Fibulin-1 Regulation of the DiGeorge Syndrome Pathogenesis Pathway

描述（由申请人提供）：小鼠靶向基因失活研究的新发现表明，细胞外基质蛋白纤维蛋白-1 （Fbln1）是心脏流出道（OFT）、咽腺、颅神经和颅骨正常发育所必需的。Fbln1缺失小鼠所表现出的发育异常再现了22q11缺失/DiGeorge综合征（DGS）的许多异常，其发生率为1:4000。Fbln1缺陷小鼠和DGS人类之间表型的相似性表明，Fbln1虽然位于22q11缺失区之外，但却是DGS中功能失调通路的修饰因子。DGS的分子途径尚未完全阐明。关键成分是转录因子Tbx1、Fgf8和Fgf8信号通路的中间体。一些证据支持Fbln1调控这一途径的假设。DNA微阵列分析表明，Fbln1是叉头盒转录因子表达的必需条件，叉头盒转录因子可调控包括二次心野（SHF）在内的咽弓区Tbx1和Fgf8的表达。qPCR分析显示，在Fbln1缺失胚的咽含弓组织中Tbx1的表达显著降低。我们还发现Fbln1与Fgf8相互作用并抑制Fgf8介导的Map激酶信号通路的激活。这些观察结果，再加上Fbln1在NCC迁移途径中表达的发现，它可以作为细胞引导和运动的调节剂，它可以相互作用并调节NCC迁移调节剂蛋白多糖的蛋白质水解，导致以下假设：Fbln1是Fgf8影响nc依赖的咽弓发育和衍生结构（包括OFT、咽腺和颅面结构）形成机制的关键组成部分。为了验证这一假设并获得机制理解，有三个具体目标：1)确定Fbln1对Fgf8信号通路的影响；2)确定Fbln1与Tbx1、Fbln1与versican之间的遗传互作；3)明确Fbln1在控制NCC增殖和诱导中的作用。这项实验的发现有望为Fbln1引导心脏OFT、大血管、头盖和咽腺正常发育的机制提供新的见解，并有助于了解涉及这些组织的先天性缺陷的病因。公共卫生相关性：本研究旨在获得与迪乔治综合征（DGS）相关的发育异常（包括心脏缺陷）的机制理解，其发生频率为4000分之一。拟议的研究旨在确定细胞外基质蛋白纤维蛋白-1在调节细胞（神经嵴细胞）的行为中的作用，这些行为有助于DGS中受影响组织的形成。研究的一个新方向源于我们的发现，即纤维蛋白1调节Fgf8的活性，Fgf8是一种生长因子，是DGS中功能失调通路的关键组成部分。

DESCRIPTION (provided by applicant): New findings from targeted gene inactivation studies in mice reveal that the extracellular matrix protein fibulin-1 (Fbln1) is required for normal development of the cardiac outflow tract (OFT), pharyngeal glands, cranial nerves and bones of the skull. The developmental anomalies displayed by mice deficient in Fbln1 recapitulate many of the anomalies associated with 22q11 deletion/DiGeorge syndrome (DGS), which occurs with a frequency of 1:4000 human births. The similarity in phenotypes between Fbln1-deficient mice and DGS humans suggests that Fbln1, although located outside of the 22q11 deletion region, is a modifier of the pathway that is dysfunctional in DGS. The molecular pathway of DGS has not been fully elucidated. Key components are the transcription factor Tbx1, Fgf8 and intermediates of the Fgf8 signaling pathway. Several lines of evidence support the hypothesis that Fbln1 regulates this pathway. DNA microarray analysis indicates that Fbln1 is required for expression of forkhead box transcription factors that regulate expression of both Tbx1 and Fgf8 in pharyngeal arch regions including the secondary heart field (SHF). qPCR analysis shows that Tbx1 expression is significantly decreased in pharyngeal arch-containing tissues from Fbln1 null embryos. We also find that Fbln1 interacts with Fgf8 and inhibits Fgf8-mediated activation of the Map kinase-signaling pathway. These observations together with findings that Fbln1 is expressed along pathways of NCC migration, that it can act as a regulator of cell guidance and motility and that it interacts and modulates proteolysis of the proteoglycan versican, a regulator of NCC migration, lead to the following hypothesis: Fbln1 is a key component of the mechanism by which Fgf8 influences NCC-dependent development of the pharyngeal arch and the formation of derivative structures including the OFT, pharyngeal glands and craniofacial structures. To test this hypothesis and gain mechanistic understanding there are three specific aims: 1) Define the effects of Fbln1 on the Fgf8 signaling pathway; 2) Identify genetic interactions between Fbln1 and Tbx1 and between Fbln1 and versican; and 3) Define the role of Fbln1 in controlling NCC proliferation and guidance. Findings from this experimentation are expected to lead to new insights into the mechanism by which Fbln1 guides the normal development of the cardiac OFT, great vessels, cranium and pharyngeal glands and to contribute to an understanding of the etiology of congenital defects involving these tissues. Public Health Relevance: This research seeks to gain a mechanistic understanding of the developmental anomalies (including heart defects) associated with DiGeorge syndrome (DGS), which occurs with a frequency of one in 4000 human births. The proposed studies are designed to determine the role of the extracellular matrix protein, fibulin-1, in regulating the behaviors of cells (neural crest cells) that contribute to the formation of tissues affected in DGS. A novel direction for the research stems from our discovery that fibulin-1 regulates the activity of Fgf8, a growth factor implicated as a key component of the pathway that is dysfunctional in DGS.