Summary Ectodysplasin A 1 promotes placodal cell fate during early morphogenesis of ectodermal appendages

A number of organs in vertebrates develop as appendages of embryonic ectoderm. The morphogenesis of these organs such as hairs, feathers, teeth and mammary glands is governed by sequential and reciprocal inductive interactions between two types of tissues, the epithelium of ectodermal origin and the mesenchyme. The mesenchyme can be derived either from the mesoderm (e.g. mammary gland and hair) or from the neural crest (e.g. tooth). Despite the diversity in their mature form and function, ectodermal organs share several common features during the early steps of development. Organogenesis begins with the appearance of local epithelial thickenings, placodes, which is followed by condensation of the underlying mesenchymal cells. In the next step, the placode develops into a bud growing into or out of the mesenchyme ensued by further growth and morphogenesis typical to each organ (Pispa and Thesleff, 2003). Although the growth of the bud is generally considered to be driven by proliferation, the cellular mechanisms governing the initial steps of placode formation have not been systematically analysed. An unanswered key question is whether placode formation is primarily a cell migration-driven event or whether it rather reflects a locally restricted burst of epithelial cell proliferation (Magerl et al., 2001; Pispa and Thesleff, 2003; Veltmaat et al., 2003). Evidently, these processes are associated with changes in cell shape as well as in their adhesive properties (Jamora et al., 2003). In most ectodermally derived organs, the mesenchyme seems to supply the first signal required to initiate organogenesis, whereas the epithelium responds according to its inherent capabilities. Classic tissue recombination experiments using mouse and chick embryonic skin from different body parts have revealed that an initial signal(s) arising in the dermis causes the formation of placodes in epidermis from non-hair bearing regions (Hardy, 1992). Similarly, mesenchyme from mouse mammary region is able to induce functional mammary epithelium when recombined with dorsal epidermis (Veltmaat et al., 2003). The issue of whether tooth initiation is determined by neural crest derived mesenchyme or by the ectoderm is controversial (Mina and Kollar, 1987; Mitsiadis et al., 2003). An epithelial placode can be considered a basal unit of ectodermal organogenesis. A characteristic feature of epithelial placodes is that many signal molecules including Wnts, bone 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 K14Eda-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.