Edar and Troy signalling pathways act redundantly to regulate initiation of hair follicle development.

The development of ectodermal organs requires signalling by ectodysplasin (Eda), a tumor necrosis factor (TNF) family member, its receptor Edar and downstream activation of the nuclear factor kappaB (NF-kappaB) transcription factor. In humans, mutations in the Eda pathway components cause hypohidrotic ectodermal dysplasia, a syndrome characterized by missing teeth, sparse hair and defects in sweat glands. It has been postulated that Eda acts redundantly with another TNF pathway to regulate ectodermal organogenesis. A potential candidate is Troy (or TNFRSF19 or Taj), a TNF receptor which is homologous with Edar in its ligand-binding domain, and is expressed in an overlapping pattern. We have characterized Troy null mice and crossed them with Eda-deficient mice. Single Troy mutants had no defects in ectodermal organs. Analysis of the double mutants revealed an essential role for Troy in hair follicle development. In mice, hair follicles develop in three different waves. Only primary hair follicles are missing in Eda single mutants, whereas the compound mutants lacked also the follicles of the second wave, as well as all hair follicles in the middle of crown leading to focal alopecia. Assessment of NF-kappaB activity with a transgenic reporter construct indicated that Eda is the main activator of NF-kappaB signalling in developing skin appendages and surprisingly that the functional overlap of Troy and Eda signalling pathways is mediated by NF-kappaB independent pathways.

The taming of the shrew milk teeth.

A characteristic feature of mammalian dentition is the evolutionary reduction of tooth number and replacement. Because mice do not replace teeth, here we used Sorex araneus, the common shrew, as a model to investigate the loss of tooth replacement. Historically, shrews have been reported to initiate the development of several, milk or deciduous teeth but these soon become rudimentary and only the replacement teeth erupt. Shrews thus offer a living example of a derived mammalian pattern where the deciduous tooth development is being suppressed. Based on histological and gene expression analyses of serial sections, we suggest that S. araneus has discernible tooth replacement only in the premolar 4 (P4) position. Both generations of teeth express Shh in the enamel knot and in the inner enamel epithelium. Nevertheless, the deciduous P4 (dP4) is reduced in size during embryogenesis and is eventually lost without becoming functional. Analysis of growth shows that P4 replaces the dP4 in a "double-wedge" pattern indicative of competitive replacement where the suppression of the deciduous tooth coincides with the initiation of its replacement. Because activator-inhibitor mechanisms have been implicated in adjacent mouse molars and in transgenic mice with continuous tooth budding, we suggest that evolutionary suppression of deciduous teeth may involve early activation of replacement teeth, which in turn begin to suppress their deciduous predecessors.

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

Ectodermal organogenesis is regulated by inductive and reciprocal signalling cascades that involve multiple signal molecules in several conserved families. Ectodysplasin-A (Eda), a tumour necrosis factor-like signalling molecule, and its receptor Edar are required for the development of a number of ectodermal organs in vertebrates. In mice, lack of Eda leads to failure in primary hair placode formation and missing or abnormally shaped teeth, whereas mice overexpressing Eda are characterized by enlarged hair placodes and supernumerary teeth and mammary glands. Here, we report two signalling outcomes of the Eda pathway: suppression of bone morphogenetic protein (Bmp) activity and upregulation of sonic hedgehog (Shh) signalling. Recombinant Eda counteracted Bmp4 activity in developing teeth and, importantly, inhibition of BMP activity by exogenous noggin partially restored primary hair placode formation in Eda-deficient skin in vitro, indicating that suppression of Bmp activity was compromised in the absence of Eda. The downstream effects of the Eda pathway are likely to be mediated by transcription factor nuclear factor-kappaB (NF-kappaB), but the transcriptional targets of Edar have remained unknown. Using a quantitative approach, we show in cultured embryonic skin that Eda induced the expression of two Bmp inhibitors, Ccn2/Ctgf (CCN family protein 2/connective tissue growth factor) and follistatin. Moreover, our data indicate that Shh is a likely transcriptional target of Edar, but, unlike noggin, recombinant Shh was unable to rescue primary hair placode formation in Eda-deficient skin explants.

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

Organs developing as appendages of the ectoderm are initiated from epithelial thickenings called placodes. Their formation is regulated by interactions between the ectoderm and underlying mesenchyme, and several signalling molecules have been implicated as activators or inhibitors of placode formation. Ectodysplasin (Eda) is a unique signalling molecule in the tumour necrosis factor family that, together with its receptor Edar, is necessary for normal development of ectodermal organs both in humans and mice. We have shown previously that overexpression of the Eda-A1 isoform in transgenic mice stimulates the formation of several ectodermal organs. In the present study, we have analysed the formation and morphology of placodes using in vivo and in vitro models in which both the timing and amount of Eda-A1 applied could be varied. The hair and tooth placodes of K14-Eda-A1 transgenic embryos were enlarged, and extra placodes developed from the dental lamina and mammary line. Exposure of embryonic skin to Eda-A1 recombinant protein in vitro stimulated the growth and fusion of placodes. However, it did not accelerate the initiation of the first wave of hair follicles giving rise to the guard hairs. Hence, the function of Eda-A1 appears to be downstream of the primary inductive signal required for placode initiation during skin patterning. Analysis of BrdU incorporation indicated that the formation of the epithelial thickening in early placodes does not involve increased cell proliferation and also that the positive effect of Eda-A1 on placode expansion is not a result of increased cell proliferation. Taken together, our results suggest that Eda-A1 signalling promotes placodal cell fate during early development of ectodermal organs.

Stimulation of ectodermal organ development by Ectodysplasin-A1.

Organs developing as ectodermal appendages share similar early morphogenesis and molecular mechanisms. Ectodysplasin, a signaling molecule belonging to the tumor necrosis factor family, and its receptor Edar are required for normal development of several ectodermal organs in humans and mice. We have overexpressed two splice forms of ectodysplasin, Eda-A1 and Eda-A2, binding to Edar and another TNF receptor, Xedar, respectively, under the keratin 14 (K14) promoter in the ectoderm of transgenic mice. Eda-A2 overexpression did not cause a detectable phenotype. On the contrary, overexpression of Eda-A1 resulted in alterations in a variety of ectodermal organs, most notably in extra organs. Hair development was initiated continuously from E14 until birth, and in addition, the transgenic mice had supernumerary teeth and mammary glands, phenotypes not reported previously in transgenic mice. Also, hair composition and structure was abnormal, and the cycling of hairs was altered so that the growth phase (anagen) was prolonged. Both hairs and nails grew longer than normal. Molar teeth were of abnormal shape, and enamel formation was severely disturbed in incisors. Furthermore, sweat gland function was stimulated and sebaceous glands were enlarged. We conclude that ectodysplasin-Edar signaling has several roles in ectodermal organ development controlling their initiation, as well as morphogenesis and differentiation.