EASI-FISH for thick tissue defines lateral hypothalamus spatio-molecular organization

Cell. 2021 Dec 22;184(26):6361-6377.e24. doi: 10.1016/j.cell.2021.11.024. Epub 2021 Dec 6.


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.

Keywords: 3D segmentation; Fluorescence in situ hybridization; RNA; brain; brain anatomy; cell type; expansion microscopy; lateral hypothalamus; transcriptomics.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biomarkers / metabolism
  • Gene Expression Profiling
  • Gene Expression Regulation
  • Hypothalamic Area, Lateral / cytology
  • Hypothalamic Area, Lateral / metabolism*
  • Imaging, Three-Dimensional
  • In Situ Hybridization, Fluorescence*
  • Male
  • Mice, Inbred C57BL
  • Neurons / metabolism
  • Neuropeptides / metabolism
  • Proto-Oncogene Proteins c-fos / metabolism
  • RNA / metabolism
  • RNA-Seq
  • Single-Cell Analysis
  • Transcription, Genetic


  • Biomarkers
  • Neuropeptides
  • Proto-Oncogene Proteins c-fos
  • RNA