Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing.
Naomi J M Kakiailatu1, Laura E Kuil2, Eric Bindels3
1Department of Clinical Genetics, Erasmus University Medical Center - Sophia Children's Hospital.
Journal of Visualized Experiments : Jove
|November 27, 2023
Summary
Researchers developed a new method to isolate live gastrointestinal cells from zebrafish larvae for single-cell RNA sequencing. This technique enables detailed study of gut cell types in both health and disease.
Area of Science:
- Developmental biology
- Gastroenterology
- Zebrafish models
Background:
- The gastrointestinal (GI) tract's development is crucial, with congenital defects leading to enteric neuromuscular disorders.
- Understanding the molecular mechanisms of GI development and dysfunction is essential for addressing these disorders.
Purpose of the Study:
- To present a novel protocol for isolating viable gastrointestinal cells from zebrafish larvae for single-cell RNA sequencing (scRNA-seq).
- To enable comprehensive analysis of GI tract cellular composition in zebrafish models.
Main Methods:
- Manual dissection of zebrafish intestines at 5 days post-fertilization.
- Enzymatic dissociation using papain to obtain single cells.
- Fluorescence-activated cell sorting (FACS) to isolate viable cells for scRNA-seq.
Main Results:
- Successful isolation of live, viable cells from the zebrafish GI tract.
- Identification of diverse intestinal cell types, including epithelial, stromal, blood, muscle, immune cells, enteric neurons, and glia.
- Demonstration of the protocol's efficacy for scRNA-seq analysis.
Conclusions:
- The developed method provides a valuable resource for studying GI tract cellular composition in zebrafish.
- This approach facilitates research into the molecular underpinnings of GI development and disease in a relevant model organism.
