Isolation and Characterization of Single Cells from Zebrafish Embryos

Summary

This protocol describes a method for isolating single cells from zebrafish embryos, enriching for cells of interest, capturing zebrafish cells in microfluidic based single cell multiplex systems, and assessing gene expression from single cells.

Abstract

The zebrafish (Danio rerio) is a powerful model organism to study vertebrate development. Though many aspects of zebrafish embryonic development have been described at the morphological level, little is known about the molecular basis of cellular changes that occur as the organism develops. With recent advancements in microfluidics and multiplexing technologies, it is now possible to characterize gene expression in single cells. This allows for investigation of heterogeneity between individual cells of specific cell populations to identify and classify cell subtypes, characterize intermediate states that occur during cell differentiation, and explore differential cellular responses to stimuli. This study describes a protocol to isolate viable, single cells from zebrafish embryos for high throughput multiplexing assays. This method may be rapidly applied to any zebrafish embryonic cell type with fluorescent markers. An extension of this method may also be used in combination with high throughput sequencing technologies to fully characterize the transcriptome of single cells. As proof of principle, the relative abundance of cardiac differentiation markers was assessed in isolated, single cells derived from nkx2.5 positive cardiac progenitors. By evaluation of gene expression at the single cell level and at a single time point, the data support a model in which cardiac progenitors coexist with differentiating progeny. The method and work flow described here is broadly applicable to the zebrafish research community, requiring only a labeled transgenic fish line and access to microfluidics technologies.

Introduction

Most current studies of cell and molecular biology are based on population averages. However, important biological events may be masked by these traditional population-based analyses since minor populations can play major roles in biological processes and disease outcome. Understanding gene expression in heterogeneous populations at the single cell level can (and has) lead to relevant biological and clinical insights^1,2. Of concern to embryonic development studies, in a larger population of cells, progenitor cells are often underrepresented, making it challenging to detect subtle changes in gene expression that ultimately initiate cell fate decisions^3<....

Protocol

This protocol requires the use of live, adult zebrafish to produce embryos. The embryos are harvested for tissue collection. It is essential to obtain approval from appropriate ethics review boards to conduct this experiment.

1. Obtain Staged Embryos

1. The day before the experiment, prepare healthy, adult zebrafish for breeding. Place one male and one female on opposite sides of a clear divider in a breeding tank.
2. Repeat 1.1 for as many breeding tanks as necessary for sufficient embryo production for the downstream application. Obtain embryos from both wild type fish and transgenic fish that express fluorescent proteins ....

Results

As proof of principle, gene expression was assessed to explore differentiation dynamics during cardiac development. In zebrafish, cardiac progenitors arise from a mesodermal population of cells that migrate to the anterior lateral plate mesoderm where they fuse to form the linear heart tube. Prior to fusion, cardiac progenitors begin to express the transcription factor nkx2.5 (NK2 homeobox 5), which is thought to be the earliest specific marker of cardiac progenitors^16,17

Discussion

The method described herein uses expression of a fluorescent protein under control of a cell-type specific promoter to enrich a population of cardiac progenitor cells from zebrafish embryos for use in microfluidic assisted single cell capture system to assess expression of a subset of cardiac genes in single cells. Provided that FACS laser excitation and emission capabilities are compatible with the fluorophore(s) of choice, this method can be used for any fluorescent reporter line. Many zebrafish reporter lines are alre.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Supplies
Dumont #5 forceps	Fine Science Tools	11254-20	For removing the chorion from embryos
Microcentrifuge tube 2 ml	GeneMate	C-3261-1
40 um cell strainer	Biobasic	SP104151
35 mm culture dish	Falcon	351008
FACS tubes topped with 35 um cell strainer	Falcon	352235
P1000 and tips	Rainin	17005089
P20 and tips	Rainin	17005091
IFC chip manufacturer's protocol	Fluidigm	100-6117	Version 100-6117 E1 was used in representative experiment
Wide bore pastuer glass pippette	VWR	14673-010	For transferring embryos
Adult wild type zebrafish	N/A		We used AB line
Adult transgenic zebrafish	N/A		We used Tg(nkx2.5:ZsYellow)
Name	Company	Catalog Number	Comments
Reagents for cell dissociation
Double distilled water	N/A
Instant Ocean Sea Salt	Instant Ocean	SS15-10
NaCl	FisherScientific	S271-3	make stock in water and use for de-yolking buffer
KCl	Sigma Aldrich	P5405	make stock in water and use for de-yolking buffer
NaHCO3	Sigma Aldrich	S6014	make stock in water and use for de-yolking buffer
Leibovitz's L-15	Gibco	21083-027
FBS	FisherScientific	03-600-511	Heat inactivate; any brand of FBS should be fine
Cell Dissociation Reagent 1 -TrypLE	Life Technologies	12605-010	Store at room temperature.
Cell Dissociation Reagent 2 - FACSmax	Genlantis	T200100	Store -20; thaw on ice; bring to room temp before use
pronase (optional)	Sigma	P5147
L/D Dye - Sytox Blue	Life Technologies	S34857	Any live/dead stain suitable for flow cytometry will work
Trypan Blue	Gibco	15250-061
Name	Company	Catalog Number	Comments
Reagents for IFC plate use and qRT-PCR
Gene-specific probes			Probes will vary by experiment
TaqMan Probe ef1a	Life Technologies	Dr03432748_m1
TaqMan Probe gata4	Life Technologies	Dr03443262_g1
TaqMan Probe nk2-5	Life Technologies	Dr03074126_m1
TaqMan Probe myl7	Life Technologies	Dr03105700_m1
TaqMan Probe vmhc	Life Technologies	Dr03431136_m1
TaqMan Probe isl1	Life Technologies	Dr03425734_m1
TaqMan Gene Expression Master Mix	Life Technologies	4369016
Reagents listed in IFC manufacturer's protocol
C1 Reagent Kit	Fluidigm	100-5319	Reagents for loading cells onto IFC plate
Ambion Single Cell-to-CTTM	Life Technologies	4458237	Reagents for reverse transcription and pre-amplification steps
Molecular Biology Quality Water	Corning	46-000CM
C1 IFC for PreAmp (5-10 um)	Fluidigm	100-5757	IFC plate for small cells
Name	Company	Catalog Number	Comments
Equipment
C1 AutoPrep machine	Fluidigm	100-5477	For IFC plate use
Hemocytometer	Sigma Aldrich	Z359629	For counting cells and assessing cell size
Dissecting microscope			For removing embryos from chorion
Tissue culture microscope			For assessing single cell digestion
FACS machine			For isolating cells of interest