Determining the spatial organization and morphological characteristics of molecularly defined cell types is a major bottleneck for characterizing the architecture underpinning brain function. We developed Expansion-Assisted Iterative Fluorescence In Situ Hybridization (EASI-FISH) to survey gene expression in brain tissue, as well as a turnkey computational pipeline to rapidly process large EASI-FISH image datasets. EASI-FISH was optimized for thick brain sections (300 μm) to facilitate reconstruction of spatio-molecular domains that generalize across brains. Using the EASI-FISH pipeline, we investigated the spatial distribution of dozens of molecularly defined cell types in the lateral hypothalamic area (LHA), a brain region with poorly defined anatomical organization. Mapping cell types in the LHA revealed nine spatially and molecularly defined subregions. EASI-FISH also facilitates iterative reanalysis of scRNA-seq datasets to determine marker-genes that further dissociated spatial and morphological heterogeneity. The EASI-FISH pipeline democratizes mapping molecularly defined cell types, enabling discoveries about brain organization.

确定分子定义的细胞类型的空间组织和形态特征是表征支撑大脑功能的结构的主要瓶颈。我们开发了扩展辅助迭代荧光原位杂交 (EASI-FISH) 来调查脑组织中的基因表达，并开发了交钥匙计算管道来快速处理大型 EASI-FISH 图像数据集。 EASI-FISH 针对厚脑切片 (300 μm) 进行了优化，以促进跨大脑泛化的空间分子域的重建。使用 EASI-FISH 流程，我们研究了下丘脑外侧区 (LHA) 中数十种分子定义的细胞类型的空间分布，该区域是一个解剖结构不明确的大脑区域。绘制 LHA 中的细胞类型图揭示了九个空间和分子定义的子区域。 EASI-FISH 还有助于对 scRNA-seq 数据集进行迭代重新分析，以确定进一步分离空间和形态异质性的标记基因。 EASI-FISH 管道使分子定义的细胞类型图谱民主化，从而能够发现大脑组织。