Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

Summary

Here, we describe a method for gut isolation from zebrafish larvae at 5 days post fertilization, for single-cell RNA sequencing analysis.

Abstract

The gastrointestinal (GI) tract performs a range of functions essential for life. Congenital defects affecting its development can lead to enteric neuromuscular disorders, highlighting the importance to understand the molecular mechanisms underlying GI development and dysfunction. In this study, we present a method for gut isolation from zebrafish larvae at 5 days post fertilization to obtain live, viable cells which can be used for single-cell RNA sequencing (scRNA-seq) analysis. This protocol is based on the manual dissection of the zebrafish intestine, followed by enzymatic dissociation with papain. Subsequently, cells are submitted to fluorescence-activated cell sorting, and viable cells are collected for scRNA-seq. With this method, we were able to successfully identify different intestinal cell types, including epithelial, stromal, blood, muscle, and immune cells, as well as enteric neurons and glia. Therefore, we consider it to be a valuable resource for studying the composition of the GI tract in health and disease, using the zebrafish.

Introduction

The gastrointestinal (GI) tract is a complex system that plays a vital role in overall health and well-being. It is responsible for the digestion and absorption of nutrients, as well as the elimination of waste products^1,2. The GI tract is composed of multiple cell types, including epithelial cells, smooth muscle cells, immune cells, and the enteric nervous system (ENS), which communicate closely together to regulate and maintain proper intestinal function^3,4,5. Defects in the development of the GI tract can have far-....

Protocol

All zebrafish husbandry and experiments were conducted according to the institutional guidelines of the Erasmus MC and Animal welfare legislation. The use of zebrafish larvae at 5 days post fertilization falls under the category of experiments that do not require formal ethical approval, as outlined by the Dutch regulations.

1. Obtaining 5 days post fertilization (dpf) wildtype and tg(phox2bb:GFP) larvae

1. Set up breeding of wildtype zebrafish and collect 50 eggs in HEPES-buffered E3 medium (hereafter referred to as E3) in a 15 cm Petri dish. Use these fish as a negative control for fluorescence-activated....

Results

With this protocol, we achieved successful isolation and dissociation of entire intestines from 5 dpf larvae. Using papain as the dissociation enzyme, we significantly enhanced cell viability, enabling the capture of 46,139 events involving single, viable cells (6.4% of all cells) out of 244 isolated guts (Figure 2A). Wildtype whole larvae were used as a control to ensure that the sorting process was optimized, enabling effective cell identification and sorting. Whole larvae could be used as.......

Discussion

Here, we present a method for isolation and dissociation of the gut of 5 dpf zebrafish larvae using FACS. With this method, different intestinal cell types were successfully collected and analyzed by scRNA-seq, using the 10x Genomics Chromium platform. We selected the tg(phox2bb:GFP) zebrafish line, as we wanted an indication that viable ENS cells would be also isolated (Figure 2D). However, it is important to note that this method can be readily extended to other zebrafish lines of.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x Trypsin (0.5%)-EDTA (0.2%)	Sigma	59418C
5 mL round bottom tube with cell-strainer cap	Falcon	352235
Agarose	Sigma-Aldrich	A9539
BD Falcon Round-Bottom Tube 5 mL (FACS tubes) snap cap	BD Biosciences	352054
Cell Ranger v3.0.2	10X Genomics	N/A
DAPI	Sigma-Aldrich	Cat#D-9542
Dissection microscope	Olympus SZX16
FACSAria III sorter machine	BD Biosciences	N/A
HBSS with CaCl2 and MgCl2	Gibco	14025050
Insect pins	Fine Science Tools	26000-25
L-Cysteine	Sigma	C7352
MS-222, Tricaine	Supelco	A5040-250G
Papain	Sigma	P4762
Seurat v3	Stuart et al. (2019)	N/A
Trypan blue	Sigma	Cat#T8154