It has been known for more than a decade that Msx1 and Pax9 are required for Bmp4 expression in tooth bud mesenchyme, but the molecular basis for this inter-relationship is largely unidentified. Potentiation of Pax9 is currently the only known molecular mechanism of Bmp4-activation by Msx1 in tooth bud mesenchyme, since Msx1 by itself cannot activate the proximal Bmp4 promoter, and other potential mediators are presently unknown. Homeodomain transcription factors usually acquire their selective functional properties through interactions with a variety of other co-factors, including paired domain transcription factors; thus, it could have been possible that Pax9 was one of these co-factors.
Here we analyzed naturally arising, human hypodontia-causing missense mutations in MSX1 to examine their interaction with Pax9 and Bmp4 on a molecular level. Most of the mutant Msx1 proteins could still potentiate Pax9-induced Bmp4 and Msx1 promoter activation, suggesting that Msx1’s ability to cause human tooth agenesis may be independent of any synergism with Pax9. The severity of the families’ phenotypes cannot be reasonably explained by the relatively mild variations observed in our functional tests, suggesting again that a different molecular mechanism must be responsible for Msx1-caused tooth agenesis. Even the A194V mutation which showed incomplete penetrance and behaved like wild-type protein in all our tests cannot yet be excluded as a disease-causing mutation.
In conclusion, we infer from the functional evaluation of the 5 MSX1
missense mutations that disturbances in the interaction with Pax9 are probably not the basis of the human phenotypes caused by MSX1
mutations, unless another molecular mechanism for the observed dependence of Bmp4 expression on combined Msx1 and Pax9 activity is discovered. A recent study by Chandler et al. (2009)
described the existence of an enhancer element far upstream of the Bmp4
promoter that drives Bmp4 expression in tooth bud mesenchyme. This enhancer could be located and tested for responsiveness to wild-type Msx1 and the Msx1 mutants, with and without Pax9 as co-activator.
Alternatively, an altogether different pathway could be involved. Early on, it had been shown that Msx1-deficient mice displayed reduced expression of not only Bmp4 but also of Lef1, syndecan-1, Runx2, and Fgf3 (Chen et al., 1996
; Bei and Maas, 1998
). In the dental mesenchyme, Fgf3 is another well-established signaling molecule besides Bmp4 that is known to promote the tooth morphogenetic process. Fgf3 appears to be expressed downstream of Wnt/Lef1 and of Runx2 (D’Souza et al., 1999
). The Msx1 pathway resulting in Bmp4 expression seems to operate in parallel with the pathway that leads to Msx1-dependent Fgf3 expression in tooth bud mesenchyme. In each of these two pathways, Msx1 is likely to cooperate with different partner factors targeting different promoters. The fact that one of the tooth agenesis MSX1
mutations (M61K) is located in a Gro/Tle binding site points to a disturbance in transcriptional repression mechanisms.
Surprisingly, Msx1 may even be completely dispensable for odontogenesis: Activation of the Wnt pathway through Apc depletion (Wang et al., 2009a
), but not β-catenin stabilization (Liu et al., 2008
), could bypass Msx1-dependent signaling pathways for the formation of supernumerary tooth-like structures with enamel knots, normal mineralization, and signs of root growth. However, these teeth were rather small and dysmorphic, suggesting that the mesenchymal Msx1-dependent pathways play a significant role in determining proper size and shape of the normal endogenous tooth organ, possibly by delaying premature differentiation.
Translating experimental evidence from mice to humans is still a challenge, especially when the organ system in question shows several remarkable differences: The mouse dentition is monophyodont and consists of only molars and continually growing incisors, while tooth agenesis resulting from Pax9 and Msx1 mutations affects mostly the secondary dentition in humans. Also, a study with human embryonic tissue revealed a few differences in gene expression patterns—for example, PAX9 is not exclusively expressed in tooth bud mesenchyme but is also found in the dental epithelium (Lin et al., 2007
). Ultimately, we will have to find valid in vitro
assays that may help to interpret mouse data on a human background.