Rescue of arrested Lef1^−/− tooth development by recombinant FGF proteins. (A,B) In vitro culture of an E13.5 Lef1^−/− mandible that contains a pair of incisor rudiments and has been associated with a bead soaked in SHH (left side, blue Affigel bead) or FGF8 (right side, brown heparin acrylic bead), respectively. A shows a live explant after 3 d of in vitro culture, and B is a graphic presentation of the structures. Note that only the right incisor anlage next to the FGF8 bead has formed a cap (and a fully developed tooth on subsequent transplantation under the kidney capsule). inc, incisor buds; lfe, lip furrow epithelium; M.c., Meckel's cartilage. (C,D) Full development of E14.5 Lef1^−/− tooth germs that have been incubated with FGF8-soaked beads (C) or apical ectodermal ridge (AER) of an E10.5 mouse hindlimb (D) in vitro for 3 d and transferred under the kidney capsule for another 9 d. Typical multicusp molars with ameloblast (am) and odontoblast (od) layers, dentin (de, blue), enamel (en, red), and dental pulp (dp) are formed. The limb ectoderm including the AER has persisted in forming a cyst with keratinized layers inside.

The Fgf4 gene is a direct target for LEF1 and Wnt signaling. (A) Schematic representation of the Fgf4 gene. Sequences in the 3′UTR that are conserved between mouse and man are boxed (gray) and the relative location and sequence of a binding site for LEF1 (stippled) is shown. (B) Electrophoretic mobility shift assay showing binding of 25 ng purified recombinant LEF1 to ³²P-labeled double-stranded oligonucleotides encompassing the wt or mt sequence from the Fgf4 3′UTR. The specificity of binding is confirmed by a competition with increasing amounts of unlabeled wt or mt oligonucleotides. (C) Schematic representation of the Fgf4LacZ reporter gene. The bacterial LacZ gene is shown in dark gray. Sequences in the 3′UTR are represented as in panel A. (D) Functional contribution of the LEF1 binding site to the activity of Fgf4 transcriptional control sequences. Reporter gene constructs carrying the wt or mt LEF1 binding sites (stippled box) in the 3′UTR were generated by linking Fgf4 promoter sequences and the Fgf4 3′UTR (open boxes) to bacterial LacZ coding sequences (hatched box). The reporter constructs were stably transfected into F9 cells and lacZ activity was determined in pools of clones. The Wnt-responsiveness of the Fgf4LacZ reporter constructs was examined by treatment of the transfected cell pools with LiCl (10 or 30 mM) as a surrogate Wnt stimulus.

Effects of mesenchymal FGF proteins on gene expression in Lef1^−/− molar explants, detected by whole mount in situ hybridization. (A) FGF7 beads fail to activate early expression of Fgf3 in the mesenchyme, but a small expression domain is seen in the epithelium after 2 d. After 4 d, Fgf3 expression is detected in the forming papilla but not around the bead. In contrast, Shh is activated in the epithelium with a similar time course as by FGF4 beads (cf. Fig. 5A). (B) FGF7 cannot induce Shh expression in dental epithelia that have been placed on nondental (dermal) mesenchyme. (C) Model of the signaling cascade governing the transition from the bud (E13) to the cap (E14) stage of tooth development. Intraepithelial Wnt10a and/or Wnt10b signals mediate, via LEF1 and β-catenin, the transcriptional activation of the Fgf4 gene in the epithelium. FGF4 signals to the subjacent mesenchyme. One of the early targets of this epithelial signaling is the Fgf3 gene in mesenchymal cells, and FGF3 together with other signals of dental mesenchyme (presumably including BMP4) in turn induce expression of Shh in the epithelium.

(A) Expression of genes encoding signaling molecules in wt and Lef1^−/− mandibular molars at E14.5. At this developmental stage, the “enamel knot,” a putative signaling center at the base of the epithelial downgrowth, and the mesenchymal papilla have been formed. Consecutive sections of the same rudiments were used for in situ hybridization with various probes. In the enamel knot of wt tooth germs, expression of Lef1, Wnt10a, Bmp4, Shh, Fgf4, and Fgf3 is detected. The same group of cells also contains Wnt10b, Fgf9, Bmp2, and Bmp7 transcripts (data not shown). With the exception of Lef1, Wnt10a, and Wnt10b (data not shown), epithelial expression of all other genes examined is abrogated in LEF1-deficient molars. In the mesenchyme of both wt and mutant tooth germs, Lef1 and Bmp4 are expressed, whereas the abundant expression of Fgf3 and Fgf10 (data not shown) in the forming papilla is detected in wt but not in mutant molars. (B) Dependence of tooth development on LEF1:β-catenin interaction. Morphology of molar tooth germs from wt and various Lef1 mutant embryos at E16. A normal cap structure of dental epithelium (de) surrounding the dental papilla (dp) is seen in sections of wt and heterozygous Lef1^+/− embryos (top). An arrested tooth bud structure is observed in Lef1^−/− and Lef1⁻/m5 embryos. Lef1⁻/m5 embryos carry one null allele and one allele with point mutations in the region of Lef1 encoding the β-catenin interaction domain of LEF1 (bottom).

Epithelial-to-mesenchyme FGF signaling. (A) Inhibition of tooth development by the FGF signaling antagonist SU5402. Frequency of tooth development (dev.) in epithelium–mesenchyme double combinations from wt (+) and Lef1^−/− (−) tooth germs. (Top panel) Transient (48 h) exposure of Lef1-deficient mesenchyme (white) to wt dental epithelium (dark gray) allows tooth development after its subsequent recombination with mutant epithelium (light gray). (Middle panel) The presence of 25 μM SU5402 during the transient presence of wt epithelium prevents subsequent tooth development under otherwise identical conditions. (Bottom panel) Control experiment indicating that 48 h of exposure to SU5402 per se does not impair subsequent tooth development. The arrest in tooth development is represented by a bud structure of the epithelium. (B) FGF4 acts on the mesenchyme. Lef1^−/− tooth germs were incubated with FGF4 beads for 24 h and after a further 48 h incubation without beads, the mutant mesenchyme (top panel) or the mutant epithelium (bottom panel) were exchanged with corresponding fresh mutant tissues (light gray). High frequency (17/18) of tooth development is observed with mutant mesenchyme, but not with mutant epithelium that had been exposed to FGF4.

Effects of FGF4 beads on the expression of selected marker genes in explanted molar rudiments of E13.5 Lef1^−/− embryos. Gene expression was monitored by whole-mount in situ hybridization after 1–4 d of incubation. (A) Application of FGF4-soaked beads or BSA-soaked control beads to the mutant tooth germs. After 1 d, strong expression of Fgf3 in the mesenchyme and weak expression of Shh in the epithelium was detected. After 4 d, Shh and Fgf9 are expressed in the epithelium, Fgf3 and Bmp4 in the mesenchyme. Note two expression domains of Fgf3: one close to the bead, the other in the mesenchymal papilla surrounded by the epithelial cap. Very rarely, weak Fgf4 expression was seen, as in one of the two explants shown here. None of these genes is activated by control beads soaked in BSA (mesenchymal expression of Bmp4 is not affected in the mutant; see Fig. 1A). (B) Time course of gene activation by FGF4 in explanted molar rudiments or in isolated molar mesenchyme of E14.5 Lef1^−/− embryos. Fgf3 transcription is activated within 3 h, even in the absence of dental epithelium. Shh expression is undetectable after 16 h (weak signals are seen after 24 h; see A), and strong signals are observed in the epithelium after 48 h. As expected, Shh transcription is not activated in the mesenchyme.