Pax9 and Msx1 encode transcription factors that are known to be essential for the switch in odontogenic potential from the epithelium to the mesenchyme. Multiple lines of evidence suggest that these molecules play an important role in the maintenance of mesenchymal Bmp4 expression, which ultimately drives morphogenesis of the dental organ. Here we demonstrate that Pax9 is able to directly regulate Msx1 expression and interact with Msx1 at the protein level to enhance its ability to transactivate Msx1 and Bmp4 expression during tooth development. In addition, we tested how a missense mutation (T62C) in the paired domain of PAX9 that is responsible for human tooth agenesis (1) affects its functions. Our data indicate that although the mutant Pax9 protein (L21P) can bind to the Msx1 protein, it fails to transactivate the Msx1 and Bmp4 promoter, presumably because of its inability to bind cognate paired domain recognition sequences. In addition, synergistic transcriptional activation of the Bmp4 promoter was lost with coexpression of mutant Pax9 and wild-type Msx1. This suggests that Pax9 is critical for the regulation of Bmp4 expression through its paired domain rather than Msx1. Our findings demonstrate the partnership of Pax9 and Msx1 in a signaling pathway that involves Bmp4. Furthermore, the regulation of Bmp4 expression by the interaction of Pax9 with Msx1 at the level of transcription and through formation of a protein complex determines the fate of the transition from bud to cap stage during tooth development.

BackgroundDental caries is a polymicrobial disease and prevalent among cleft lip and palate (CLP) patients, although their oral hygiene is well maintained. Dysbiosis, the state of imbalance within the dental plaque microbiota, may cause caries prevalence among these patients. However, little is known about how dysbiosis occurs and affects cariogenicity. To find dysbiotic signs, here we conducted a metatranscriptomic analysis for the plaque microbiota in six CLP patients and four controls.MethodsTotal bacterial RNA was extracted from each sample and sequenced. Bacterial composition and functional profiles were estimated from 16S rRNA and mRNA reads, respectively. The mRNA reads were further used for estimating bacterial composition. Species listed in both rRNA-based and mRNA-based bacterial composition were identified as viable taxa with in situ function (VTiF), and the VTiF with a high mRNA-to-rRNA ratio were considered to be transcriptionally active. A network was constructed for each group by connecting two VTiF if their mRNA abundances were positively correlated.ResultsThe bacterial composition and functional profiles themselves did not provide remarkable signs of dysbiosis in the CLP group. However, the group-specific active taxa were identified, including streptococcal and Prevotella species in the CLP group. Moreover, the network structure was different between groups; Actinomyces johnsonii and several species in the CLP group were the active taxa, which were connected based on positive correlations with statistical significance.ConclusionsFunctional dysbiosis within the plaque microbiota was observed such as difference of the network structure between groups, and may be associated with cariogenicity. The observed functional dysbiosis was an invisible change within the microbiota in the oral cavity of CLP patients. This may emphasize the importance of maintaining good oral hygiene of the patients with cleft anomalies.Electronic supplementary materialThe online version of this article (10.1186/s40510-019-0265-1) contains supplementary material, which is available to authorized users.

Pax9 is a paired domain-containing transcription factor that plays an essential role in the patterning of murine dentition. In humans, mutations in PAX9 are associated with unique phenotypes of familial tooth agenesis that mainly involve posterior teeth. Among these, a frameshift mutation (219InsG) within the paired domain of PAX9 produces a protein product associated with a severe form of molar agenesis in a single family. The objectives of this study were to gain new insights into the molecular pathogenesis of the 219InsG mutation and its role in tooth agenesis. Here we describe functional defects in DNA binding and transactivation of mutant 219InsGPax9. Although wild type Pax9 binds to the high affinity paired domain recognition sequences, e5 and CD19 -2(A-ins), the 219InsGPax9 mutant protein was unable to bind to these cognate DNA-binding sites. In co-transfection assays, wild type Pax9 acti- PAX9 belongs to the PAX family of transcription factors that play pivotal roles in embryonic patterning and in disease (1). Related to the paired gene in Drosophila melanogaster, proteins of this family are characterized by a highly conserved 128-amino acid motif that encodes the paired domain. The paired domain is structurally composed of two distinct helixturn-helix motifs that mediate sequence-specific interaction with DNA, primarily with target genes containing the core GTTC motif (2). In addition to the paired domain, the 341-amino acid polypeptide chain encoding the PAX9 gene possesses, at least, another functionally distinct domain that functions putatively as a transactivation domain. Although detailed characterization of the transactivation domain of PAX9 is currently unavailable, sequence homology with potent transactivator proteins localizes the putative transactivation function to the proline-, serine-, and threonine-rich C-terminal domain of PAX9. In humans, the identification of several novel PAX9 mutations in individuals with posterior teeth agenesis confirms that PAX9 protein is critical for the patterning of dentition (3).In mouse embryos, Pax9 is an early marker of tooth development, appearing in odontogenic mesenchyme before ectodermal thickening and prior to the expression of other tooth signaling genes. High levels of Pax9 expression persist in the mesenchyme during the bud and cap stages and are down-regulated at the late bell stage of developing dentition (4). Functional deletion of Pax9 in mice results in neonatal lethality and an arrest of tooth development at the bud stage (5). The expression of Pax9 also modulates the expression of crucial developmental regulatory genes, such as Msx1 and Bmp4, and antagonistic interactions between Fgf8 and Bmp2 or Bmp4 control the initiation of Pax9 expression in mandibular mesenchyme (6, 7). Pax9-dependent odontogenic activities constitute part of a complex process that is not yet fully understood at the molecular level. How and which of the Pax9-regulated processes are altered by mutations in the gene is an issue of current biological importance. Clearly, fu...

Mutations in the paired-domain transcription factor PAX9 are associated with non-syndromic tooth agenesis that preferentially affects posterior dentition. Of the 18 mutations identified to date, eight are phenotypically well-characterized missense mutations within the DNA-binding paired domain. We determined the structural and functional consequences of these paired domain missense mutations and correlated our findings with the associated dental phenotype variations. In vitro testing included subcellular localization, protein-protein interactions between MSX1 and mutant PAX9 proteins, binding of PAX9 mutants to a DNA consensus site and transcriptional activation from the Pax9 effector promoters Bmp4 and Msx1 with and without MSX1 as co-activator. All mutant PAX9 proteins were localized in the nucleus of transfected cells and physically interacted with MSX1 protein. Three of the mutants retained the ability to bind the consensus paired domain recognition sequence; the others were unable or only partly able to interact with this DNA fragment and also showed a similarly impaired capability for activation of transcription from the Msx1 and Bmp4 promoters. For seven of the eight mutants, the degree of loss of DNA-binding and promoter activation correlated quite well with the severity of the tooth agenesis pattern seen in vivo. One of the mutants however showed neither reduction in DNA-binding nor decrease in transactivation; instead, a loss of responsiveness to synergism with MSX1 in target promoter activation and a dominant negative effect when expressed together with wild-type PAX9 could be observed. Our structure-based studies, which modeled DNA binding and subdomain stability, were able to predict functional consequences quite reliably.

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