A vertebrate model of viral and hereditary microcephaly

项目摘要/摘要 小头畸形症严重抑制了头部和大脑的发育，可能导致智力极度低下。 残疾。小头畸形有许多不同的原因，包括基因突变、病原体感染和化学物质。 曝光。虽然小头畸形症的原因可能很广泛，但可能有共同的分子机制 牵涉其中。最近发表的结果显示寨卡病毒非结构蛋白4A(NS4A)如何抑制大脑 抑制被基因突变破坏功能的同一个ANKLE2蛋白在果蝇中的发育 是一种遗传性小头畸形症。这表明在分子上可能有关键的相似之处 导致病毒性和遗传性小头畸形的机制。研究这些分子上的相似性 脊椎动物的模型对于理解人类的疾病是必不可少的，因为脊椎动物的大脑不同 控制大脑发育的结构和基因。然而，研究这一点的工具，即强壮的脊椎动物 模式体系，并不存在。 拟议项目的主要目标是通过开发脊椎动物来填补这些主要的知识空白。 通过ANKLE2途径作用的病毒性和遗传性小头畸形模型。对于遗传模型， Ankle2将使用CRISPR/Cas9基因组编辑进行突变。对于病毒模型，寨卡病毒NS4A蛋白将是 用Tol2转基因表达。发育中的缺陷，包括各种外部测量的形态 指标，大脑的大小，结构和细胞缺陷的增殖和生存将分析使用解剖学 测量和免疫荧光显微镜。建成后，这一新的模式系统将为 在分子和细胞水平上了解ANKLE2在脊椎动物脑中的功能 以及它是如何被基因突变和病毒感染破坏的。从长远来看，它将使高- 通过对小头症的化学敏感剂和抑制剂的筛选，允许探索脊椎动物- 体内大脑发育的特定机制，以及行为研究。

PROJECT SUMMARY/ABSTRACT Microcephaly, in which head and brain development are severely inhibited, can result in extreme intellectual disability. Microcephaly has many different causes, including gene mutation, pathogen infection and chemical exposure. While the causes of microcephaly may be broad, there may be shared molecular mechanisms involved. Recently published results show how Zika virus non-structural protein 4A (NS4A) inhibits brain development in fruit flies by inhibiting the same ANKLE2 protein whose function is disrupted by gene mutation in a hereditary form of microcephaly. This suggests that there may be key similarities in the molecular mechanisms contributing to a viral and hereditary form of microcephaly. Studying these molecular similarities in vertebrate models is essential to understanding disease in humans since vertebrates have different brain structure and genes that regulate brain development. However, the tools to study this, namely a strong vertebrate model system, do not exist. The primary goal of the proposed project is to fill these major gaps in knowledge by developing a vertebrate model of viral and hereditary microcephaly acting through the ANKLE2 pathway. For the hereditary model, ankle2 will be mutated using CRISPR/Cas9 genome editing. For the viral model, Zika virus NS4A protein will be expressed using Tol2 transgenesis. Defects in development, including various externally measured morphology metrics, brain size, structure and cellular defects in proliferation and survival will be assayed using anatomical measurement and immunofluorescence microscopy. When completed, this new model system will lay the foundation for a molecular and cellular level understanding of ANKLE2 function during vertebrate brain development and how it is disrupted by gene mutation and viral infection. In the long-term, it will enable high- throughput screening of chemical sensitizers and inhibitors of microcephaly, allow the exploration vertebrate- specific mechanisms of brain development in vivo, and behavioral studies.