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.

Activation of the bone morphogenetic protein signaling pathway induces inhibin beta(B)-subunit mRNA and secreted inhibin B levels in cultured human granulosa-luteal cells.

During the human menstrual cycle the circulating levels of inhibin B, a dimer of inhibin alpha- and beta(B)-subunits, fluctuate in a fashion distinct from that of inhibin A, the alpha-beta(A)-subunit dimer. This suggests that human inhibin subunits are each regulated in a distinct manner in human ovarian granulosa cells by endocrine and local factors. We have previously shown using cultures of human granulosa-luteal (hGL) cells that gonadotropins stimulate the steady state mRNA levels of inhibin alpha- and beta(A)-subunits, but not those of the beta(B)-subunit, which, on the other hand, are up-regulated by, for instance, activin and TGF beta. We recently identified the TGF beta gene family member bone morphogenetic protein-3 (BMP-3) as a granulosa cell-derived growth factor, but whether BMP-3 or other structurally related BMPs regulate human granulosa cell inhibin production is not known. We show here that hGL cells express mRNAs for distinct serine/threonine kinase receptors (BMP-RIA and BMP-RII) and Smad signaling proteins (Smad1, Smad4, and Smad5) involved in the mediation of cellular effects of BMPs. Subsequently, we determined in hGL cell cultures the effects of distinct members of the BMP family previously found to be expressed in mammalian ovaries. Recombinant BMP-2 induces potently in a time- and concentration-dependent manner the expression of the inhibin beta(B)-subunit mRNAs in hGL cells without affecting the levels of alpha- or beta(A)-subunit mRNAs. BMP-6 has a similar, but weaker, effect than BMP-2, whereas BMP-3 and its close homolog, BMP-3b (also known as growth differentiation factor-10) had no effect on inhibin subunit mRNA expression. hCG treatment of hGL cells was previously shown to abolish the stimulatory effect of activin on beta(B)-subunit mRNA levels, and here hCG is also shown to suppress the effect of BMP-2. Furthermore, BMP-2 stimulates hGL cell secreted dimeric inhibin B levels in a concentration-dependent manner. Depending on the experiment, maximal increases in inhibin B levels of 6- to 28-fold above basal levels were detected during a 72-h culture period. We conclude that activation of the BMP-signaling pathway in hGL cells stimulates inhibin beta(B)-subunit mRNA levels and leads at the protein level to a dramatic stimulation of secreted inhibin B dimers. Our results are consistent with the suggestion that in addition to the distinct activin- and TGF beta-activated signaling pathways, the BMP-activated pathway is likely to be implicated in the complex regulation of inhibins in the human ovary.