The mammalian palate is anatomically divided into the anterior bony hard palate and the posterior muscular soft palate. However, how the anterior hard palate is patterned and how the palatal osteogenesis is controlled remain unknown. It was recently demonstrated that in the developing branchial arches, the TALE superclass homeodomain proteins particularly Meis proteins set up a ground state that is common to all the arches and their derivatives whereas Hox transcription factors act as tissue-specific cofactor to specify the arch identity. However, this raises a fundamental question as what factors interact with TALE factors to specify and pattern the Hox-free first arch and its derivatives including the palate. The homeobox gene Shox2 is expressed specifically in the anterior palatal mesenchyme, overlapping with the future bony hard palate domain. We have shown previously that Shox2 mutation leads to not only a rare type anterior clefting of the secondary palate, but also the significantly reduced bone formation in the hard palate, which, together with -/- the virtual loss of the stylopod in Shox2 limb, indicates an essential role for Shox2 in organ patterning and skeletogenesis. Our preliminary studies present evidence that Shox2 mutation leads to premature/ectopic + expression of Runx2 in Shox2-expressing palatal cells. RNA-Seq on Shox2 cells from E13 palatal shelves and limbs demonstrates a genome-wide elevated expression of osteogenic genes in the absence of Shox2, consistent with the observation that Shox2 overexpression in cranial neural crest lineage cells inhibits osteogenesis. Moreover, Shox2 ChIP-Seq on the developing palate and limb reveals genome-wide preferential occupation of Shox2 on the responsive cis-regulatory elements of genes bound by Hox and TALE proteins, suggesting that in the Hox-free developing palate, Shox2 functions together with TALE factors to pattern the hard palate and regulates osteogenesis. Based on the abovementioned observations, we hypothesize that in the Hox-free palatal shelf, Shox2 interacts with TALE factors to establish the hard palate identity by antagonizing Meis transcriptional output to prevent premature osteogenesis. In this application, three specific aims are proposed to test this novel hypothesis rigorously: 1) to test the hypothesis that Shox2 expression prevents premature osteogenesis in the developing palate; 2) to test the hypothesis that Shox2 antagonizes the transcription output of Meis and to establish the functional mechanisms for Shox2; 3) to determine the tissue specific chromatin landscape and identify specific enhancer elements underlying palate formation. The results obtained will provide novel knowledge for understanding of palate development and cleft palate formation, and provide solid foundation for future tissue engineering of palatal bone for clinical treatment/repair of cleft palate defects.

Public Health Relevance

Cleft palate represents a common structural birth defect affecting around 1 out 700 children in US and worldwide. Gene mutations and/or environmental perturbations during pregnancy are known to cause cleft palate, but the underlying molecular mechanisms are far from understanding. This research project uses unique genetically modified mouse strains combined with state-of-the-art molecular biology, genomics, and genome editing approaches to investigate molecular mechanisms that pattern the hard palate and control bone formation during palatogenesis. Information gained from the proposed studies will advance our understanding of cleft palate and palatal bone formation, which will provide novel insight into future development of effective treatment and repair of cleft palate as well as regeneration of palatal bones.

National Institute of Health (NIH)
National Institute of Dental & Craniofacial Research (NIDCR)
Research Project (R01)
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Special Emphasis Panel (ZRG1)
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Stein, Kathryn K
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Tulane University
Anatomy/Cell Biology
Schools of Arts and Sciences
New Orleans
United States
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Yuan, Guohua; Zhan, Yunyan; Gou, Xiaohui et al. (2018) TGF-? signaling inhibits canonical BMP signaling pathway during palate development. Cell Tissue Res 371:283-291