Function and regulation of Lhx genes in craniofacial development

描述（由申请人提供）：涉及第一鳃弓衍生物（包括颌骨、腭和牙齿）的颅面畸形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。我的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，并确定它们的层次关系，来表征控制第一鳃弓发育的转录网络。先前的基因表达分析表明，Lhx 6和Lhxd基因，编码同源结构域转录因子，可能是第一鳃弓发育的主要调节因子，但支持这一观点的功能证据很少。我对缺乏Lhxd和Lhx 8活性的小鼠突变体的初步分析表明，它们对腭和牙齿的正常发育至关重要。因此，我的短期目标是使用小鼠突变模型以及生物化学和分子生物学方法进一步确定Lhx 6和LhxS在第一鳃弓中的功能和调节。该提案的具体目的是1）在解剖学、细胞和分子水平上表征Lhx 6-/-8-/-小鼠突变体的表型。根据我的初步数据，我将重点放在第二腭和牙齿的发展。2）、使用全基因组无偏倚的方法，结合联合收割机转录谱分析和染色质免疫沉淀，鉴定Lhxd和LhxS的下游靶点。我将使用微阵列分析比较野生型和Lhx 6-/-;8-/-第一鳃弓中的基因表达。此外，我将进行染色质免疫沉淀，然后使用Lhx 6抗体进行DMA芯片分析（ChIP芯片），以确定Lhx 6的直接靶基因和Lhx 6作用的顺式调节元件。3)为了鉴定控制Lhxd和Lhxd在第一鳃弓中表达的增强子元件，我将根据序列的进化保守性来鉴定候选增强子。本研究将在鸡神经嵴电穿孔系统和转基因小鼠中验证其活性。然后，我将使用生物信息学工具来识别这些增强子的候选调节蛋白，以进行进一步分析。*.我的研究结果将提供Lhxd和LhxS基因功能和表达的破坏如何导致人类颅面缺陷的关键信息。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整体认识，从而促进对人类颅面出生缺陷背后遗传缺陷的识别和理解。

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.