Mechanisms of shape variation in a mouse model of craniofacial birth defects

项目总结 虽然颅面畸形的遗传基础正在被发现，但我们不明白为什么 表型变异会发生，尤其是由相同的突变引起的。一个例子是 全脑畸形(HPE)是一种常见疾病，每250个妊娠中就有1个受到影响，但只有10,000个活产中有1个受到影响 由于宫内致命性。HPE的独特之处在于其畸形范围从轻微的面部低眼压到眼球旋转 在人类身上。Sonic Hedgehog(SHH)和该途径的其他成员的突变会导致某些人的HPE 病人。然而，Shh突变的小鼠模型不能重现观察到的 病人。这项建议的目的是确定导致面部形状变化的机制。 新的HPE小鼠模型，可产生轻度到重度HPE的全谱表型，并确定 这些结果可以逆转到接近更正常的表型的程度。 我们实验室以前的研究已经确定信号分子(即FGF8和SHH)具有非线性 表型变异与基因剂量的关系。具体地说，这表明存在一个阈值 基因剂量的微小变化产生的影响很小或没有影响，但在 临界点。此外，改变基因剂量的下游效应只会改变特定的下游靶点。 连接到信号通路。在这里，我建议使用一种新的小鼠模型，它具有一氧化氮的失活 合酶相互作用蛋白(NOSIP)。这种突变通过改变 蛋白磷酸酶2A(PP2A)的单一泛素化，这似乎影响SHH和Wnt信号。在这 在这项工作中，我将量化基因到表型的映射，以提供对导致 畸形及其变异。 此应用程序旨在测试NOSIP失活会产生一系列连续谱的假设 面部中部表型与在人类HPE患者中观察到的表型相似。此外，我将评估 导致脸型变化的机制。在目标1中，我将通过以下方式描述NOSIP突变体 量化脸型、Shh和Wnt信号、基因表达水平和细胞过程(增殖、 细胞凋亡、极性)。目标1的这一结果不仅将为 一般的表型变异，以及所涉及的关键细胞过程，也会决定是否会发生变化 是由于特定的途径失调，还是更具全球性的影响。目标2将决定在何种程度上 改变NOSIP活性的直接和间接靶点(即PP2A、SHH和WNT)可以减少表型 Nosip突变体的变异，并恢复更正常的表型。这一目标将为 每种调节剂对表型变异的贡献，以及HPE可能的治疗靶点。 总而言之，这两个目标将大大增加我们对表型变异是如何产生的理解 并可能为发展未来的治疗研究提供线索。

PROJECT SUMMARY While the genetic basis of craniofacial malformations is being discovered, we do not understand why phenotypic variation occurs, especially when caused by the same mutation. One example is holopresencephaly (HPE), a common disease that affects 1 in 250 conceptions, but only 1 in 10,000 live births due to intrauterine lethality. HPE is unique in that malformations range from mild facial hypotelorism to cyclopia in humans. Mutations in Sonic hedgehog (SHH), and other members of this pathway, cause HPE in some patients. However, mouse models of Shh mutations do not recreate the spectrum of phenotypes observed in patients. The objective of this proposal is to determine the mechanisms that cause facial shape variation in a novel mouse model of HPE that produces a full spectrum of mild to severe HPE phenotypes, and to determine the extent to which these outcomes can be reversed to approximate a more normal phenotype. Previous research in our lab has determined that signaling molecules (i.e., FGF8 and SHH) have a non-linear relationship between phenotypic variation and gene dosage. Specifically, this indicates that there is a threshold effect where small changes in gene dosage produce little to no effect, but produces wide variation below a threshold. Additionally, the downstream effects of altering gene dosage only alter downstream targets specific to the signaling pathway. Here, I propose to use a novel mouse model that has inactivation of nitric oxide synthase interacting protein (NOSIP). This mutation affects key developmental events through changes in mono-ubiquitination of protein phosphatase 2A (PP2A), which appear to affect SHH and Wnt signaling. In this work, I will quantify the genotype to phenotype map to provide novel insight on mechanisms that cause malformations and their variance. This application aims to test the hypothesis that inactivation of NOSIP produces a continuous spectrum of midfacial phenotypes mimicking those observed in human patients with HPE. Further, I will assess mechanisms contributing to face shape variation. In Aim 1, I will characterize the NOSIP mutants by quantifying face shape, Shh and Wnt signaling, gene expression levels, and cellular processes (proliferation, apoptosis, polarity). This results from Aim 1 will not only provide important mechanistic insight to the cause of general phenotypic variation, and the key cellular processes involved, but will also determine if the changes are due to specific pathway dysregulation or a more global effect. Aim 2 will determine the extent to which altering direct and indirect targets of NOSIP activity (i.e., PP2A, SHH, and Wnt) can reduce the phenotypic variance of Nosip mutants and restore a more normal phenotype. This aim will provide specific insight to the contribution of each regulator towards phenotypic variation, as well as possible therapeutic targets for HPE. Collectively, the two Aims will add significantly to our understanding of how phenotypic variation is produced and may provide clues for developing future therapeutic studies.