Imaging Subcellular Structures in the Living Zebrafish Embryo

Summary

Imaging the dynamic behavior of organelles and other subcellular structures in vivo can shed light on their function in physiological and disease conditions. Here, we present methods for genetically tagging two organelles, centrosomes and mitochondria, and imaging their dynamics in living zebrafish embryos using wide-field and confocal microscopy.

Abstract

In vivo imaging provides unprecedented access to the dynamic behavior of cellular and subcellular structures in their natural context. Performing such imaging experiments in higher vertebrates such as mammals generally requires surgical access to the system under study. The optical accessibility of embryonic and larval zebrafish allows such invasive procedures to be circumvented and permits imaging in the intact organism. Indeed the zebrafish is now a well-established model to visualize dynamic cellular behaviors using in vivo microscopy in a wide range of developmental contexts from proliferation to migration and differentiation. A more recent development is the increasing use of zebrafish to study subcellular events including mitochondrial trafficking and centrosome dynamics. The relative ease with which these subcellular structures can be genetically labeled by fluorescent proteins and the use of light microscopy techniques to image them is transforming the zebrafish into an in vivo model of cell biology. Here we describe methods to generate genetic constructs that fluorescently label organelles, highlighting mitochondria and centrosomes as specific examples. We use the bipartite Gal4-UAS system in multiple configurations to restrict expression to specific cell-types and provide protocols to generate transiently expressing and stable transgenic fish. Finally, we provide guidelines for choosing light microscopy methods that are most suitable for imaging subcellular dynamics.

Introduction

In vivo imaging provides direct visualization of cellular behaviors in the most physiological context. The transparency of zebrafish embryos, their rapid and external development and a rich array of genetic tools that permit fluorescent labeling have all contributed to the growing use of in vivo microscopy to elucidate the dynamics of key developmental events. Imaging studies of nervous system development in zebrafish have for example greatly expanded our knowledge of the behavior of neural progenitor cells and the fate of their progeny including their subsequent migration, differentiation and circuit integration^1-8.

Protocol

All animal experiments were performed in accordance with local regulations of the government of Upper Bavaria (Munich, Germany). 

1. Labeling Organelles and Other Subcellular Structures

NOTE: Here genetic reporter constructs that fluorescently tag centrosomes, mitochondria and cell membranes are described.

1. Use conventional cloning methods²⁹ to generate fusion proteins that fluorescently label centrosomes and mitochondria. Clone the coding sequence of zebrafish centrin4 (cetn4) in-frame with a fluorescent protein² (FP) such as Yellow fluorescent protein to generat....

Results

Here the use of wide-field and confocal microscopy to image mitochondria and centrosomes in living zebrafish embryos is directly compared and contrasted. Depending on the location of the cells in which organelle dynamics are to be examined and the inherent frequency of the specific subcellular events, generally either wide-field or confocal microscopy is the better choice. We imaged organelles in RB neurons located on the surface of the embryo and in retinal cells located deeper. The supe.......

Discussion

Here, we demonstrate the versatility of the Gal4-UAS expression system to fluorescently tag mitochondria, centrosomes and the cellular membranes of specific cell-types in vivo in zebrafish embryos. Many fluorescent fusion proteins that label other organelles or subcellular structures can be found in the published literature and can be obtained from the respective laboratory, commercial sources or non-commercial plasmid depositories (e.g., Addgene). To design a new fluorescent fusion protein, several par.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Agarose (2-hydroxyethylagarose)	Sigma-Aldrich	A4018-10G	Low-gelling temperature Type VII
Block heater	Eppendorf		Thermomixer compact
Ca(NO3)2 Calcium nitrate hydrate, 99.996%	Aldrich	202967-50g	To prepare 30x Danieau's
CCD camera	Qimaging	Retiga Exi Fast 1394
Ceramic Coated Dumont #5 Forceps	Dumont - Fine Science Tools	11252-50	#5 Forceps
Confocal laser-scanning microscope	Olympus	FV1000 Fluoview
Culture dish heater	Warner Instrument Corporation	DH-35	Heating ring
Ethyl 3-aminobenzoate methanesulfate salt	PharmaQ	Tricaine PharmaQ-25g	Tricaine (anesthetic)
Fluorescence dissecting microscope	Leica	M205 FA
GeneClean kit	MP Biomedicals	111001200
Glass Bottom Culture Dishes	MatTek Corporation	P35G-0-14-C	35 mm petri dish, 14 mm microwell, No. 0 coverglass
Glass needles	World Precision Instruments Inc.	TW100F-4	For microinjections
HEPES	Sigma	H3375-250g	To prepare 30x Danieau's
High vacuum grease	Dow Corning	DCC000001242 150g	Silicon dioxide grease
KCl 99%	Sigma-Aldrich	S7643-5kg	To prepare 30x Danieau's
MgSO4.7H2O   Magnesium sulfate heptahydrate 98+% A.C.S reagent	Sigma-Aldrich	230291-500g	To prepare 30x Danieau's
Microinjector	Eppendorf		FemtoJet
Microloader tips	Eppendorf	930001007	0.5-20 ul
Micromanipulator	Maerzhaeuser Wetzlar	MM33 Rechts/00-42-101-0000/M3301R
Micropipette holder	Intracel	P/N 50-00XX-130-1
mMESSAGE mMACHINE SP6 Transcription Kit	Ambion	AM1340	To transcribe PCS-Transposase
NaCl BioXtra >99.5%	Sigma-Aldrich	P9541-1kg	To prepare 30x Danieau's
Nanophotometer			To measure DNA/RNA concentration
Needle puller	Sutter Instrument	P-1000	Flaming/Brown
NIR Apo 40x/0.80W	Nikon		Water-dipping-cone objective
N-Phenylthiourea Grade I, approx. 98%	Sigma	P7629-10G	PTU (prevents pigmentation)
Petri dishes	Sarstedt AG	821472	92 x 16mm
Plastic molds	Adaptive Science Tools	TU-1	For microinjections
Plexiglas cover-with a hole	Custom-made		The hole in the Plexiglas cover should be 3 mm larger than the diameter of the water-dipping-cone objective
Tea-strainer (Plastic)			To collect zebrafish eggs
Temperature controller	Warner Instrument Corporation	TC-344B	Dual Automatic Temperature Controller
Transfer pipettes	Sarstedt AG	86.1171	3.5mL plastic transfer pipettes
UMPlanFI 100x/1.00W	Olympus		Water-dipping-cone objective
UMPlanFLN 20x/0.50W	Olympus		Water-dipping-cone objective
Widefield microscope	Olympus	BX51WI
PTU (50x Stock)			Dissolve 76 mg PTU in 50 ml distilled water Stir vigorously at room temperature  Store at -20 oC in 1 ml aliquots Use at 1x working solution
Tricaine (20x Stock)			Dissolve 200 mg Tricaine in 48 ml distilled water Add 2 ml 1M Tris base (pH9) Adjust to pH 7  Store at -20 oC in 1 ml aliquots Use at 1x working solution
Danieau's Solution (30x Stock)			1,740 mM  NaCl  21 mM      KCl  12 mM      MgSO4.7H2O  18 mM      Ca (NO3)2 150 mM    HEPES buffer  Distilled water upto 1 L  Store at 4 oC  Use at 0.3x working solution