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First published online December 12, 2006
doi: 10.1242/10.1242/dev.02708

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Ectodysplasin has a dual role in ectodermal organogenesis: inhibition of Bmp activity and induction of Shh expression

¹ Institute of Biotechnology, Developmental Biology Program, University of Helsinki, 00014 Helsinki, Finland.
² Department of Biochemistry, University of Lausanne, 1066 Epalinges, Switzerland.

View larger version (60K): [in this window] [in a new window]   Fig. 1. Eda-A1 suppresses BMP activity in developing incisors. Whole-mount in situ hybridization analysis of ameloblastin expression in E15 incisors cultured with protein-releasing beads for 1 day. (A) Some endogenous expression of ameloblastin is present in explants cultured with a BSA bead. (B) Ameloblastin was strongly induced by a Bmp4-releasing bead. (C) BSA-releasing beads (small and blue) were placed at the same time with the Bmp4 bead (large and white). (D) Eda-A1 beads (blue) had no major effect on Bmp4-induced expression of ameloblastin when applied simultaneously with the Bmp4 bead (white). (E) BSA beads (blue) applied 6 hours before the Bmp4 bead (white) had no effect on ameloblastin expression. (F) Introduction of Eda-A1-releasing beads (blue) 6 hours before the BMP bead (white) strongly inhibited the induction of ameloblastin. (G) The endogenous expression of ameloblastin was reduced in the presence of Eda-A1 (compare with A).

View larger version (105K): [in this window] [in a new window]   Fig. 2. Expression patterns of Edar and BMP inhibitors in wild-type molar teeth. In situ hybridization with probes specific to Edar (A,B), noggin (C,D), gremlin (E,F), Dan (G,H) and bambi (I,J) at the placode stage (A,C,E,G,I) and at the cap stage (B,D,F,H,J) of tooth development. Edar is expressed in the dental placode (arrowhead) at E12, and in the enamel knot (arrow) at E14. Noggin, gremlin and Dan were detected only in the mesenchyme, whereas bambi was also expressed in the epithelium overlapping the enamel knot at E14.

View larger version (77K): [in this window] [in a new window]   Fig. 3. Noggin partially restores hair placode formation in Eda-deficient skin. E13 skin explants were cultured for 1 day and hair placode induction was detected by expression of placode marker Shh. (A) Eda-deficient explants cultured in the control medium lack the primary hair follicles. (B,C) Explants cultured in the presence of 0.5 µg/ml exogenous noggin (B) partially restored hair placode induction, whereas a more prominent rescue was seen with 2 µg/ml of noggin (C). (D,E) Eda-A1 at 0.1 µg/ml restored the expression of Shh (D), whereas 2 µg/ml Eda-A1 caused a prominent enlargement of placodes in Eda-/- skin (E). (F,G) noggin at 2 µg/ml caused enlargement of hair placodes in wild-type skin (G) compared with untreated explants (F).

View larger version (27K): [in this window] [in a new window]   Fig. 4. Expression of Ccn2 and follistatin is induced by Eda-A1. Eda-deficient (A,B) or wild-type (C) E14 individual skin explants were cut in two halves along the midline and cultured in the absence or presence of 2 µg/ml Eda-A1, and gene expression was analysed by quantitative RT-PCR. (A) A timecourse of expression of Ccn2, Smad7, follistatin and I-B in Eda-deficient skin upon Eda-A1 stimulus. Upregulation of Ccn2 is detected already after 1 hour of Eda treatment and peaks at 3-4 hours. I-B is induced with kinetics similar to Ccn2. (B) Comparison of Ccn2 induction by Eda-A1 in skin explants cultured in a medium with or without serum. Ccn2 transcripts are maintained at high level for a longer time in the absence of serum. (C) Induction of Ccn2, Smad7 and follistatin gene expression in wild-type skin after exposure to Eda-A1 in the absence of serum.

View larger version (95K): [in this window] [in a new window]   Fig. 5. Edar and Ccn2 colocalize during early hair and tooth development. Whole-mount in situ hybridization with a probe specific to Ccn2 (A-G,H,K,M) and Edar (I,J,L). (A-C') Ccn2 is expressed in primary hair placodes of wild-type embryos at E14, and is often concentrated at the circumference of the placode (arrowheads in C). (D) No localized expression of Ccn2 is detected in Eda-null skin at E14. (E-G) Recombinant Eda-A1-induced localized upregulation of Ccn2 in Eda-/- E13 skin explants cultured for 24 hours. (H,I) Vibratome section of whole mounts confirmed the colocalization of Edar and Ccn2 in primary hair placodes of E14 wild-type embryos. (J,K) Edar and Ccn2 are coexpressed also in wild-type molar (arrow) and incisor (arrowhead) placodes at E12. (L,M) In contrast to primary hair placodes, expression of Ccn2 is unaffected in Eda-deficient tooth placodes. Mc, Meckel's cartilage. Scale bar: 1 mm in A; 0.5 mm in B-D,J-M; 50 µm in H,I.

View larger version (36K): [in this window] [in a new window]   Fig. 6. Eda-A1 upregulates transcription of Shh, but recombinant Shh is unable to rescue hair placode induction in Eda-deficient skin. (A) Eda-deficient skin explants were cultured in the absence or presence of Eda-A1, as depicted in Fig. 4A, and Shh expression was analysed by quantitative RT-PCR. A 2.5-fold induction of Shh was evident already after 1 hour of treatment, and was highest after 3-4 hours of treatment. (B) The number of Shh transcripts was increased in E14 K14-Eda-A1 skin compared with control littermates. (C-F) Eda-deficient E13 skin explants cultured for 1 day in the control medium are devoid of hair follicles and no rescue is detected with 0.1 µg/ml, 0.5 µg/ml or 1.0 µg/ml recombinant Shh.

View larger version (18K): [in this window] [in a new window]   Fig. 7. A schematic representation of the outcomes of Edar activation in primary hair follicles compared to signalling in secondary hair follicles. Upon ligand engagement, Edar activates NF-B, resulting in the upregulation of Ccn2, follistatin and Shh expression. We suggest that Ccn2 and follistatin act locally to inhibit Bmps expressed in the epithelium and/or the condensed mesenchyme, thereby allowing the expansion of the nascent placode. After the placode stage, upregulation of Shh promotes invagination of the hair bud. In secondary hair follicles, noggin expressed in the condensed mesenchyme suppresses Bmp activity within the placode, whereas other signals, possibly Wnts, induce the expression of Shh.