polr1c and polr1d mutant zebrafish as new models for Treacher Collins syndrome

描述（由申请人提供）：了解控制脊椎动物头部发育的机制是一个重要的问题，因为颅面畸形占所有出生缺陷的三分之一以上。Treacher柯林斯综合征（TCS）是一种颅面疾病，影响约1/50000活产婴儿。众所周知，TCOF 1突变通过破坏称为神经嵴（NC）细胞的迁移干细胞和祖细胞群的发育而成为TCS发病机制的基础1。最近，两个新的基因POLR 1C和POLR 1D的突变也在TCS 2患者中被发现。POLR 1C和POLR 1D编码RNA聚合酶I和III的亚基，但对这些基因在NC细胞和颅面发育中的作用一无所知。由于大多数颅面异常主要归因于NC细胞的形成、增殖、迁移和/或分化缺陷，因此推测POLR 1C和POLR 1D的突变可能破坏NC细胞中的核糖体生物合成并促成TCS的发病。这项工作将研究如何在全球进程，如核糖体RNA转录和核糖体生物合成中断可以引起非常具体的先天性缺陷。这些研究结果将为研究TCS提供新的模型，并为polr 1c和polr 1d在NC和颅面发育中的作用提供新的信息。在本提案的目标1中，我们将确定polr 1c和polr 1d在胚胎发育过程中的作用，以研究TCS发病机制的潜在机制，并确定新的预防途径。我们假设Polr 1c和Polr 1d功能动态胚胎发生过程中，我们将证明通过原位杂交polr 1c和polr 1d。此外，我们假设polr 1c和polr 1d的功能丧失将干扰核糖体生物合成，导致NC细胞形成，增殖，迁移或分化的缺陷。本建议的第二个目的是，我们将研究两种预防与TCS相关的颅面畸形的方法。首先，我们将通过TUNEL和Western印迹分析证明p53依赖性细胞凋亡有助于polr 1c和polr 1d突变斑马鱼的颅面异常。我们推测，p53基因在polr 1c和polr 1d斑马鱼的抑制将防止颅面畸形的发病机制。作为预防的第二种途径，我们假设核糖体生物合成的营养刺激可以防止polr 1c和polr 1d突变斑马鱼的颅面异常。我们将确定救援的机制，并假设p53抑制将防止神经上皮细胞的损失和恢复NC的形成，迁移和分化。我们假设，饮食亮氨酸将刺激核糖体生物合成和增殖，同时减少神经上皮细胞死亡，恢复NC细胞群。总的来说，这将为polr 1c和polr 1d突变导致颅面畸形的机制提供新的信息。此外，这些目标将确定用于治疗人类TCS的新疗法，这可以减轻对广泛矫正手术的需求。

DESCRIPTION (provided by applicant): Understanding the mechanisms that control vertebrate head development is an important problem since craniofacial anomalies account for over one third of all birth defects. Treacher Collins Syndrome (TCS) is a craniofacial disorder affecting approximately 1 in 50000 live births. Mutations in TCOF1 are well known to underlie the pathogenesis of TCS through disrupting the development of a migratory stem and progenitor cell population called neural crest (NC) cells1. Recently, mutations in two new genes POLR1Cand POLR1D were also identified in patients with TCS2. POLR1C and POLR1D encode subunits of RNA polymerase I and III, but nothing is known about the role of these genes in NC cell and craniofacial development. As most craniofacial anomalies are largely attributed to defects in the formation, proliferation, migration, and/or differentiation of NC cell, it is hypothesized that mutations in POLR1C and POLR1D may disrupt ribosome biogenesis in NC cells and contribute to the pathogenesis of TCS. This work will examine how disruptions in global processes such as ribosomal RNA transcription and ribosome biogenesis can elicit very specific congenital defects. Results from these studies will provide new models for studying TCS and new information on the roles of polr1c and polr1d in NC and craniofacial development. In Aim 1 of this proposal, we will determine the role of polr1c and polr1d during embryonic development in order to investigate the mechanisms underlying the pathogenesis of TCS and identify new avenues for prevention. We hypothesize Polr1c and Polr1d function dynamically during embryogenesis which we will demonstrate via in situ hybridization for polr1c and polr1d. In addition, we hypothesize that polr1c and polr1d loss of function will perturb ribosome biogenesis leading to defects in NC cell formation, proliferation, migration, or differentiation. I the second aim of this proposal, we will investigate two avenues for prevention of the craniofacial malformations associated with TCS. First, we will demonstrate through TUNEL and Western blot analysis that p53-dependent apoptosis contributes to craniofacial anomalies in polr1c and polr1d mutant zebrafish. We hypothesize that genetic inhibition of p53 in polr1c and polr1d zebrafish will prevent the pathogenesis of craniofacial anomalies. As a second avenue for prevention, we hypothesize that nutritional stimulation of ribosome biogenesis can prevent the craniofacial anomalies in polr1c and polr1d mutant zebrafish. We will determine the mechanisms of rescue and hypothesize that p53 inhibition will prevent the loss of neuroepithelial cells and restore NC formation, migration, and differentiation. We hypothesize that dietary leucine will stimulate ribosome biogenesis and proliferation while reducing neuroepithelial cell death, restoring the NC cell population. Collectively, this will provide new information on the mechanisms by which mutations in polr1c and polr1d result in craniofacial malformations. Additionally, these aims will identify new therapies for the treatment of TCS in humans which could alleviate the need for extensive corrective surgeries.