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.

The stage is now set to investigate the subcellular dynamics underlying these cellular behaviors. Indeed, zebrafish are already being exploited as tools for in vivo cell biology. It is now possible to visualize mitochondria^9-11, centrosomes^2,8,12-14, Golgi¹⁵, the microtubule⁴ and actin¹⁶ cytoskeleton, endosomes¹⁷ and components of the apical membrane complex^1,18, among other subcellular structures in zebrafish embryos in vivo. So far, much of what is known about the function of these organelles comes from studying their behavior in cultured cells. While in vitro studies have yielded tremendous insight into cell biology, cells in culture do not fully represent the complexity of the in vivo situation and therefore do not necessarily reflect the function and dynamics of subcellular organelles in vivo. Zebrafish embryos offer a viable in vivo alternative to examining subcellular dynamics.

As vertebrates, zebrafish possess many organ systems (e.g., neural retina) that are homologous to those found in mammalian species. Additionally, zebrafish embryos are increasingly being used to model human diseases^19,20, including those related to centrosomal function (e.g., microcephaly²¹ and Leber's congenital amaurosis²²) and to mitochondrial function (e.g., Parkinson's disease²³, tauopathies^10,24 and Barth syndrome²⁵). In vivo imaging at the cellular and subcellular level in these instances will permit a better understanding of the cell biology underlying these pathological states.

The overall goal of the methods described here is to provide a comprehensive guide to investigate organelles and other subcellular structures in zebrafish embryos using in vivo light microscopy. The entire work-flow involved in visualizing and tracking subcellular structures in vivo is described – from genetic labeling approaches, to generating transiently expressing and stable transgenic fish, and finally to imaging using wide-field and confocal microscopy. While each of these procedures is used by numerous zebrafish laboratories, the protocols described are optimized and streamlined for investigating the dynamics of subcellular structures. Two specific aspects of the work described here warrant mention: First, the use of the Gal4-UAS expression system in multiple configurations to genetically label organelles in specific cell-types. Second, a direct comparison of wide-field and confocal microscopy to image subcellular structures in vivo.

Current strategies to genetically label organelles and other subcellular structures in zebrafish either make use of capped mRNA^1,4,8 or DNA based constructs where promoter elements directly drive the expression of fusion proteins^9,14,15. In vitro transcribed capped RNA results in rapid and broad expression, that is not tissue-specific however. Additionally, expression levels diminish over time as the capped RNA is diluted or degraded. Thus the use of RNA based constructs to examine organelle dynamics at later stages in development is limited (usually up to 3 days post-fertilization).

These limitations can be overcome by using DNA constructs, where spatial and temporal control of expression is determined by specific promoter elements. When DNA based constructs are used in the context of the Gal4-UAS system significant improvements to transgene expression levels are observed^26,27. In this bipartite expression system, cell-type specific promoter elements drive the expression of a transcriptional activator Gal4, while reporter genes are cloned downstream of the Gal4-binding upstream activating sequence (UAS). By combining UAS reporters with appropriate Gal4 drivers, expression can be restricted to specific cell-types, circumventing the need to clone reporter genes behind different promoters every time a specific expression pattern is desired. Furthermore, the expression of multiple UAS reporter genes can be driven by a single Gal4 activator. The Gal4-UAS system thus provides a versatile and flexible genetic approach for subcellular labeling.

Wide-field and confocal microscopes are the workhorses of most laboratories. Wide-field systems typically use an arc lamp as a light source and detect the emitted light with a sensitive camera that is placed at the end of the light path. This imaging modality is typically restricted to thin samples as out-of focus light obscures in-focus information in thicker samples. Confocal microscopes differ from wide-field systems in that they are built to favor signals that originate from the focal plane over those that originate out of focus (i.e., "optical sectioning")²⁸. To achieve optical sectioning a pinhole is placed in the emission path in a conjugate position to the point light source. Lasers are used as light sources and signals are detected with photomultiplier tubes (PMTs). Practically, a laser beam is swiped over the sample point-by-point and the fluorescence emission at each spot (pixel) is detected by the PMT.

Here we image the very same subcellular structures in living zebrafish embryos using both wide-field and confocal microscopy to provide a direct comparison of both microscopy modalities. The underlying aim of providing such comparisons is to offer guidelines for choosing the most appropriate microscopy technique for the specific question at hand.

Using the approaches described here we demonstrate Gal4-UAS based genetic labeling of mitochondria and centrosomes. These organelles are imaged in different cell-types of the nervous system and in muscle cells using wide-field and confocal microscopy to demonstrate the suitability of each imaging modality. The methods described here can easily be adapted for investigating other organelles and subcellular structures in the living zebrafish embryo.

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. Use conventional cloning methods29 to generate fusion proteins that fluorescently label centrosomes and mitochondria….

Representative 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

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	Fluka Analytical	A5040-100G	Tricaine (anesthetic)
Fluorescence dissecting microscope	Leica	M205 FA
GeneClean kit	MP Biomedicals	111001200
Glass Bottom Culture Dishes	MatTek Corporation	P35G-0-14-C	35mm petri dish, 14mm 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
Incubator	Thermo Scientific	Heraeus	To maintain zebrafish embryos at 28.5⁰ C
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	0.5-20uL
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)			1740 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