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Single Molecule-Based fliFISH Validates Radial and Heterogeneous Gene Expression Patterns in Pancreatic Islet β-Cells
Fangjia Li1, Dehong Hu1, Cailin Dieter2
1Environmental Molecular Sciences Laboratory, Pacific Northwest National Laboratory, Richland, WA.
Diabetes
|March 9, 2021
Summary
Single-cell RNA-sequencing (scRNA-Seq) reveals islet cell heterogeneity, but fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) offers superior transcript quantification and spatial context. This method precisely maps gene expression patterns in intact pancreatic islets, highlighting cell maturation states.
Area of Science:
- Endocrinology
- Molecular Biology
- Genomics
Background:
- Single-cell RNA-sequencing (scRNA-Seq) has advanced understanding of pancreatic islet cell transcriptomes, revealing beta-cell heterogeneity.
- Limitations of scRNA-Seq include inconsistencies in gene expression patterns and inability to resolve spatial organization of heterogeneity.
- Accurate quantification of low-abundance transcripts and spatial transcriptomic analysis remain challenges in islet research.
Purpose of the Study:
- To introduce and validate fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) for precise transcript quantification in mouse pancreatic islets.
- To compare gene expression patterns of key beta-cell maturation markers (Ins2, Mafa, Ucn3) and Rgs4 using fliFISH.
- To investigate the spatial distribution of heterogeneous gene expression within intact pancreatic islets.
Main Methods:
- Utilized fluctuation localization imaging-based fluorescence in situ hybridization (fliFISH) on mouse pancreatic islet sections.
- Quantified transcripts for Insulin 2 (Ins2), Mafa, Ucn3, and Rgs4 at the single-cell level.
- Evaluated transcript heterogeneity and spatial expression patterns within the intact islet architecture.
Main Results:
- fliFISH accurately quantified transcripts across a wide dynamic range (single copies to >100 copies/cell).
- Confirmed significant heterogeneity in gene expression levels within individual islets.
- Identified relative and radial expression patterns of transcripts, suggesting spatial organization related to beta-cell maturation.
Conclusions:
- fliFISH provides a powerful tool for high-resolution spatial transcriptomics in pancreatic islets.
- Beta-cell heterogeneity is spatially organized within islets, reflecting distinct maturation states along the radial axis.
- This spatial transcriptomic approach enhances our understanding of islet biology and beta-cell function.
