Abstract
Dact proteins belong to the Dapper/Frodo protein family and function as cytoplasmic attenuators in Wnt and TGFβ signaling. Previous studies show that Dact1 is a potent Wnt signaling inhibitor by promoting degradation of β-catenin. We report a new mechanism for Dact2 function as an inhibitor of the canonical Wnt signaling pathway by interacting with PITX2. PITX2 is a downstream transcription factor in Wnt/β-catenin signaling, and PITX2 synergizes with Lef-1 to activate downstream genes. Immunohistochemistry verified the expression of Dact2 in the tooth epithelium, which correlated with Pitx2 epithelial expression. Dact2 loss of function and PITX2 gain of function studies reveal a feedback mechanism for controlling Dact2 expression. Pitx2 endogenously activates Dact2 expression and Dact2 feeds back to repress Pitx2 transcriptional activity. A Topflash reporter system was employed showing PITX2 activation of Wnt signaling, which is attenuated by Dact2. Transient transfections demonstrate the inhibitory effect of Dact2 on critical dental epithelial differentiation factors during tooth development. Dact2 significantly inhibits PITX2 activation of the Dlx2 and amelogenin promoters. Multiple lines of evidence conclude the inhibition is achieved by the physical interaction between Dact2 and Pitx2 proteins. The loss of function of Dact2 also reveals increased cell proliferation due to up-regulated Wnt downstream genes, cyclinD1 and cyclinD2. In summary, we have identified a novel role for Dact2 as an inhibitor of the canonical Wnt pathway in embryonic tooth development through its regulation of cell proliferation and differentiation.
Highlights
The mouse tooth is an advantageous model to study organogenesis by analyzing molecular signaling networks that regulate cell differentiation and proliferation
Pitx2 is one of the earliest transcription markers observed during tooth development, and it is restricted to the epithelium of the developing tooth
Pitx2 is regulated by the Wnt/b-catenin pathway and functions in the pathway by recruiting and independently interacting with Lef-1 and b-catenin to synergistically activate target genes, and many of these target genes are critical for tooth development [5,6]
Summary
The mouse tooth is an advantageous model to study organogenesis by analyzing molecular signaling networks that regulate cell differentiation and proliferation. The importance of signaling pathways including Wnts in the reciprocal interactions between oral epithelium and mesenchyme were proved in previous studies [1,2]. Several transcription factors including Pitx, Dlx, FoxJ1 and amelogenin (Amelx) have hierarchical expression during tooth development [3]. Together with the upstream signaling pathways, these mechanisms play critical roles in the dental crown and root formation [4]. Pitx is regulated by the Wnt/b-catenin pathway and functions in the pathway by recruiting and independently interacting with Lef-1 and b-catenin to synergistically activate target genes, and many of these target genes are critical for tooth development [5,6]
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