Microfluidics-Enabled High-Throughput Studies on Early Embryonic Development

描述（由申请人提供）：产前酒精暴露导致一系列发育障碍，通常被称为胎儿酒精谱系障碍（FASDs）。fasd最严重的病例被称为胎儿酒精综合征（FAS），其特征是生长缺陷、颅面畸形以及结构和功能中枢神经系统（CNS）异常。技术限制降低了实验通量，加上生物过程固有的随机性，使我们无法定量了解乙醇相关发育障碍的发生和进展。因此，关于乙醇暴露如何表现为fasd的机制还不太清楚。在这里，我们的目标是开发一种基于微流体的系统，使高通量和高含量的研究能够阐明乙醇暴露对胚胎发生的影响。模式生物黑胃果蝇（Drosophila melanogaster）已被证明是FASDs的模型，因为当胚胎在乙醇溶液中培养时，果蝇表现出与人类相似的表型缺陷。目前尚不清楚乙醇暴露如何影响胚胎发生过程中导致fasd广泛症状的发育过程。在此之前的技术差距是一种方法，可以将发育中的苍蝇胚胎暴露在乙醇中，同时也允许高通量实时成像和数据采集。该项目的目标是开发全胚胎成像技术，以揭示胚胎乙醇暴露影响最终导致fasd的形态发生和发育模式的机制。为了解决技术上的差距，我们正在开发一种微流体系统，可以以高度并行的方式培养和暴露活的果蝇胚胎于乙醇中。该系统还将实现胚胎发生的实时成像，以便我们更好地了解乙醇暴露如何影响形态发生。此外，微流体系统将与组织清除技术相结合，这将使整个胚胎成像，从而使我们能够可视化整个胚胎基因表达模式。在这项研究中，我们将研究关键酶的突变体，以更好地阐明它们在FASD中的作用。使用这个设备，我的目的是更好地了解与fasd有关的物理和基因表达变化。微流体系统将与计算机自动化程序集成，这将提高数据采集率。通过计算机视觉算法的发展，数据分析将被简化，这将有助于识别与乙醇暴露相关的不明显的、可能被人工注释遗漏的表型变化。该项目不仅将扩大发育生物学的工具库，而且将对乙醇致畸学产生重大影响。

DESCRIPTION (provided by applicant): Prenatal alcohol exposure causes a range of developmental disorders that are commonly referred to as fetal alcohol spectrum disorders (FASDs). The most severe case of FASDs is known as fetal alcohol syndrome (FAS) and is characterized by growth deficiencies, craniofacial malformations, and both structural and functional central nervous system (CNS) abnormalities. The combination of technological limitations that decrease experimental throughput and the inherent stochasticity of biological processes eliminate the ability to gain a quantitative understanding about the onset and progression of ethanol-related developmental disorders. As a result there is not much known mechanistically about how ethanol exposure manifests as FASDs. Here, we aim to develop a microfluidic-based system that enables high-throughput and high-content studies to elucidate the effects of ethanol exposure on embryogenesis. The model organism Drosophila melanogaster (Drosophila) has been shown to be a model of FASDs, because Drosophila exhibits similar phenotypical defects as humans when embryos are raised on ethanol solutions. It has yet to be seen how ethanol exposure affects developmental processes that occur during embryogenesis that lead to the wide ranging symptoms of FASDs. The technological gap that precedes this is a method that can robustly expose developing fly embryos to ethanol while also allowing high-throughput live imaging and data acquisition. The objective of this project is to develop enabling technologies for whole embryo imaging to uncover the mechanisms through which embryonic ethanol exposure affects morphogenesis and developmental patterning that ultimately lead to FASDs. In order to address the technological gap we are developing a microfluidic system that can culture and expose live Drosophila embryos to ethanol in a highly-parallelized manner. This system will also enable live imaging of embryogenesis, so that we can better understand how ethanol exposure affects morphogenesis. Furthermore, the microfluidic system will be integrated with tissue clearing techniques that will enable whole embryo imaging and thus allow us to visualize whole embryo gene expression patterns. In this study, we will study mutants of key enzymes to better elucidate their roles in FASD. Using this device, I aim to better our understanding about the physical and gene expression changes involved with FASDs. Microfluidic systems will be integrated with computer automation programs that will increase data acquisition rates. Data analysis will be streamlined through the development of computer vision algorithms that will help identify phenotypical changes associated with ethanol exposure that are non-obvious and likely to be missed by manual annotation. This project will not only enlarge the repertoire of tools available for developmental biology in general, but also will have significant impact on ethanol teratology.