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良好的组织相互作用发生的。在.期间 上颌骨的发育，前脑、面部外胚层和面部外胚层之间的信号相互作用 中间的神经脊细胞调节上颌的发育。我们的研究表明，信号 大脑和神经脊细胞共同作用诱导Sonic hedgehog(SHH)在脑内的表达 额鼻外胚层(FEZ)，位于额鼻表面外胚层的信号中枢 引导上颌骨生长的过程(FNP)。我们的初步数据还表明， 前B细胞白血病同源框(PBX)转录因子参与诱导和维持这种模式 通过相互作用的遗传网络在FEZ中表达SHH。这项申请中提出的研究是 旨在揭示由来自前脑和大脑的信号激活的基因调节机制 以及研究PBX转录因子在调节SHH表达中的作用。 非斯。具体地说，我们假设：1)来自前脑的SHH信号诱导表层的“能力” 外胚层细胞表达SHH；2)随后，神经脊细胞在到达时诱导SHH在 3)PBX转录因子参与调节SHH的表达。 非斯。我们将使用鸟类和小鼠的胚胎在三个特定的目标上检验这一假设。在每个目标中，我们都会 使用小鸡胚胎，因为它们允许我们操纵大脑和 FEZ(目标1)、神经脊细胞和外胚层(目标2)或Pbx1和PBX3的表达(目标3)。我们 将使用多种基因组方法(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，我们将用小鼠 利用Selleri实验室已经产生的数据。工作的成果 在本申请中提出的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途径中的等位基因变异 成员与人类头面部复合体的畸形在疾病中有关，如 无前颅脑畸形和唇裂，但无腭裂。最近，pbx基因被确认为风险基因。 颅面畸形患者的变异。因此，我们的创新方法结合了研究和 扎实的发育生物学基础和染色质分析将产生直接的信息 适用于理解人类畸形学。

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.