MSX1 Drives Tooth Morphogenesis Through Controlling Wnt Signaling Activity.

Tooth agenesis is a common structural birth defect in humans that results from failure of morphogenesis during early tooth development. The homeobox transcription factor Msx1 and the canonical Wnt signaling pathway are essential for "bud to cap" morphogenesis and are causal factors for tooth agenesis. Our recent study suggested that Msx1 regulates Wnt signaling during early tooth development by suppressing the expression of Dkk2 and Sfrp2 in the tooth bud mesenchyme, and it demonstrated partial rescue of Msx1-deficient molar teeth by a combination of DKK inhibition and genetic inactivation of SFRPs. In this study, we found that Sostdc1/Wise, another secreted Wnt antagonist, is involved in regulating the odontogenic pathway downstream of Msx1. Whereas Sostdc1 expression in the developing tooth germ was not increased in Msx1-/- embryos, genetic inactivation of Sostdc1 rescued maxillary molar, but not mandibular molar, morphogenesis in Msx1-/- mice with full penetrance. Since the Msx1-/-;Sostdc1-/- embryos exhibited ectopic Dkk2 expression in the developing dental mesenchyme, similar to Msx1-/- embryos, we generated and analyzed tooth development in Msx1-/-;Dkk2-/- double and Msx1-/-;Dkk2-/-;Sostdc1-/- triple mutant mice. The Msx1-/-;Dkk2-/- double mutants showed rescued maxillary molar morphogenesis at high penetrance, with a small percentage also exhibiting mandibular molars that transitioned to the cap stage. Furthermore, tooth development was rescued in the maxillary and mandibular molars, with full penetrance, in the Msx1-/-;Dkk2-/-;Sostdc1-/- mice. Together, these data reveal 1) that a key role of Msx1 in driving tooth development through the bud-to-cap transition is to control the expression of Dkk2 and 2) that modulation of Wnt signaling activity by Dkk2 and Sostdc1 plays a crucial role in the Msx1-dependent odontogenic pathway during early tooth morphogenesis.

An Explanation for How FGFs Predict Species-Specific Tooth Cusp Patterns.

Species-specific cusp patterns result from the iterative formation of enamel knots, the epithelial signaling centers, at the future cusp positions. The expressions of fibroblast growth factors (FGFs), especially Fgf4, in the secondary enamel knots in the areas of the future cusp tips are generally used to manifest the appearance of species-specific tooth shapes. However, the mechanism underlying the predictive role of FGFs in species-specific cusp patterns remains obscure. Here, we demonstrated that gerbils, which have a lophodont pattern, exhibit a striped expression pattern of Fgf4, whereas mice, which have a bunodont pattern, have a spotted expression pattern, and these observations verify the predictive role of Fgf4 in species-specific cusp patterns. By manipulating FGFs' signaling in the inner dental epithelium of gerbils, we provide evidence for the intracellular participation of FGF signaling, specifically FGF4 and FGF20, in Rac1- and RhoA-regulated cellular geometry remolding during the determination of different cusp patterns. Our study presents a novel explanation of how different FGF expression patterns produce different cusp patterns and implies that a conserved intracellular FGF-GTPase signaling module might represent an underlying developmental basis for evolutionary changes in cusp patterns.

Activin and Bmp4 Signaling Converge on Wnt Activation during Odontogenesis.

Previous studies show that both activin and Bmp4 act as crucial mesenchymal odontogenic signals during early tooth development. Remarkably, mice lacking activin-ßA ( Inhba-/-) and mice with neural crest-specific inactivation of Bmp4 ( Bmp4ncko/ncko) both exhibit bud-stage developmental arrest of the mandibular molar tooth germs while their maxillary molar tooth germs completed morphogenesis. In this study, we found that, whereas expression of Inhba and Bmp4 in the developing tooth mesenchyme is independent of each other, Bmp4ncko/nckoInhba-/- compound mutant mice exhibit early developmental arrest of all tooth germs. Moreover, genetic inactivation of Osr2, a negative regulator of the odontogenic function of the Bmp4-Msx1 signaling pathway, rescues mandibular molar morphogenesis in Inhba-/- embryos. We recently reported that Osr2 and the Bmp4-Msx1 pathway control the bud-to-cap transition of tooth morphogenesis through antagonistic regulation of expression of secreted Wnt antagonists, including Dkk2 and Sfrp2, in the developing tooth mesenchyme. We show here that expression of Dkk2 messenger RNAs was significantly upregulated and expanded into the tooth bud mesenchyme in Inhba-/- embryos in comparison with wild-type littermates. Furthermore, in utero treatment with either lithium chloride, an agonist of canonical Wnt signaling, or the DKK inhibitor IIIC3a rescued mandibular molar tooth morphogenesis in Inhba-/- embryos. Together with our finding that the developing mandibular molar tooth bud mesenchyme expresses significantly higher levels of Dkk2 than the developing maxillary molar tooth mesenchyme, these data indicate that Bmp4 and activin signaling pathways converge on activation of the Wnt signaling pathway to promote tooth morphogenesis through the bud-to-cap transition and that the differential effects of loss of activin or Bmp4 signaling on maxillary and mandibular molar tooth morphogenesis are mainly due to the differential expression of Wnt antagonists, particularly Dkk2, in the maxillary and mandibular tooth mesenchyme.

Deletion of Osr2 Partially Rescues Tooth Development in Runx2 Mutant Mice.

Tooth organogenesis depends on genetically programmed sequential and reciprocal inductive interactions between the dental epithelium and neural crest-derived mesenchyme. Previous studies showed that the Msx1 and Runx2 transcription factors are required for activation of odontogenic signals, including Bmp4 and Fgf3, in the early tooth mesenchyme to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 acts downstream of Msx1 to activate Fgf3 expression. Recent studies identified Osr2 as a repressor of tooth development and showed that inactivation of Osr2 rescued molar tooth morphogenesis in the Msx1(-/-) mutant mice as well as in mice with neural crest-specific inactivation of Bmp4. Here we show that Runx2 expression is expanded in the tooth bud mesenchyme in Osr2(-/-) mutant mouse embryos and is partially restored in the tooth mesenchyme in Msx1(-/-)Osr2(-/-) mutants in comparison with Msx1(-/-) and wild-type embryos. Whereas mandibular molar development arrested at the bud stage and maxillary molar development arrested at the bud-to-cap transition in Runx2(-/-) mutant mice, both mandibular and maxillary molar tooth germs progressed to the early bell stage, with rescued expression of Msx1 and Bmp4 in the dental papilla as well as expression of Bmp4, p21, and Shh in the primary enamel knot in the Osr2(-/-)Runx2(-/-) compound mutants. In contrast to the Msx1(-/-)Osr2(-/-) compound mutants, which exhibit nearly normal first molar morphogenesis, the Osr2(-/-)Runx2(-/-) compound mutant embryos failed to activate the expression of Fgf3 and Fgf10 in the dental papilla and exhibited significant deficit in cell proliferation in both the dental epithelium and mesenchyme in comparison with the control embryos. These data indicate that Runx2 synergizes with Msx1 to drive tooth morphogenesis through the bud-to-cap transition and that Runx2 controls continued tooth growth and morphogenesis beyond the cap stage through activation of Fgf3 and Fgf10 expression in the dental papilla.