Regulation of Nodal Signaling in Holoprosencephaly

描述（由申请人提供）：无前脑畸形是一种常见的出生缺陷（活产1:16 000，死产1:250），具有广泛的颅面畸形，从令人痛苦的独眼到单个中央门牙的轻微症状。它是由腹侧前脑（前轴中线的一部分）的结构缺陷引起的，这随后导致大脑不完全分离为左右半球。最近的研究表明，节点信号在控制中线发育中起着核心作用，因此，我们将重点关注节点信号在小鼠胚胎发生中的调节，并对轴向中线形成特别感兴趣。Nodal是转化生长因子β (tgf - β)超家族的成员，该家族利用激活素I型和II型受体、Smad2和4以及FoxH1（FAST）定义的信号通路。重要的是，EGF-CFC细胞外蛋白家族的成员，如小鼠Cripto，是Nodal的重要辅助因子。我们之前报道了一个Cripto无效等位基因，最近我们通过遗传操作产生了一个Cripto亚胚等位基因Cripto3-loxP。大约50%的Cripto3-loxP/CriptoNull小鼠表现出广泛的轴向中线缺陷，类似于前脑畸形。相反，TGIF是一个同源盒基因，编码一种核蛋白，通过阻断Smad2功能来对抗tgf - β信号。有趣的是，人类TGIF基因突变与前脑畸形相关，提示其在轴向中线形成中起作用，可能通过调节Nodal/Smad2信号通路。基于这些结果，我们将在拟开展的研究中实现以下具体目标：1)通过从形态学和分子水平上详细分析Cripto3-loxP/CriptoNull小鼠的缺陷，对作为腹侧前脑缺陷和HPE模型系统的Cripto3-loxP/CriptoNull小鼠进行分析；II)通过鉴定Cripto起作用的组织和细胞以及Cripto下游靶基因，探讨Cripto在小鼠轴向中线形成中的作用机制；III)通过生成无TGIF胚胎和检测TGIF对节点信号的调节，研究TGIF在小鼠轴向中线发育中的功能。这些研究将提高我们对哺乳动物中线形成和人类前脑无裂畸形的认识。

DESCRIPTION (provided by applicant): Holoprosencephaly represents a common birth defect (1:16,000 in live births and 1:250 in stillbirths) with a broad spectrum of craniofacial malformations ranging from distressful cyclopia to mild symptom of a single central incisor. It is caused by defects in the specification of the ventral forebrain (a part of the anterior axial midline), which subsequently lead to incomplete separation of the brain into the left and right hemispheres. Recent studies indicated that Nodal signaling plays a central role in controlling midline development, we therefore will focus on the regulation of Nodal signaling in mouse embryogenesis with a special interest in anterior axial midline formation. Nodal is a member of the transforming growth factor beta (TGF-beta) superfamily that utilizes a signaling pathway defined by Activin type I and II receptors, Smad2 and 4, and FoxH1(FAST). Importantly, members of the EGF-CFC family of extracellular proteins such as mouse Cripto are essential co-factors for Nodal. We previously reported a Cripto null allele, and recently we generated a Cripto hypomorphic allele, Cripto3-loxP, by genetic manipulation. Approximately 50% of the Cripto3-loxP/CriptoNull mice displayed a wide range of axial midline defects resembling holoprosencephaly. In contrast, TGIF is a homeobox gene encoding a nuclear protein that antagonizes TGF-beta signaling by blocking Smad2 function. Interestingly, mutations in human TGIF gene are associated with holoprosencephaly, suggesting its function in axial midline formation, presumably through regulating Nodal/Smad2 signaling pathway. Based on these results, we will pursue the following specific aims in the proposed research: I) Analysis of the Cripto3-loxP/CriptoNull mice as a model system for ventral forebrain defects and HPE by detailed analysis of the defects in Cripto3-loxP/CriptoNull mice at morphology and molecular levels; II) Investigation of mechanisms underlying Cripto function in mouse axial midline formation by identifying the tissues and cells where Cripto is functioning and downstream target genes of Cripto; III) Investigation of TGIF function in mouse axial midline development by generating TGIF null embryos and examining the modulation of Nodal signaling by TGIF. These studies should improve our understanding of mammalian axial midline formation and human holoprosencephaly.