Microfluidics-Enabled High-Throughput Studies on Early Embryonic Development

描述(由申请人提供)：产前酒精暴露会导致一系列发育障碍，通常被称为胎儿酒精谱系障碍(FASD)。最严重的FASDS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上的中枢神经系统(CNS)异常。减少实验吞吐量的技术限制和生物过程固有的随机性相结合，消除了对与酒精相关的发育障碍的发生和发展的定量了解的能力。因此，关于酒精暴露是如何表现为FASD的，从机理上来说并不是很清楚。在这里，我们的目标是开发一种基于微流控的系统，使高通量和高含量的研究能够阐明乙醇暴露对胚胎发育的影响。模式生物黑腹果蝇已被证明是FASD的模型，因为当胚胎在乙醇溶液中饲养时，果蝇表现出与人类相似的表型缺陷。目前还不清楚乙醇暴露如何影响胚胎发生期间发生的发育过程，从而导致FASD的广泛症状。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强有力地暴露在乙醇中，同时还可以进行高通量的实时成像和数据采集。该项目的目标是开发全胚胎成像的使能技术，以揭示胚胎酒精暴露影响最终导致FASD的形态发生和发育模式的机制。为了解决这一技术差距，我们正在开发一种微流控系统，它可以以高度并行的方式培养活的果蝇胚胎，并将其暴露在乙醇中。该系统还将使胚胎发生的实时成像成为可能，这样我们就可以更好地了解酒精暴露如何影响形态发生。此外，微流控系统将与组织透明技术相结合，这将使我们能够进行全胚胎成像，从而使我们能够可视化整个胚胎的基因表达模式。在这项研究中，我们将研究关键酶的突变，以更好地阐明它们在FASD中的作用。使用这个设备，我的目标是更好地了解FASD涉及的物理和基因表达变化。微流控系统将与计算机自动化程序集成，以提高数据采集速度。将通过开发计算机视觉算法来简化数据分析，这些算法将有助于识别与酒精接触有关的不明显且可能被手动注释遗漏的表型变化。该项目不仅将扩大发育生物学可用工具的总体范围，而且还将对乙醇畸形学产生重大影响。

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.