Regulation of spinal cord development and the Wnt signaling pathway by Oto

描述（申请人提供）：Wnt信号是控制脊椎动物中枢神经系统生长和模式的关键途径之一。Wnt信号通路成分的突变经常导致出生缺陷和影响神经系统的肿瘤。因此，了解Wnt在神经系统发育过程中的调控机制对我们理解和治疗疾病至关重要。本研究的重点是阐明Oto（一种新的Wnt蛋白调节剂）在脊髓发育和Wnt信号通路中的作用。耳头畸形突变体具有多种表型，似乎是由于Wnt信号的过量或早期发作，包括全前额畸形、agnathia和脊髓过度生长。Oto位点编码wnt的糖基磷脂酰肌醇（GPI）修饰所需的酶。这为Wnt调控机制提供了潜在的新见解。wnt是一种分泌的远距离信号分子，在某些生理条件下可能是gpi锚定在脂质膜上的。gpi连接蛋白靶向脂筏，并在分泌途径中从一般的非gpi连接蛋白中分离出来。GPI修饰所赋予的复杂生物学特性为GPI相关wnt的活性提供了多个调控点。我推测Oto的功能是调节wnt的细胞分选、分泌和/或组织迁移，从而调节wnt的可用性和/或信号传导效率。为了解决这个假设，我将在多个层面上检查Oto功能。我将研究已知受Wnt控制的脊髓发育的良好描述特征，包括祖细胞增殖和背-腹侧模式，并将潜在的发现与Wnt信号转导的变化联系起来。我还将研究Oto在培养细胞系统中通过分泌途径使Wnt蛋白成熟中的作用。进一步，我将直接分析Oto在小鸡Wnt1蛋白在小鸡脊髓分布建立中的作用。脊髓实验将采用小鼠遗传学和鸡胚电穿孔两种方法。建议的研究应该扩大我们对脊髓发育和Wnt信号调控的理解。许多人类疾病和畸形都与Wnt通路中的发育缺陷有关，包括前脑畸形、脑肿瘤和神经管缺陷。这些综合征中的许多都与影响Wnt通路的突变有遗传基础，但增强Wnt通路的药物的致畸作用也是人类神经管缺陷的重要原因。这一建议将使人们对引起这些重要综合征的分子和发育事件有新的认识。

DESCRIPTION (provided by applicant): Wnt signaling is one of the key pathways controlling growth and patterning of the vertebrate central nervous system. Mutations in components of the Wnt signaling pathway often lead to birth defects and tumors affecting the nervous system. Thus, understanding the mechanisms of Wnt regulation in the context of nervous system development is vital for our understanding and treatment of disease. The focus of this study is to elucidate the role of Oto, a novel regulator of Wnt proteins, in spinal cord development and the Wnt signaling pathway. The otocephaly mutant has a variety of phenotypes that appear to be due to either excess or early onset of Wnt signaling, including holoprosenchephaly, agnathia and spinal cord overgrowth. The Oto locus encodes an enzyme required for proper glycosylphosphatidylinositol (GPI)-modification of Wnts. This provides potential new insights into the mechanisms of Wnt regulation. Wnts, which are secreted and considered to be long-range signaling molecules, may be GPI-anchored to lipid membranes in some physiological conditions. GPI-linked proteins are targeted to lipid rafts and segregate from the general population of nonGPI-linked proteins in the secretory pathway. The complex biology conferred by GPI modification provides multiple points of regulation for the activity of GPI-linked Wnts. I hypothesize that Oto functions to regulate cellular sorting, secretion and/or tissue mobility of Wnts, thus regulating the availability and/or signaling efficiency of Wnts. To address this hypothesis, I will examine Oto function at multiple levels. I will examine well-described features of spinal cord development known to be under Wnt control, including progenitor proliferation and dorsal-ventral patterning, and correlate potential findings to changes in Wnt signal transduction. I will also examine the function of Oto in the maturation of Wnt proteins through the secretory pathway in cultured cell systems. Furthermore, I will directly analyze the function of Oto in the establishment of chick Wnt1 protein distribution in the chick spinal cord. For the spinal cord experiments, both mouse genetics and in ovo chick embryo electroporation will be utilized. The proposed studies should expand our understanding of spinal cord development and Wnt signaling regulation. A number of human diseases and malformations are associated with developmental defects in the Wnt pathway, including holoprosencephaly, brain tumors and neural tube defects. Many of these syndromes have a genetic basis in mutations affecting the Wnt pathway, but teratogenic effects of drugs augmenting the Wnt pathway are also an important cause of neural tube defects in humans. This proposal will allow new understanding of the molecular and developmental events causing these important syndromes.