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

描述(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 Collins综合征(TCS)是一种头面部疾病，大约每50000名活产儿中就有一名受到影响。众所周知，TCOF1的突变通过干扰称为神经脊(NC)细胞的迁移性干细胞和祖细胞群体的发育而成为TCS发病的基础。最近，在TCS2患者中也发现了两个新基因POLR1C和POLR1D的突变。POLR1C和POLR1D编码RNA聚合酶I和III的亚基，但这些基因在NC细胞和颅面发育中的作用尚不清楚。由于大多数颅面畸形主要归因于NC细胞的形成、增殖、迁移和/或分化方面的缺陷，推测POLR1C和POLR1D突变可能扰乱NC细胞核糖体的生物合成，并参与TCS的发病。这项工作将研究核糖体RNA转录和核糖体生物发生等全球过程的中断如何导致非常特定的先天性缺陷。这些研究结果将为研究TCS提供新的模型，并为Polr1c和polr1d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polr1c和polr1d在胚胎发育过程中的作用，以研究TCS的发病机制并寻找新的预防途径。我们假设Polr1c和Polr1d在胚胎发育过程中动态发挥作用，我们将通过polr1c和polr1d的原位杂交来证明这一点。此外，我们假设polr1c和polr1d功能的丧失将扰乱核糖体的生物发生，导致NC细胞形成、增殖、迁移或分化方面的缺陷。I这项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，在polr1c和polr1d突变斑马鱼中，p53依赖的细胞凋亡与头面部异常有关。我们假设，在Polr1c和Polr1d斑马鱼中，P53的遗传抑制将防止颅面畸形的发生。作为预防的第二条途径，我们假设核糖体生物发生的营养刺激可以预防polr1c和polr1d突变斑马鱼的头面部畸形。我们将确定挽救机制和假设，即抑制P53将防止神经上皮细胞的丢失，并恢复NC的形成、迁移和分化。我们假设饮食中的亮氨酸将刺激核糖体的生物生成和增殖，同时减少神经上皮细胞的死亡，恢复NC细胞群。总而言之，这将为polr1c和polr1d突变导致颅面畸形的机制提供新的信息。此外，这些目标将确定治疗人类TCS的新疗法，这些疗法可能会减少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.