Deciphering neural crest-specific TFAP2 pathways in midface development and dysplasia

项目总结：颅面畸形是出生时出现的第二大结构畸形， 最常见的形式出现在中面部和上面部区域。然而，我们有限的理解 控制其发展的分子机制阻碍了有效治疗的策略， 预防几项研究已经确定了胚胎时期下面部形成的潜在途径 开发（例如，颌和牙齿）。然而，较少的重点放在中面，和独特的 人们对该区域发展的协调机制知之甚少。这项研究的长期目标是 是剖析控制面中部发育的分子基础以及它们的失调如何导致 发育不良我们和其他人已经独立地发现了在神经嵴中起作用的关键中面部基因， 一种胚胎干细胞群，可以产生面部骨骼和软骨。例如，删除Alx 1/3/4 (Alx)，其缺失导致额鼻发育不良综合征形式，导致小鼠严重的面中部裂。我们 实验室发现，神经嵴中Tfap 2的缺失会损害Alx基因的表达，并表现出相同的 面中部裂的胚胎。因此，我们在这项研究提案中的总体目标是利用 这种新的动物模型用于额鼻发育不良，以揭示TFAP 2转录因子如何在细胞内运作。 独特的基因调控网络（GRN）。为了达到这一目标，我们测试了整体假设 TFAP 2转录因子直接调节Alx基因表达以获得适当的神经嵴存活， 面中部融合和骨骼形成。在AIM 1中，我们将采用全基因组和表观基因组分析， (CUT&RUN，ATAC-seq）来破译TFAP 2如何靶向Alx并塑造基因组景观以调节其表达。 表达水平。我们将利用斑马鱼报告策略来测试TFAP 2结合的Alx非编码增强子 中面部特异性。在AIM 2中，我们将把复杂的小鼠遗传学与免疫荧光结合起来， 骨骼分析，以表征Tfap 2和Alx如何在遗传上相互作用以控制神经嵴活力、行为 和骨骼元素的形成。这些目标的完成将填补我们在知识上的一个重要空白。 了解这些基因和途径是如何连接到塑造发展中的中面。这种知识 将有助于制定治疗面中部疾病甚至预防这些疾病的策略。的 本研究的首席研究员Nguyen先生在小鼠遗传学、胚胎学和基因方面接受过广泛的培训。 表达谱分析技术强调了这一建议。在货车Otterloo博士的共同指导下 和Amendt，Nguyen先生将发展全基因组分子测定，下一代测序 方法，生物信息学技术，以及免疫荧光和共聚焦显微镜方法;总的来说， 极大地推进了他的博士培训，扩大了他的研究工具包。培训计划，建立团队， 本研究建议中概述的制度环境不仅为改善 他不仅致力于颅面治疗和健康，而且还促进了他成为独立研究人员的职业生涯。

PROJECT SUMMARY: Craniofacial anomalies are the 2nd leading structural malformation to appear at birth, with the most common forms occurring in the mid- and upper-face regions. However, our limited understanding in the molecular mechanisms controlling their development has hindered strategies for effective treatment and prevention. Several studies have identified the pathways underlying lower face formation during embryonic development (e.g., jaw and teeth). Yet, less emphasis has been placed on the midface, and the unique mechanisms orchestrating the development of this region are poorly understood. The long-term goal of this study is to dissect the molecular underpinnings governing midface development and how their dysregulation causes dysplasia. We and others have independently uncovered key midface genes that function within the neural crest, an embryonic stem cell population that gives rise to facial bone and cartilage. For example, deletion of Alx1/3/4 (Alx), whose loss causes syndromic forms of Frontonasal Dysplasia, lead to a severe midfacial cleft in mice. Our lab has found that loss of Tfap2 in the neural crest compromises expression of Alx genes and presents the same midface cleft as the Alx knockout embryos. Thus, our overall objective in this research proposal is to leverage this new animal model for Frontonasal Dysplasia to uncover how TFAP2 transcription factors operate within the midface-unique gene regulatory networks (GRN’s). To approach this objective, we test the overall hypothesis that TFAP2 transcription factors directly regulate Alx gene expression for appropriate neural crest survival, midface fusion, and skeletal formation. In AIM 1, we will employ genome- and epigenome-wide assays (CUT&RUN, ATAC-seq) to decipher how TFAP2 targets Alx and shapes the genome landscape to regulate their expression levels. We will leverage zebrafish reporter strategies to test TFAP2-bound Alx noncoding enhancers for midface specificity. In AIM 2, we will couple sophisticated mouse genetics with immunofluorescence and skeletal analyses to characterize how Tfap2 and Alx genetically interact to control neural crest viability, behavior, and formation of the skeletal elements. Completion of these aims will fill a critical knowledge gap in our understanding for how these genes and pathways are linked to shape the developing midface. Such knowledge will contribute to the development of strategies in treating midfacial disorders or even preventing them. The principal investigator of this study, Mr. Nguyen, has extensive training in mouse genetics, embryology, and gene expression profiling techniques highlighted in this proposal. With combined mentorship from Drs. Van Otterloo and Amendt, Mr. Nguyen will develop skills in genome-wide molecular assays, next-generation sequencing approaches, bioinformatic techniques, and immunofluorescent and confocal microscopy methods; collectively, greatly advancing his doctoral training and expanding his research toolkit. The training plan, established team, and institutional environment outlined in this research proposal will not only pave the way to improving craniofacial treatment and health, but also catalyze his career towards becoming an independent researcher.