Quantitative microscopy-based rapid phenotyping and screening

 描述：突触是神经系统最基本的功能。线虫由于其基因组测序以及丰富的分子生物学工具和突变体，是寻找基因和阐明途径的优秀遗传模型系统。由于其神经系统的简单，线虫在了解神经系统模式和突触发育的分子机制方面已经取得了许多突破。目前的瓶颈是人工和非定量技术，如视觉屏幕，限制了实验的吞吐量和人们可以检查的表型。我们的长期目标是开发技术，了解基因、年龄和环境如何共同定义并继续重塑有机体的神经系统。在过去的资助期间，我们在硬件系统设计(包括微技术和自动化技术)和表型定量表征软件方面取得了很大进展。这一后续项目的目标是进一步设计用于大规模实时成像和定量成像技术的高级微型设备，并结合遗传学和基因组方法的力量来研究这个体内系统中的突触发育；从这项研究中出现的基因和通路可能成为神经疾病治疗的靶点。我们在该项目的前一阶段已经证明，基于定量显微镜的方法确实能够识别传统方法无法识别的新基因和途径。在后续阶段，我们将进一步优化芯片上快速和高含量的活体成像技术，同时进一步开发分析这些高含量数据的算法和定量措施；我们将根据人眼无法观察到的新的合成表型进行筛选；我们还将利用强大的基因组技术来识别塑造突触形态的基因座和潜在的多基因相互作用。这些实验方法将识别由于表型分析相关的困难而无法以其他方式识别的基因，但使用我们这里的工程技术进行处理。这种方法是创新的，因为这里开发的技术极大地提高了实验的吞吐量、灵敏度和准确性，并真正使极其强大的遗传和基因组方法的实用成为可能。这项拟议的研究意义重大，因为它满足了高通量和高内容筛选以及识别新基因和途径的迫切需求。此外，除了对特定神经生物学的贡献外，这些技术还广泛适用于发育细胞生物学等领域，以及其他小生物，如苍蝇幼虫和斑马鱼胚胎。

 DESCRIPTION: Synapses are most fundamental to the function of a nervous system. C. elegans is an excellent genetic model system for finding genes and elucidating pathways because of its sequenced genome and the abundance of molecular biology tools and mutants. Due to the simplicity of its nervous system, many breakthroughs have been made in C. elegans for understanding molecular mechanisms in the patterning of the nervous system and synapse development. The current bottlenecks are in the manual and non-quantitative techniques such as visual screens, limiting both the throughput of the experiments and the phenotypes one can examine. Our long-term objective is to develop technologies and to understand how genes, age, and the environment together define and continue to remodel the nervous system of an organism. In the last funding period, we have made large progress in hardware system design (including microtechnologies and automation technologies) and software for quantitative characterization of phenotypes. The objective of this continuation project is to further engineer superior micro devices for large-scale live imaging and quantitative imaging technologies, and combine with the power of genetic and genomic approaches to study synapse development in this in vivo system; genes and pathways emerging from this study could potentially become targets of therapeutics in neurological disorders. We have shown in the previous phase of the project that quantitative microscopy-based approaches can indeed enable identification of novel genes and pathways that conventional approaches cannot. In the continuation phase, we will further optimize on-chip rapid and high-content in vivo imaging techniques, and in parallel further develop algorithms and quantitative measures for the analysis of such high-content data; we will screen based on novel synthetic phenotype unobservable by eye; we will also exploit powerful genomic techniques to identify loci and potential multigenic interactions that shape the synapse morphology. These experimental approaches will identify genes that cannot have been identified otherwise because of the difficulties associated with the phenotypical profiling, but addressed using our engineered techniques here. The approach is innovative because the technology developed here dramatically increases the throughput, sensitivity, and accuracy of the experiments, and truly enables the utility of extremely powerful genetic and genomic methods. The proposed research is significant because it fills the urgent need in high-throughput and high-content screens as well as identifying novel genes and pathways. In addition, besides the contribution to the specific neurobiology, the technologies are widely applicable to areas such as developmental cell biology, and to other small organisms such as fly larvae and zebrafish embryos.