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

解读中面部发育和发育不良中神经嵴特异性 TFAP2 通路

• 批准号: 10676016
• 负责人: Timothy Nguyen
• 金额: $ 4.34万
• 依托单位: UNIVERSITY OF IOWA
• 依托单位国家: 美国
• 财政年份: 2023
• 资助国家: 美国
• 起止时间: 2023-07-01 至 2025-06-30
• 项目状态: 未结题
• 来源: https://reporter.nih.gov/project-details/10676016
• 关键词: ATAC-seq; Address; Aftercare; Animal Model; Apoptosis; Behavior; Binding; Binding Sites; Bioinformatics; Biological Assay; Birth; Cartilage; Cell Count; Cell Death; Cell Survival; Cell physiology; Cephalic; Chromatin; Code; Confocal Microscopy; Coupling; Craniofacial Abnormalities; DNA; Data; Defect; Deformity; Development; Disease; Dysplasia; Elements; Embryo; Embryology; Embryonic Development; Enhancers; Environment; Etiology; Face; Financial Hardship; Frontonasal dysplasia; Future; Gene Deletion; Gene Expression; Gene Expression Profiling; Genes; Genome; Goals; Health; Human; Immunofluorescence Immunologic; Impairment; Institution; Jaw; Knock-out; Knowledge; Lead; Link; Mentorship; Methods; Modeling; Molecular; Morphogenesis; Mus; Mutation; Neural Crest; Neural Crest Cell; Newborn Infant; Operative Surgical Procedures; Oral health; Orbital separation excessive; Pathogenesis; Pathology; Pathway interactions; Pattern; Phenocopy; Phenotype; Positioning Attribute; Prevention; Principal Investigator; Proliferating; Public Health; Reporter; Research; Research Personnel; Research Proposals; Resources; Shapes; Shorthand; Signal Transduction; Skeleton; Specificity; Stains; Structural defect; TFAP2A gene; TFAP2B gene; Techniques; Testing; Tissues; Tooth structure; Training; United States; Untranslated RNA; Zebrafish; bone; career; cell motility; cost; craniofacial; craniofacial complex; craniofacial disorder; effective therapy; embryonic stem cell; epigenome; face bone structure; gene function; gene regulatory network; genome-wide; improved; interest; malformation; mouse genetics; mutant; neuroregulation; next generation sequencing; novel; novel strategies; orofacial cleft; prevent; research study; single-cell RNA sequencing; skeletal; skills; stem cell population; transcription factor; treatment strategy

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.

项目摘要：头面部畸形是出生时出现的第二大结构畸形， 最常见的形态发生在面部中部和上方区域。然而，我们有限的理解 在控制其发展的分子机制中阻碍了有效治疗和 预防。几项研究已经确定了胚胎时期面部下部形成的途径。 发育(例如，颌骨和牙齿)。然而，人们对面部中部和独特的 人们对协调该地区发展的机制知之甚少。这项研究的长期目标是 是剖析支配面部中部发育的分子基础，以及它们的失调如何导致 发育不良。我们和其他人独立地发现了关键的面中部基因，这些基因在神经脊内发挥作用， 形成面部骨骼和软骨的胚胎干细胞群体。例如，删除ALX1/3/4 (ALX)的丢失会导致额鼻发育不良的综合征形式，会导致小鼠面中部严重裂隙。我们的 实验室发现，Tfap2在神经脊中的缺失会影响Alx基因的表达，并呈现相同的情况 面中部裂开为Alx基因敲除的胚胎。因此，我们在这项研究提案中的总体目标是利用 这一新的额鼻发育不良动物模型揭示了TFAP2转录因子如何在 中面-独特的基因调控网络(GRN‘s)。为了达到这一目标，我们检验了总体假设 TFAP2转录因子直接调节Alx基因的表达，以维持适当的神经脊存活， 面中部融合和骨骼形成。在目标1中，我们将使用基因组和表观基因组范围的分析 (Cut&Run，ATAC-seq)破译TFAP2如何针对Alx并塑造基因组图景以调节其 表达级别。我们将利用斑马鱼报告策略来测试TFAP2结合的Alx非编码增强子 对于面部中部的特异性。在AIM 2中，我们将把复杂的小鼠遗传学与免疫荧光和 骨骼分析，以表征Tfap2和Alx如何在遗传上相互作用，以控制神经脊的活性，行为， 和骨架元素的形成。完成这些目标将填补我们的关键知识缺口 了解这些基因和途径是如何联系在一起塑造发育中的面中部的。这样的知识 将有助于制定治疗面中部疾病甚至预防面中部疾病的策略。这个 这项研究的首席研究员阮先生在小鼠遗传学、胚胎学和基因方面接受了广泛的培训 在这项提案中重点介绍了表情分析技术。在Van Otterloo博士的共同指导下 然后，Nguyen先生将在全基因组分子分析、下一代测序方面发展技能 方法、生物信息学技术以及免疫荧光和共聚焦显微镜方法；统称为， 大大推进了他的博士培训，扩大了他的研究工具包。训练计划，成熟的团队， 而这项研究提案中概述的制度环境不仅将为改善 他不仅从事头面部治疗和健康方面的研究，而且还促进了他成为一名独立研究人员的事业。