Transcriptional regulatory landscapes underlying FEZ Formation

摘要： 脊椎动物组织和器官的形态发生是通过精心策划的组织相互作用而发生的。期间 上颌的发育，前脑、面部外胚层和下颌之间的信号相互作用 干预神经嵴细胞调节上颌的发育。我们的研究表明，信号 来自大脑的神经嵴细胞共同作用，诱导 Sonic Hedgehog (SHH) 在 额鼻外胚层区 (FEZ)，位于额鼻表面外胚层的信号中心 指导上颌原基图案化生长的过程（FNP）。我们的初步数据还表明 前 B 细胞白血病同源盒 (PBX) 转录因子参与诱导和维持该模式 通过相互作用的遗传网络影响 FEZ 中 SHH 的表达。本申请中提出的研究是 旨在揭示由前脑和大脑信号激活的基因调控机制 神经嵴，以及研究 PBX 转录因子在调节 SHH 表达中的作用 自由经济区。具体来说，我们假设 1) 来自前脑的 SHH 信号诱导表面的“能力” 外胚层细胞表达SHH； 2）随后，到达后，神经嵴细胞诱导SHH转录 FEZ 感受态细胞，3) PBX 转录因子参与调节 SHH 的表达 自由经济区。我们将使用鸟类和小鼠胚胎在三个特定目标中测试这一假设。在每个目标中，我们将 使用鸡胚胎，因为它们使我们能够操纵大脑和大脑之间的信号相互作用 FEZ（目标 1）、神经嵴细胞和外胚层（目标 2）或 PBX1 和 PBX3 的表达（目标 3）。我们 将使用各种基因组方法（ATAC-seq、ChIP-seq、GRO-seq）来评估染色质的变化 并激活 SHH 基因座的转录调控元件 (TRE)。在目标 3 中，我们还将评估 改变 PBX 基因表达的形态、细胞和分子结果，我们将使用小鼠 胚胎以利用 Selleri 实验室已生成的数据。工作成果 本申请中提出的方法将阐明面部发育的分子方面，并将有助于 了解该地区发生的疾病过程。 SHH 或 SHH 通路中的等位基因变异 成员，与以下疾病中的人类颅面复合体畸形有关 前脑无裂畸形和唇裂，但没有腭裂。最近，PBX基因被确定为风险基因 颅面畸形患者的变异。因此，我们的创新方法将研究与 通过染色质分析奠定发育生物学的坚实基础将产生直接的信息 适用于理解人类畸形。

Summary: Morphogenesis of vertebrate tissues and organs occurs through well-orchestrated tissue interactions. During development of the upper jaw, signaling interactions among the forebrain, the facial ectoderm, and the intervening neural crest cells regulate development of the upper jaw. Our research has revealed that signals from the brain and the neural crest cells act together to induce expression of Sonic hedgehog (SHH) in the Frontonasal Ectodermal Zone (FEZ), a signaling center located in the surface ectoderm of the Frontonasal Process (FNP) that directs patterned growth of the upper jaw anlagen. Our preliminary data also suggest that Pre-B-cell leukemia homeobox (PBX) transcription factors are involved in inducing and maintaining the pattern of SHH expression in the FEZ by an interacting genetic network. The research proposed in this application is designed to uncover the gene regulatory mechanisms that are activated by signals from the forebrain and the neural crest, as well as, to investigate the role of PBX transcription factors in regulating SHH expression in the FEZ. Specifically, we hypothesize that 1) SHH signaling from the forebrain induces “competence” in surface ectoderm cells to express SHH; 2) subsequently, upon arrival, neural crest cells induce transcription of SHH in competent cells of the FEZ, and 3) PBX transcription factors participate in regulating expression of SHH in the FEZ. We will test this hypothesis in three specific aims using avian and murine embryos. In each Aim, we will use chick embryos because they allow us to manipulate the signaling interactions between the brain and the FEZ (Aim 1), the neural crest cells and the ectoderm (Aim 2), or expression of PBX1 and PBX3 (Aim 3). We will use a variety of genomic approaches (ATAC-seq, ChIP-seq, GRO-seq) to evaluate changes in chromatin and activated transcriptional regulatory elements (TREs) at the SHH locus. In Aim 3, we will also assess morphological, cellular, and molecular outcomes of altering PBX gene expression, and we will use mouse embryos to take advantage of data already generated in the Selleri laboratory. The results of the work proposed in this application will shed light on the molecular aspects of facial development, and will aid understanding of disease processes that occur in this region. Allelic variants in SHH, or SHH pathway members, are associated with human dysmorphologies of the craniofacial complex in diseases such as Holoprosencephaly and cleft lip with our without cleft palate. Recently, PBX genes have been identified as risk variants in patients with craniofacial dysmorphology. Hence, our innovative approach combining studies with a solid foundation in developmental biology with chromatin analyses will yield information that is directly applicable to understanding human dysmorphology.