Marja L. Mikkola

p63 regulates multiple signalling pathways required for ectodermal organogenesis and differentiation

Heterozygous germline mutations in p63, a transcription factor of the p53 family, result in abnormal morphogenesis of the skin and its associated structures, including hair follicles and teeth. In mice lacking p63, all ectodermal organs fail to develop, and stratification of the epidermis is absent. We show that the ectodermal placodes that mark early tooth and hair follicle morphogenesis do not form in p63-deficient embryos, although the multilayered dental lamina that precedes tooth placode formation develops normally. The N-terminally truncated isoform of p63 (ΔNp63) was expressed at high levels in embryonic ectoderm at all stages of tooth and hair development, and it was already dominant over the transactivating TAp63 isoform prior to epidermal stratification. Bmp7, Fgfr2b, Jag1 and Notch1 transcripts were co-expressed withΔ Np63 in wild-type embryos, but were not detectable in the ectoderm of p63 mutants. In addition, β-catenin and Edar transcripts were significantly reduced in skin ectoderm. We also demonstrate that BMP2, BMP7 and FGF10 are potent inducers of p63 in cultured tissue explants. Hence, we suggest that p63 regulates the morphogenesis of surface ectoderm and its derivatives via multiple signalling pathways.

The role of growth factors in tooth development

Growth factors and other paracrine signal molecules regulate communication between cells in all developing organs. During tooth morphogenesis, molecules in several conserved signal families mediate interactions both between and within the epithelial and mesenchymal tissue layers. The same molecules are used repeatedly during advancing development, and several growth factors are coexpressed in epithelial signaling centers. The enamel knots are signaling centers that regulate the patterning of teeth and are associated with foldings of the epithelial sheet. Different signaling pathways form networks and are integrated at many levels. Many targets of the growth factors have been identified, and mutations in several genes within the signaling networks cause defective tooth formation in both humans and mice.

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.

Reciprocal Requirements for EDA/EDAR/NF-κB and Wnt/β-Catenin Signaling Pathways in Hair Follicle Induction

Wnt/beta-catenin and NF-kappaB signaling mechanisms provide central controls in development and disease, but how these pathways intersect is unclear. Using hair follicle induction as a model system, we show that patterning of dermal Wnt/beta-catenin signaling requires epithelial beta-catenin activity. We find that Wnt/beta-catenin signaling is absolutely required for NF-kappaB activation, and that Edar is a direct Wnt target gene. Wnt/beta-catenin signaling is initially activated independently of EDA/EDAR/NF-kappaB activity in primary hair follicle primordia. However, Eda/Edar/NF-kappaB signaling is required to refine the pattern of Wnt/beta-catenin activity, and to maintain this activity at later stages of placode development. We show that maintenance of localized expression of Wnt10b and Wnt10a requires NF-kappaB signaling, providing a molecular explanation for the latter observation, and identify Wnt10b as a direct NF-kappaB target. These data reveal a complex interplay and interdependence of Wnt/beta-catenin and EDA/EDAR/NF-kappaB signaling pathways in initiation and maintenance of primary hair follicle placodes.

RNA HELICASE ACTIVITY OF SEMLIKI FOREST VIRUS REPLICASE PROTEIN NSP2

Semliki Forest virus replicase protein nsP2 shares sequence homology with several putative NTPases and RNA helicases. NsP2 has RNA‐dependent NTPase activity. Here we expressed polyhistidine‐tagged nsP2 in Escherichia coli, purified it by metal‐affinity chromatography, and used it in RNA helicase assays. RNA helicase CI of plum pox potyvirus was used as a positive control. Unwinding of α‐32P‐labelled partially double‐stranded RNA required nsP2, Mg2+ and NTPs. NsP2 with a mutation, K192N, in the NTP‐binding sequence GVPGSGK192SA could not unwind dsRNA and had no NTPase activity. This is the first demonstration of RNA helicase activity within the large alphavirus superfamily.

Ectodysplasin signaling in development

Ectodysplasin (Eda), a signaling molecule belonging to the tumor necrosis factor family, is required for normal development of several ectodermally derived organs in humans and mice. Two closely related isoforms of ectodysplasin, Eda-A1 and Eda-A2, have been described which bind to and activate two different receptors, Edar and X-linked Eda-A2 receptor (Xedar), respectively. Mutations in Eda, Edar or other molecules of this signaling pathway cause ectodermal dysplasias characterized by defective development of teeth, hairs, and several exocrine glands such as sweat glands presumably due to impaired NF-kappaB response. Studies with mice either lacking the functional proteins of Edar pathway or overexpressing the ligand or receptor suggest that Eda-A1-Edar signaling has multiple roles in ectodermal organ development regulating their initiation, morphogenesis, and differentiation.

Molecular aspects of hypohidrotic ectodermal dysplasia.

Hypohidrotic (anhidrotic) ectodermal dysplasia (HED) is a congenital syndrome characterized by sparse hair, oligodontia, and reduced sweating. It is caused by mutations in any of the three Eda pathway genes: ectodysplasin (Eda), Edar, and Edaradd which encode a ligand, a receptor, and an intracellular signal mediator of a single linear pathway, respectively. In rare cases, HED is associated with immune deficiency caused by mutations in further downstream components of the Eda pathway that are necessary for the activation of the transcription factor NF‐κB. Here I present a brief research update on the molecular aspects of this evolutionarily conserved pathway. The developmental role of Eda will be discussed in light of loss‐ and gain‐of‐function mouse models with emphasis on the past few years.

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.

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-κB (NF-κB), 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, a protein required for epithelial morphogenesis, is a novel TNF homologue and promotes cell-matrix adhesion.

In the mouse Tabby (Ta) mutant and human X-linked anhidrotic ectodermal dysplasia (EDA) syndrome development of several ectodermal organs such as hair, teeth, and sweat glands is impaired. The gene behind Tabby and EDA has been cloned, and several alternative transcripts have been isolated. The protein product named ectodysplasin had no obvious function or prominent homology to other known gene products apart from a short collagen-like sequence. We have isolated two novel Ta transcripts which are variants of the longest isoform of Tabby, named Ta-A. In situ hybridizations revealed Ta-A to be the major transcript in the developing embryo. It was detected in the endoderm of early embryos and subsequently in specific locations in the neuroepithelium and ectoderm. Unexpectedly, sequence analysis of the most C-terminal domain of Ta revealed that ectodysplasin is a novel member of the tumor necrosis factor (TNF) ligand superfamily. Mouse ectodysplasin was biochemically and functionally characterized, and shown to be a glycosylated, oligomeric type II membrane protein (N-terminus inside), all characteristics typical to TNF-like proteins. Members of the TNF family are critically involved in host defence and immune response often mediating either apoptosis or cell survival. Expression of Ta in several epithelial cell lines did not result in prominent changes in cell morphology and did not promote apoptosis. Instead, it was shown to promote cell adhesion to extracellular matrix, a function consistent with its postulated role in epithelial-mesenchymal interactions regulating the development of ectodermal appendages. Ectodysplasin is the first TNF-like signaling molecule described known to be required for epithelial morphogenesis.