smiFISH and embryo segmentation for single-cell multi-gene RNA quantification in arthropods

Area of Science:

• Molecular Biology
• Developmental Biology
• Genetics

Background:

• Advances in single-cell RNA visualization are crucial for understanding transcriptional regulation.
• Single-molecule inexpensive fluorescent in-situ hybridization (smiFISH) offers high-resolution RNA detection.
• Existing protocols require adaptation for complex multicellular organisms like whole embryos.

Purpose of the Study:

• To adapt and validate the smiFISH protocol for whole arthropod embryos and non-embryonic tissues.
• To develop an imaging and analysis pipeline for 3D cell segmentation and single-cell RNA quantification.
• To propose and compare novel statistical measures for single-cell gene expression variability.

Main Methods:

• Adaptation of the smiFISH protocol for whole embryos across diverse arthropod species.
• Simultaneous detection of up to eight different RNA species using multi-spectral fluorophores and white light laser confocal microscopy.
• Integration of smiFISH with cell membrane immunofluorescence for combined spatial and expression analysis.
• Development of a computational pipeline for 3D cell segmentation and single-cell RNA counting.

Main Results:

• Successful application of smiFISH to whole arthropod embryos and other tissues.
• Simultaneous detection and quantification of multiple RNA molecules from up to eight genes within individual cells.
• Establishment of a robust pipeline for 3D imaging and analysis of single-cell RNA expression in whole blastoderm embryos.
• Proposal of two new variability measures and comparison with the Fano factor for single-cell expression data.

Conclusions:

• The adapted smiFISH protocol provides high-resolution, multi-gene RNA detection in whole embryos.
• The developed pipeline enables comprehensive 3D analysis of single-cell gene expression.
• Novel variability measures offer new perspectives on interpreting single-cell expression heterogeneity.