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

polr1c 和 polr1d 突变斑马鱼作为 Treacher Collins 综合征的新模型

基本信息

批准号：
8596630
负责人：
Kristin Emily Noack Watt
金额：
$ 3.03万
依托单位：
STOWERS INSTITUTE FOR MEDICAL RESEARCH
依托单位国家：
美国
项目类别：
财政年份：
2013
资助国家：
美国
起止时间：
2013-07-01 至 2016-06-30
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8596630
关键词：
Accounting Address Affect Alcian Blue Animal Model Antibodies Apoptosis Biogenesis Budgets Cartilage Cell Death Cell Differentiation process Cell Proliferation Cleft Lip Cleft Palate Congenital Abnormality Connective Tissue Cranial Nerves Data Defect Deformity Development Diagnosis Diet Dietary Supplementation Disease Ear Embryo Embryonic Development Etiology Face Genes Genetic Genetic Transcription Head Healthcare Histone H3 Human Immigration In Situ Hybridization In Situ Nick-End Labeling Individual Infant Mortality Intervention Lead Leucine Live Birth Malignant Neoplasms Mandibulofacial Dysostosis Methods Modeling Mutation Neural Crest Neural Crest Cell Neuroepithelial Neuroepithelial Cells Nutritional Operative Surgical Procedures Pathogenesis Pathway interactions Patients Pattern Phenotype Play Polyribosomes Population Prevention Process RNA Polymerase I Reverse Transcriptase Polymerase Chain Reaction Ribosomal RNA Ribosomes Role Staining method Stains Stem cells Stress Study models Transcript Translations Western Blotting Work Zebrafish bone craniofacial face bone structure human FRAP1 protein insight loss of function malformation migration mutant novel novel strategies novel therapeutics prevent progenitor public health relevance stem transcriptome sequencing tumorigenesis

项目摘要

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.

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 Collins综合征（TCS）是一种颅面疾病，大约每5万例活产婴儿中就有1例受到影响。众所周知，TCOF1的突变通过破坏被称为神经嵴（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，在TCS2患者中也发现了两个新基因polr1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。由于大多数颅面异常在很大程度上归因于NC细胞的形成、增殖、迁移和/或分化缺陷，因此假设POLR1C和POLR1D的突变可能破坏NC细胞中的核糖体生物发生，并参与TCS的发病机制。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引发非常具体的先天性缺陷。这些研究结果将为TCS的研究提供新的模型，并为polr1c和polr1d在NC和颅面发育中的作用提供新的信息。在本提案的目的1中，我们将确定polr1c和polr1d在胚胎发育中的作用，以探讨TCS发病机制的潜在机制并确定新的预防途径。我们假设Polr1c和Polr1d在胚胎发生过程中动态起作用，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功能的丧失会扰乱核糖体的生物发生，导致NC细胞形成、增殖、迁移或分化的缺陷。本提案的第二个目的，我们将探讨预防与TCS相关的颅面畸形的两种途径。首先，我们将通过TUNEL和Western blot分析证明p53依赖性凋亡导致polr1c和polr1d突变斑马鱼颅面异常。我们假设斑马鱼polr1c和polr1d基因中p53的基因抑制将阻止颅面异常的发病机制。作为预防的第二种途径，我们假设营养刺激核糖体生物生成可以预防polr1c和polr1d突变斑马鱼的颅面异常。我们将确定挽救的机制，并假设p53抑制将防止神经上皮细胞的损失，恢复NC的形成、迁移和分化。我们假设膳食亮氨酸会刺激核糖体的生物发生和增殖，同时减少神经上皮细胞的死亡，恢复NC细胞群。总的来说，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，从而减轻大面积矫正手术的需要。