-A1 transgenic embryonic E14 skin than in wild-type skin, as visualised by whole-mount in situ hybridisation by marker genes ß-catenin (A,B) and Edar (C,D). E-H are higher magnification views of A-D. Fusions (arrow in B and F) and the irregular shape (arrows in D and H) of the early hair follicles were common in transgenic skin. In radioactive in situ hybridisation analysis, the expression of Lef1 was seen throughout wild-type (I) and transgenic (J) skin. Placodal epithelial and mesenchymal marker Ptch1 also showed expansion of the hair follicles in the transgenic skin (arrow in L) when compared with wild type (K). Expression of Bmp4 mRNA localised to the mesenchyme under the forming follicles in wild-type (M) and transgenic (N) skin. Enlarged hair follicles with irregular shape in the transgenic ectoderm (P) were evident in thin plastic sections of the E14 skin, whereas wild-type follicles were smaller and had a regular round shape (O). Scanning electron microscopy images also revealed irregularities in shape and increased sizes of the hair placodes of the transgenic embryos (R) and fusions of their follicles (T), when compared with the wild-type embryos with smaller regular placodes (Q,S).

View larger version (26K): [in a new window]   Fig. 2. Production of recombinant Eda-A1 proteins. (A) Schematic drawing of the Eda-A1 protein with a transmembrane domain (TM), a furin cleavage site (asterisk), collagen like Gly-X-Y repeats (COL) and the globular TNF homology domain (TNF-DOMAIN). Figures refer to amino acid numbering of endogenous Eda-A1. Recombinant Eda-A1 starts at amino acid residue 180 and contains an N-terminal 6x His tag. Control protein contained Y343C mutation that abolishes receptor binding. Recombinant Fc-Eda-A1 consists of an N-terminal HA tag, an Fc-domain and the TNF-domain of Eda-A1. (B) Cos cells were transfected with the soluble, truncated form of wild-type Rec Eda-A1 (lane 2) or mutated Y343C Eda-A1 construct (lane 3), or were mock transfected (lane 1). Cell supernatants were analysed by western blot using an anti-Eda polyclonal antibody. (C) Purified Fc-Eda-A1 (5 µg) was analysed by SDS-PAGE under reducing conditions and stained with Coomassie Blue. The doublet band results from glycosylation. Molecular weight markers are indicated on the left.

View larger version (98K): [in a new window]   Fig. 3. Eda-A1 recombinant protein increased the size and caused fusions of the hair follicles in in vitro cultures of embryonic skin. Hair follicles of the cultured back skin samples were analysed by whole-mount in situ hybridisation with ß-catenin probe. E13 skin explants showed bigger and fused hair follicles after 1 day in culture with the conditioned media from cells producing recombinant Eda-A1 protein (A), compared with normal hair follicles seen in cultures with Y343 control media (B). Similar effect was seen also after two days of culture (C,D). Older explants of E14 skin showed no effects after 1 day in culture with Eda-A1 medium (E): the hair follicles were of a similar size to those of the control cultures (F). After 3 days of culture with Rec Eda-A1 media (G), the hair follicles were clearly bigger than in control cultures (H).

View larger version (114K): [in a new window]   Fig. 4. Ectopic Eda-A1 protein rescued the early Tabby skin phenotype and caused enlargement of the placodal fields in a quantitative manner when analysed with placode marker genes Shh or ß-catenin. Eda-deficient Tabby skin lacks the first hair follicles as shown by the absence of placode marker Shh whole-mount in situ (A). Shh expression in placodes was partially rescued by culturing the Tabby skin explants with 0.05 µg/ml of Fc-Eda-A1 protein (B). Full rescue was obtained with 0.1 µg/ml (C) to 0.5 µg/ml (D) of Fc-Eda-A1. Higher concentration (2 µg/ml) caused enlargement and fusion of the follicles as shown by Shh expression (E). Similarly, ß-catenin expression analysis showed rescue with 0.5 µg/ml of Fc-Eda-A1 (F) and fused follicles with 2 µg/ml (G). Wild-type E13 skin sample cultured for 1 day showed ß-catenin localisation to the forming hair placodes (H), while 2 µg/ml concentration of Fc-Eda-A1 protein (I) caused expansion and fusion of the ß-catenin expression domains of the wild-type skin. Skin explants prepared at E12 and cultured for 1 day did not show Shh expression (J) and no indication of placode formation was detected upon addition of 2 µg/ml Fc-Eda-A1 to the culture medium (K).

View larger version (157K): [in a new window]   Fig. 5. Bmp4 inhibits placode formation, despite the presence of Eda-A1. E13 wild-type skin explants were grown for 24 hours in the presence of 2 µg/ml of Eda-A1 and analysed for placode formation by whole-mount in situ hybridisation with ß-catenin (data not shown) or Shh probe. BSA control beads (A; 0/12 explants) had no effect, whereas two agarose beads soaked in 20 ng/µl (B; 11/12 explants), 75 ng/µl (C; 10/10 explants) or 500 ng/µl (D; 13/13 explants) of Bmp4 placed on top of each explant inhibited placode formation and enlargement in the vicinity of the bead.

View larger version (70K): [in a new window]   Fig. 6. Eda-A1 overexpression caused enlargement of the first molar placode at E12 and an extra molar placode in E13 lower jaws. (A-F) Top views of lower jaws. The odontogenic molar (red arrows in A and B) and incisor (blue arrows in A and B) fields were visualised in E11 embryonic lower jaws by Pitx2 expression. No difference was seen between wild-type (A) and K14-Eda-A1 transgenic (B) embryos. At E12, the dental placodes were visualised by Shh expression (C,D). The molar placodes were larger in transgenic embryos when compared with controls (arrows in C,D). At E13, extra placodes (blue arrow) were located in front of the transgenic molar placodes (red arrow in F). However, a small Shh-positive placode was also detected in wild-type embryos (blue arrow in E). The size measurements of E12 mandibular molar placodes of K14-Eda-A1 and control mice are presented as a box plot presentation (G). The size measure is a product of width and length of a placode. The height of the box presents 50% of the results. Mean is shown as a horizontal line inside the box and 95% confidence limits are shown by the lines outside the box. The difference between groups is statistically significant (P=0.03) according to the Mann-Whitney U-test. Measurements were made on three litters of mice. Number of placodes measured: K14-Eda-A1 n=23; wild-type littermates, n=18. Scale bars: 1 mm.

View larger version (103K): [in a new window]   Fig. 7. K14-Eda-A1 transgenic mice had ectopic mammary placodes, as visualised by whole-mount in situ hybridisation with Lef1 probe. At E11 the early placodes were initiated in both the wild-type (A) and transgenic (B) embryos. Mammary placodes 3 and 4 were seen as clear dots (M3, M4). Placode 2 (M2) had still a comet shaped appearance at this developmental stage. Placodes 1 and 5 were hidden by the developing limbs. At E11.5, M2-M4 are seen as clear dots in wild-type embryos (C), whereas supernumerary placodes (arrows) between M3 and M4 are evident in transgenic animals (D). At E12, the extra placodes were still visible in the transgenics (arrow in F) in contrast to the normal five placodes of wild-type animals (E).

View larger version (23K): [in a new window]   Fig. 8. Overexpression of Eda-A1 in embryonic ectoderm did not increase proliferation in hair placodes or developing skin. The amount of BrdU positive S-phase proliferating cells (arrow) in E14 hair placodes was smaller than in the interplacodal areas in both the wild-type (A) and transgenic skin (B) sections.

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