Molecular patterning of the hard palate during palatogenesis

摘要/摘要 哺乳动物的上颚在解剖学上分为前部骨性硬腭和后部肌肉。 口感柔和。然而，前硬腭是如何形成图案的，以及腭部成骨是如何控制的 仍然不为人所知。最近的研究表明，在发育中的鳃弓中，童话超类 同源结构域蛋白，特别是Meis蛋白，建立了所有拱门和 它们的衍生物，而HOX转录因子作为组织特异性的辅助因子来指定ARCH的身份。 然而，这提出了一个基本问题，因为哪些因素与故事因素相互作用，以具体说明和 设计无HOX的第一弓及其衍生产品，包括上颚。同源盒基因Shox2是 在前腭间充质中特异表达，与未来的骨性硬腭重叠 域。我们以前已经证明，Shox2基因突变不仅会导致一种罕见的前裂 但也显著减少了硬腭骨的形成，这与 -/- 在Shox2肢体中花柱足类的虚拟丧失，表明Shox2在器官构型和 骨骼发育。我们的初步研究提供了Shox2突变导致早产/异位的证据 Runx2在表达Shox2的腭部细胞中的表达E13腭架Shox2细胞的RNA序列分析 和肢体显示在缺乏Shox2的情况下成骨基因在全基因组范围内的表达增加， 与Shox2在脑神经脊系细胞中过表达抑制作用的观察一致 成骨作用。此外，发育中的腭部和四肢上的Shox2芯片序列揭示了全基因组 Shox2对Hox和Hox结合的基因顺式调控元件的优先占位 TALL蛋白，表明在无HOX的上颌发育中，Shox2与TALE一起发挥作用 影响硬腭形态和调节成骨的因素。基于上述观察，我们 假设在无HOX的腭架中，Shox2与故事因子相互作用来建立硬腭 通过拮抗MEIS的转录输出来防止过早成骨。在此应用程序中， 提出了三个具体目标来严格检验这一新假说：1)检验Shox2的假说 表达可防止发育中的腭部过早成骨；2)检验Shox2的假说 拮抗MEI的转录输出并建立Shox2的作用机制；3) 确定组织特定的染色质景观并确定特定的腭部增强剂成分 队形。所获得的结果将为理解腭部发育和 为未来腭骨组织工程的临床应用提供了坚实的基础 唇腭裂的治疗/修复。

Abstract/Summary 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.