This work was funded by the friends of Sophia Foundation (SSWO WAR-63).

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
<ol>
	<li>Set up breeding of wildtype zebrafish and collect 50...

Processing Gut Cells from Zebrafish Larvae for Single-Cell RNA Sequencing

<ol>
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N., Pack, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Unique+and+conserved+aspects+of+gut+development+in+zebrafish.">Unique and conserved aspects of gut development in zebrafish.</a> Developmental Biology. 255 (1), 12-29 (2003).</li><li>Harrison, C., Wabbersen, T., Shepherd, I. T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=In+vivo+visualization+of+the+development+of+the+enteric+nervous+system+using+a+Tg(-8.3bphox2b:Kaede)+transgenic+zebrafish.">In vivo visualization of the development of the enteric nervous system using a Tg(-8.3bphox2b:Kaede) transgenic zebrafish.</a> Genesis. 52 (12), 985-990 (2014).</li><li>Kuil, L. E., Chauhan, R. K., Cheng, W. W., Hofstra, R. M. W., Alves, M. 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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...

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 products1,2. 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 function3,4,5. Defects in the development of the GI tract can have far-reaching effects on various aspects such as nutrient absorption, microbiota composition, the gut-brain axis, and the ENS, leading to several enteric neuromuscular disorders, such as Hirschsprung disease and Chronic intestinal pseudo-obstruction6,7. These disorders are characterized by severe gut dysmotility caused by alterations in various key cells, such as the interstitial cells of Cajal, smooth muscle cells, and the ENS6,8,9. However, the molecular mechanisms underlying GI development and dysfunction are still poorly understood.
The zebrafish is a valuable model organism for studying GI development and dysfunction due to its rapid embryonic development, transparency during embryonic and larval stages, and genetic tractability10,11,12,13,14. Numerous transgenic zebrafish lines expressing fluorescent proteins are available. An example of such a line is the tg(phox2bb:GFP) zebrafish, commonly used to study the ENS, as all phox2bb+ cells, including enteric neurons, are labeled15,16. Here, using the tg(phox2bb:GFP) zebrafish line, we present a method for intestinal isolation of 5 days post fertilization (dpf) larvae for single-cell RNA sequencing (scRNA-seq) analysis (Figure 1).

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr>
			<td>10x Trypsin (0.5%)-EDTA (0.2%)</td>
			<td>Sigma</td>
			<td>59418C</td>
			<td></td>
		</tr>
		<tr>
			<td>5 mL round bottom tube with cell-strainer cap</td>
			<td>Falcon</td>
			<td>352235</td>
			<td></td>
		</tr>
		<tr>
			<td>Agarose</td>
			<td>Sigma-Aldrich</td>
			<td>A9539</td>
			<td></td>
		</tr>
		<tr>
			<td>BD Falcon Round-Bottom Tube 5 mL (FACS tubes) snap cap</td>
			<td>BD Biosciences</td>
			<td>352054</td>
			<td></td>
		</tr>
		<tr>
			<td>Cell Ranger v3.0.2</td>
			<td>10X Genomics</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>DAPI</td>
			<td>Sigma-Aldrich</td>
			<td>Cat#D-9542</td>
			<td></td>
		</tr>
		<tr>
			<td>Dissection microscope</td>
			<td>Olympus SZX16</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>FACSAria III sorter machine</td>
			<td>BD Biosciences</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>HBSS with CaCl2 and MgCl2</td>
			<td>Gibco</td>
			<td>14025050</td>
			<td></td>
		</tr>
		<tr>
			<td>Insect pins</td>
			<td>Fine Science Tools</td>
			<td>26000-25</td>
			<td></td>
		</tr>
		<tr>
			<td>L-Cysteine</td>
			<td>Sigma</td>
			<td>C7352</td>
			<td></td>
		</tr>
		<tr>
			<td>MS-222, Tricaine</td>
			<td>Supelco</td>
			<td>A5040-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Papain</td>
			<td>Sigma</td>
			<td>P4762</td>
			<td></td>
		</tr>
		<tr>
			<td>Seurat v3</td>
			<td>Stuart et&#160;al. (2019)</td>
			<td>N/A</td>
			<td></td>
		</tr>
		<tr>
			<td>Trypan blue&#160;</td>
			<td>Sigma&#160;</td>
			<td>Cat#T8154</td>
			<td></td>
		</tr>
	</tbody>
</table>

gut isolation from zebrafish larvae for single-cell rna sequencing

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,&#160;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.

Author Spotlight: Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development 

Gut Isolation from Zebrafish Larvae for Single-cell RNA Sequencing

Our research aims to investigate the transcriptomic changes occurring during gastrointestinal development and dysfunction in zebrafish larvae. Here, we provide a new protocol that allows isolation of different cell types within the zebrafish intestine, which has potential applications in understanding gastrointestinal health and disease. In recent years, there has been growing interest in exploring the interaction between the enteric nervous system and other intestinal cell types during development and disease.Understanding these complex dynamics using advanced techniques like single-cell RNA sequencing, holds the potential to reveal critical insights into enteric neuromyopathies, leading to more precise diagnostics and treatments. Using this protocol, we successfully identified various intestinal cell types, including epithelial, stromal, bloods, muscle, immune cells, and even enteric neurons and glia, which have not been captured before at this amount using similar approaches. Thus, we provide a reliable technique to study the gastrointestinal composition during development and dysfunction.

With this protocol, we achieved successful isolation and dissociation of entire intestines from 5 dpf&#160;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 us...

Watch this Scientific Journal Video about Isolating and Analyzing Intestinal Cells of Zebrafish Larvae for Investigating Transcriptomic Aspects of Gastrointestinal Development at JoVE.com

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