Optimizing MERFISH to allow multiplexed measurement of developmental and tonotopicgene expression gradients in the cochlea

项目总结 成体耳蜗给研究其正常功能的研究人员带来了许多技术障碍 组成细胞类型，以及这些细胞类型如何对损伤、衰老和退化作出反应。因为 耳蜗包裹在坚固的骨骼中，与其他感觉器官相比，其细胞数量相对较少。 器官，如视网膜，分离细胞以表征其结构、生理或基因表达 很有挑战性。此外，使耳蜗更容易获得的组织学方法，如脱钙，是 可以降解信使核糖核酸或蛋白质的严厉治疗。在这项试点R21提案中，我们将评估 一种最近发展起来的技术，称为MerFish-Multiplexed Error-Robust原位荧光技术 杂交--同时检测和量化数百个基因在耳蜗中的表达。我们 我相信这项技术足够灵敏，可以揭示基因表达的数量差异 沿耳蜗基底部-顶轴存在的发育梯度和紧张度梯度。开发一个 一次检测、量化和空间定位数百个基因的可靠协议具有转化的能力 我们对耳蜗正常功能的理解，以及基因表达如何在耳蜗内和 噪音暴露、毛细胞丢失、衰老和用药后的螺旋神经节。这项提议的目标是 为了证明MerFish在新生儿和成人耳蜗中的可行性，并开发方案以允许 供听力和平衡社区使用的墨鱼。

PROJECT SUMMARY The adult cochlea presents many technical hurdles to researchers studying the normal function of its constituent cell types, and how these cell types respond to injury, aging and degeneration. Because the cochlea is encased in strong bone and contains relatively small numbers of cells compared to other sensory organs such as the retina, isolating cells to characterize their structure, physiology or gene expression is challenging. Moreover, histological methods to make the cochlea more accessible, such as decalcification, are harsh treatments that can degrade mRNA or protein. In this pilot R21 proposal, we will assess the feasibility of a recently-developed technique known as MERFISH – Multiplexed Error-Robust Fluorescence In Situ Hybridization – to simultaneously detect and quantify the expression of hundreds of genes in the cochlea. We believe this technique is sensitive enough to reveal quantitative differences in gene expression along the developmental and tonotopic gradients that exist along the basal-apical axis of the cochlea. Developing a robust protocol to detect, quantify and spatially localize hundreds of genes at once has the ability to transform our understanding of the normal function of the cochlea, and how gene expression changes in the cochlea and spiral ganglion after noise exposure, hair cell loss, aging and drug administration. The goal of this proposal is to demonstrate the feasibility of MERFISH in the neonatal and adult cochlea and to develop protocols to allow MERFISH to be used by the hearing and balance community.