Tuija Mustonen

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.

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, Edar and TNFRSF19 are expressed in complementary and overlapping patterns during mouse embryogenesis

Ectodysplasin (Eda), a member of the tumor necrosis factor (TNF) superfamily, and its receptor Edar are necessary components of ectodermal organ development. Analysis of their expression patterns and mutant phenotypes has shown that during mouse hair and tooth development they may be involved in signalling between separate epithelial compartments. Here we have analysed ectodysplasin and Edar expression in other embryonic mouse tissues, and show that Edar mRNA is confined to the epithelium. Ectodysplasin and Edar are expressed in separate epithelial compartments in the developing brain and the lacrimal gland. In the salivary gland ectodysplasin is expressed in the mesenchyme and Edar in the epithelium. This is the first indication of ectodysplasin-Edar signalling between the epithelium and the mesenchyme. We also studied the expression pattern of a related TNF receptor, TNFRSF19, and show that it is expressed in an overlapping domain with Edar in the tooth, mammary gland, whiskers, and limb bud suggesting a potentially redundant role.

Tooth patterning and enamel formation can be manipulated by misexpression of TNF receptor Edar

Signaling by Edar, a tumor necrosis factor receptor, is required for the development of ectodermal organs. Mutations in Edar or other molecules of the same signaling pathway cause ectodermal dysplasias in humans and mice. In these diseases, teeth are missing or malformed, and the development of hairs and several glands is hypoplastic. During tooth and hair development, Edar expression becomes patterned to ectodermal placodes and signaling centers. This localization has been suggested to be required for organogenesis. We have expressed Edar throughout the ectoderm using the keratin 14 promoter and show that this misexpression disrupts tooth patterning and differentiation. Tooth shape and cusp number are differentially affected, depending on the amount of transgene expression. In addition, tooth enamel formation is defective in a dose‐dependent manner. We speculate that the tooth patterning defects are caused by ectopic Edar activity outside the signaling centers. Developmental Dynamics 231:432–440, 2004.

Regulation of Calvarial Osteogenesis by Concomitant De-repression of GLI3 and Activation of IHH Targets

Loss-of-function mutations in GLI3 and IHH cause craniosynostosis and reduced osteogenesis, respectively. In this study, we show that Ihh ligand, the receptor Ptch1 and Gli transcription factors are differentially expressed in embryonic mouse calvaria osteogenic condensations. We show that in both Ihh−/− and Gli3Xt−J/Xt−J embryonic mice, the normal gene expression architecture is lost and this results in disorganized calvarial bone development. RUNX2 is a master regulatory transcription factor controlling osteogenesis. In the absence of Gli3, RUNX2 isoform II and IHH are upregulated, and RUNX2 isoform I downregulated. This is consistent with the expanded and aberrant osteogenesis observed in Gli3Xt−J/Xt−J mice, and consistent with Runx2-I expression by relatively immature osteoprogenitors. Ihh−/− mice exhibited small calvarial bones and HH target genes, Ptch1 and Gli1, were absent. This indicates that IHH is the functional HH ligand, and that it is not compensated by another HH ligand. To decipher the roles and potential interaction of Gli3 and Ihh, we generated Ihh−/−;Gli3Xt−J/Xt−J compound mutant mice. Even in the absence of Ihh, Gli3 deletion was sufficient to induce aberrant precocious ossification across the developing suture, indicating that the craniosynostosis phenotype of Gli3Xt−J/Xt−J mice is not dependent on IHH ligand. Also, we found that Ihh was not required for Runx2 expression as the expression of RUNX2 target genes was unaffected by deletion of Ihh. To test whether RUNX2 has a role upstream of IHH, we performed RUNX2 siRNA knock down experiments in WT calvarial osteoblasts and explants and found that Ihh expression is suppressed. Our results show that IHH is the functional HH ligand in the embryonic mouse calvaria osteogenic condensations, where it regulates the progression of osteoblastic differentiation. As GLI3 represses the expression of Runx2-II and Ihh, and also elevates the Runx2-I expression, and as IHH may be regulated by RUNX2 these results raise the possibility of a regulatory feedback circuit to control calvarial osteogenesis and suture patency. Taken together, RUNX2-controlled osteoblastic cell fate is regulated by IHH through concomitant inhibition of GLI3-repressor formation and activation of downstream targets.

Rab23 deficient mice exhibit lambdoid suture craniosynostosis through aberrant Fgf signaling

Adolescent idiopathic scoliosis (IS), a disease characterized by 3D spinal curvatures, afflicts 3% of children worldwide. However, the underlying developmental basis for this disease has remained unknown. Recently, the teleost fish have emerged as robust models of IS. Using zebrafish, we have discovered that spinal curves are caused by loss of motile cilia function, which in turn results in defective CSF flow. Human IS can be caused by mutations in PTK7, and we find that cilia/CSF flow defects are also the basis of spinal curves in ptk7 zebrafish mutants. Thus, we propose that IS can be caused by disruptions to CSF flow during the growth phase, providing a novel mechanism for this prevalent disease. We also give evidence that spinal curves can be partially rescued by restoring cilia motility after curve onset, opening potential therapeutic avenues. Overall, our study provides new insight into spine formation and the maintenance of spine linearity.

09-P087 FGF signaling is required for the specification of GnRH neurons

like) exhibit a pleiotropic phenotype including abnormalities of cardiac, skeletal and limb development, in addition to renal agenesis. Here we report abnormalities of gonad development in homozygous mutants. From early stages, Vcc/Vcc embryos contain gonads longer than controls and by 13.5 dpc this increased length is evidenced by an increase in the number of testis cords. The gene mutated in Vcc, Pcsk5, encodes a proprotein convertase that is thought to co-ordinately regulate the expression of several paralogous Hox genes. Utilising a variety of approaches, we are investigating the possibility that abnormal HOX-dependent patterning of the urogenital ridge in Vcc/Vcc embryos expands the gonadal field, resulting in the striking lengthening of the mutant gonads. Studies of this mutation may shed light on the cellular processes underlying gonad formation and subsequent testis cord formation in males, and their genetic regulation.