Pax genes have been shown to play important roles in mammalian development and organogenesis. Pax9, a member of this transcription factor family, is expressed in somites, pharyngeal pouches, mesenchyme involved in craniofacial, tooth, and limb development, as well as other sites during mouse embryogenesis. To analyze its function in vivo, we generated Pax9 deficient mice and show that Pax9 is essential for the development of a variety of organs and skeletal elements. Homozygous Pax9-mutant mice die shortly after birth, most likely as a consequence of a cleft secondary palate. They lack a thymus, parathyroid glands, and ultimobranchial bodies, organs which are derived from the pharyngeal pouches. In all limbs, a supernumerary preaxial digit is formed, but the flexor of the hindlimb toes is missing. Furthermore, craniofacial and visceral skeletogenesis is disturbed, and all teeth are absent. In Pax9-deficient embryos tooth development is arrested at the bud stage. At this stage, Pax9 is required for the mesenchymal expression of Bmp4, Msx1, and Lef1, suggesting a role for Pax9 in the establishment of the inductive capacity of the tooth mesenchyme. In summary, our analysis shows that Pax9 is a key regulator during the development of a wide range of organ primordia.

Targeted inactivation of the murine gene encoding the transcription factor LEF-1 abrogates the formation of organs that depend on epithelial-mesenchymal tissue interactions. In this study we have recombined epithelial and mesenchymal tissues from normal and LEF-1-deficient embryos at different stages of development to define the LEF-1-dependent steps in tooth and whisker organogenesis. At the initiation of organ development, formation of the epithelial primordium of the whisker but not tooth is dependent on mesenchymal Left gene expression. Subsequent formation of a whisker and tooth mesenchymal papilla and completion of organogenesis require transient expression of Lefl in the epithelium. These experiments indicate that the effect of Lefl expression is transmitted from one tissue to the other. In addition, the finding that the expression of Lefl can be activated by bone morphogenetic protein 4 (BMP-4) suggests a regulatory role of this transcription factor in BMP-mediated inductive tissue interactions.[Key Words: Lefl; BMP-4; epithelial-mesenchymal interactions; tooth and whisker development]

Lymphoid enhancer factor (LEF1), a nuclear mediator of Wnt signaling, is required for the formation of organs that depend on inductive interactions between epithelial and mesenchymal tissues. In previous tissue recombination experiments with normal and Lef1 −/− tooth germs, we found that the effect of LEF1 expression in the epithelium is tissue nonautonomous and transferred to the subjacent mesenchyme. Here we examine the molecular basis for LEF1 function and find that the epithelium of the developmentally arrested Lef1 −/− tooth rudiments fails to express Fgf4, Shh, and Bmp4, but not Wnt10a. We identify the Fgf4 gene as a direct transcriptional target for LEF1 and show that beads soaked with recombinant FGF4 protein can fully overcome the developmental arrest of Lef1 −/− tooth germs. In addition, we find that FGF4 beads induce rapidly the expression of Fgf3 in dental mesenchyme and that both epithelial and mesenchymal FGF proteins induce the delayed expression of Shh in the epithelium. Taken together, these data indicate that a single target of LEF1 can account for the function of LEF1 in tooth development and for a relay of a Wnt signal reception to a cascade of FGF signaling activities, allowing for a sequential and reciprocal communication between epithelium and mesenchyme.

Little is known about the regulation of cell fate decisions that lead to the formation of five pairs of mammary placodes in the surface ectoderm of the mouse embryo. We have previously shown that fibroblast growth factor 10 (FGF10) is required for the formation of mammary placodes 1, 2, 3 and 5. Here, we have found that Fgf10 is expressed only in the somites underlying placodes 2 and 3, in gradients across and within these somites. To test whether somitic FGF10 is required for the formation of these two placodes, we analyzed a number of mutants with different perturbations of somitic Fgf10 gradients for the presence of WNT signals and ectodermal multilayering, markers for mammary line and placode formation. The mammary line is displaced dorsally, and formation of placode 3 is impaired in Pax3 ILZ/ILZ mutants, which do not form ventral somitic buds. Mammary line formation is impaired and placode 3 is absent in Gli3Xt-J/Xt-J and hypomorphic Fgf10 mutants, in which the somitic Fgf10 gradient is shortened dorsally and less overall Fgf10 is expressed, respectively. Recombinant FGF10 rescued mammogenesis in Fgf10 -/-and Gli3Xt-J/Xt-J flanks. We correlate increasing levels of somitic FGF10 with progressive maturation of the surface ectoderm, and show that full expression of somitic Fgf10, co-regulated by GLI3, is required for the anteroposterior pattern in which the flank ectoderm acquires a mammary epithelial identity. We propose that the intra-somitic Fgf10 gradient, together with ventral elongation of the somites, determines the correct dorsoventral position of mammary epithelium along the flank.