Function and regulation of Lhx genes in craniofacial development

描述(申请人提供)：涉及第一鳃弓衍生的颅面畸形，包括颌骨、腭部和牙齿，是人类出生缺陷的主要类别。了解颅面发育背后的分子遗传学机制对于设计诊断、预防和治疗人类缺陷的创新方法至关重要。我研究的长期目标是通过识别在这一过程中重要的转录因子和顺式调控元件，并确定它们的等级关系，来表征控制第一鳃弓发育的转录网络。以往的基因表达分析表明，编码同源结构域转录因子的Lhx6和Lhxd基因可能是第一鳃弓发育的主要调控基因，但支持这一观点的功能证据很少。我对同时缺乏Lhxd和Lhx8活性的小鼠突变体的初步分析表明，它们对腭部和牙齿的正常发育是必不可少的。因此，我的短期目标是利用小鼠突变模型以及生化和分子生物学方法，进一步明确Lhx6和LhxS在第一鳃弓中的功能和调控。这项建议的具体目的是1)在解剖、细胞和分子水平上表征Lhx6-/-8-/-小鼠突变体的表型。根据我的初步数据，我将重点研究二次腭部和牙齿的发育。2)：使用结合转录图谱和染色质免疫沉淀的全基因组、无偏见的方法来识别Lhxd和LhxS的下游靶点。我将使用基因芯片分析比较野生型和Lhx6-/-；8-/-第一鳃弓中的基因表达。此外，我将进行层析免疫沉淀，然后使用Lhx6抗体进行DMA芯片分析(ChlP-CHIP)，以确定Lhx6的直接靶基因和Lhx6作用的顺式调控元件。3)寻找控制Lhxd和Lhxd在第一鳃弓表达的增强子元件。我将根据序列的进化保守性来确定候选增强子。我将在鸡神经脊电穿孔系统和转基因小鼠中验证它们的活性。然后，我将使用生物信息学工具来确定这些增强剂的候选调控蛋白，以便进一步分析。*。我的研究结果将为Lhxd和LhxS基因功能和表达中断如何导致人类头面部缺陷提供关键信息。此外，这些结果将显著提高我们对颅面和牙齿发育过程中基因表达调控的全面了解，从而有助于识别和理解人类颅面出生缺陷背后的遗传缺陷。

DESCRIPTION (provided by applicant): Craniofacial abnormalities involving the first branchial arch derivatives, including the jaw, palate and teeth, are a major class of birth defects in humans. Understanding the molecular genetic mechanisms behind craniofacial development is vital to devising innovative methods for the diagnosis, prevention and treatment of human defects. The long-term goal of my research is to characterize the transcriptional network that governs development of the first branchial arch, by identifying transcription factors and cis-regulatory elements important in this process, and determining their hierarchical relationship. Previous gene expression analysis suggested that the Lhx6 and Lhxd genes, which encode homeodomain transcription factors, may be major regulators of first branchial arch development, but the functional evidence to support this idea was scarce. My preliminary analysis of mouse mutants lacking both Lhxd and Lhx8 activity indicates that they are essential for the normal development of the palate and teeth. Therefore, my short-term goal is to further define the function and regulation of Lhx6 and LhxS in the first branchial arch using the mouse mutant model, as well as biochemical and molecular biology methods. The specific aims of this proposal are 1) To characterize the phenotypes of Lhx6-/-8-/- mouse mutants at the anatomical, cellular and molecular level. Based on my preliminary data, I will focus on the secondary palate and tooth development. 2): To identify downstream targets of Lhxd and LhxS using genome-wide, unbiased approaches which combine transcriptional profiling and chromatin-immunoprecipitation. I will compare gene expression in the wild type and Lhx6-/-;8-/- first branchial arch using microarray analysis. In addition, I will perform chromatinimmunoprecipitation followed by DMA chip analysis (ChlP-chip) using an Lhx6 antibody to identify direct target genes of Lhx6 and the cis-regulatory elements that Lhx6 acts upon. 3) To identify enhancer elements that control Lhxd and Lhxd expression in the first branchial arch. I will identify candidate enhancers based on the evolutionary conservation of sequences. I will verify their activity in chick neural crest electroporation system and transgenic mice. I will then use bioinformatic tools to identify candidate regulatory proteins for these enhancers for further analysis. *. The results of my research will provide critical information on how disruption in Lhxd and LhxS gene function and expression can contribute to human craniofacial defects. In addition, the results will significantly enhance our overall knowledge on gene expression regulation during craniofacial and dental development, and thus facilitate identification and understanding of genetic defects behind human craniofacial birth defects.