A Network of Transcription Factors Operates during Early Tooth Morphogenesis

Abstract

dImproving the knowledge of disease-causing genes is a unique challenge in human health. Although it is known that genes causing similar diseases tend to lie close to one another in a network of protein-protein or functional interactions, the identification of these protein-protein networks is difficult to unravel. Here, we show that Msx1, Snail, Lhx6, Lhx8, Sp3, and Lef1 interact in vitro and in vivo, revealing the existence of a novel context-specific protein network. These proteins are all expressed in the neural crest-derived dental mesenchyme and cause tooth agenesis disorder when mutated in mouse and/or human. We also identified an in vivo direct target for Msx1 function, the cyclin D-dependent kinase (CDK) inhibitor p19 ink4d , whose transcription is differentially modulated by the protein network. Considering the important role of p19 ink4d as a cell cycle regulator, these results provide evidence for the first time of the unique plasticity of the Msx1-dependent network of proteins in conferring differential transcriptional output and in controlling the cell cycle through the regulation of a cyclin D-dependent kinase inhibitor. Collectively, these data reveal a novel protein network operating in the neural crest-derived dental mesenchyme that is relevant for many other areas of developmental and evolutionary biology.

Identification of a Face Enhancer Reveals Direct Regulation of LIM Homeobox 8 (Lhx8) by Wingless-Int (WNT)/β-Catenin Signaling

“…Because of the modest transfection efficiency of PMAC, we turned to C3H10T1/2, an immortalized cell line of undifferentiated mesenchyme cells. C3H10T1/2 endogenously expresses many transcription factors that are found in craniofacial mesenchyme, including LHX8 (81). Transfection with ␤-catenin siRNA efficiently inhibited both WNT/␤-catenin signaling and Lhx8 expression in C3H10T1/2 (Fig.…”

Homeobox protein MSX‐1 inhibits expression of bone morphogenetic protein 2, bone morphogenetic protein 4, and lymphoid enhancer‐binding factor 1 via Wnt/β‐catenin signaling to prevent differentiation of dental mesenchymal cells during the late bell stage

“…Lymphoid enhancer‐binding factor 1 is a critical gene downstream of Msx1 . An interaction between LEF‐1 and MSX‐1 proteins has recently been demonstrated . Therefore, we also investigated the regulation of Lef1 expression by MSX‐1.…”

“…Accordingly, the late bell stage is ideal for examining whether MSX‐1 prevents differentiation of tooth‐germ mesenchyme by inhibiting expression of Bmp2 and Bmp4 . Furthermore, in view of the previous findings that MSX‐1 interacts with LEF‐1 and that LEF‐1 promotes odontoblast differentiation during tooth‐germ development , with its expression also increasing at late bell stage , we investigated whether LEF‐1 participates in MSX‐1‐mediated suppression of cytodifferentiation. The LEF‐1 protein is a critical transcription factor in the Wnt/ β ‐catenin signaling pathway, and Wnt/ β ‐catenin signaling is essential for tooth‐germ differentiation through promoting expression of Bmp4 and Lef1 at the late bell stage .…”

Msx1andTbx2antagonistically regulateBmp4expression during the bud-to-cap stage transition in tooth development

“…Since Msx1 functions primarily as a repressor (Catron et al, 1995;Zhang et al, 1996), it is generally believed that Msx1 activates Bmp4 indirectly. However, recent studies indicate that certain Msx1 protein-protein interactions can result in synergistic activation of target promoters directly (Ogawa et al, 2006;Zhao et al, 2013). Furthermore, O'Connell et al (O'Connell et al, 2012) have shown that epithelial Bmp4 can diffuse to the mesenchyme and signal back to the epithelium.…”

“…As a control, incubation with GST protein or beads alone did not retain any 35 S-Tbx2. The co-immunoprecipitation analysis utilized C3H10T1/2 cells, a mammalian mesenchymal cell line that is appropriate for Msx1 protein-protein interaction studies (Miletich et al, 2011;Zhao et al, 2013). C3H10T1/2 cells behave as early odontogenic precursor cells and endogenously express most of the early odontogenic proteins including Msx1 and Tbx2 (Fig.…”