Department of Biological Sciences, Smith College
Deschene, E. R., Barresi, M. J. Tissue Targeted Embryonic Chimeras: Zebrafish Gastrula Cell Transplantation. J. Vis. Exp. (31), e1422, doi:10.3791/1422 (2009).
Part 1: Alexa 594 Labeled Embryos
1.1 Microinjection Plate and Needle Preparation
1.2 Embryo Collection and Microinjection Apparatus Setup
1.3 Microinjections of Fluorescent Alexa 594
Part 2: Gastrula Stage Transplantations
2.1 Dechorionating and Transplant Plate Preparation
2.2 Embryo and Transplant Apparatus Setup
2.3 Gastrula Stage Cell Transplants
2.4 Visualizing and Imaging Embryos
In an effort to address the role of astroglial cells in the forebrain it is necessary to to both visualize these cells and target different genetic manipulations to small clusters of diencephalic and telencephalic cells. To generate these types of chimeric embryos, in which some portion of the astroglial population within the embryo is different whether by genotype or phenotype, we used a multifaceted approach that combines the use of GFP transgenic embryos, which labels astroglia, with the use of gastrula-staged cell transplantation. To specifically target our clones to the diencephalon, we utilized the zebrafish gastrula fate map to selectively extract cells at the midline equidistant from the animal pole and the shield 5 (Fig. 1B). These extracted tg[gfap:gfp] cells were then transplanted into the same location in a wild type host gastrula, which was then raised to 30hpf, and immunolabeled for all axons (α-Acetylated Tubulin) as reference landmarks for forebrain anatomy. As an example shown here, confocal imaging of one host embryo reveals isolated GFP+ cells throughout the telencephalon and diencephalon (Fig. 2A). The full morphology of these GFP+ cells can be observed in this clonal assay, revealing that most of these cells take on the characteristic radial glial morphology, where the soma is located adjacent to the ventricular zone and a large end foot terminates a radial process at the pial surface (Fig. 2A, inset). Three-dimensional rendering of the Z-stack of these collected optical slices clearly showed the position of these cells with respect to the labeled axons (Movie 1).
Another approach commonly used for visualizing transplanted cells is to initially microinject a fluorescent cell lineage dye, such as Alexa 594, into the yolk of a one-cell staged wild type or GFP transgenic embryo. As an example we injected Alexa 594 into wild type embryos at the one-cell stage. We allowed them to develop to shield-stage gastrula and then carried out forebrain targeted transplantation as mentioned above, but instead we transplanted into tg[gfap:nuc-gfp] transgenic host gastrula. Imaging of the dorsal telencephalon by Laser Scanning Confocal microscopy revealed fluorescent red clusters of donor cells amongst GFP+ nuclei within the forebrain (Fig. 2B). We further analyzed this host by collecting Z-stacks every three minutes over the course of two hour with the Laser Scanning Confocal. 4-dimensional rendering of this timelapse using Volocity software (Improvision) shows dynamic cellular movements of the rhodamine cell membranes and the GFP+ nuclei (Movie 2).
Click her to see a larger version of figure 1.
Figure 1: Transplant apparatus and schematic of experimental methods. A) Apparatus used to conduct gastrula-stage transplants. The Zeiss Lumar fluorescent stereo microscope was used to conduct these transplants. It has joystick controlled focus and zoom (left red circle). The Transferman NK was used to manipulate the position of the capillary which is also joystick controlled (right red circle). During setup 60-80 embryos are placed into individual agar wells previously made in a 100mm petri dish with a plastic mold (purple outlined box). The transplant capillary (TransferTip) manufactured by eppendorf has a 15μm inner diameter opening and a 20 degree angled tip (green outlined box). The aspiration of cells is controlled with Eppendorf's AirTram (red outlined box). B) Illustration of gastrula stage transplantation procedure from donor into host embryos. Cells are extracted from tg[gfap:GFP] embryos (shown here) or Alexa 594 injected (as described in protocol) donor embryos. Cells are removed from the midline halfway between the shield and animal pole, and transplanted into the same region of host embryos. Hosts are fixed and imaged using Laser Scanning Confocal Microscopy.
Please click here to see a larger version of figure 2.
Figure 2. Example of gastrula stage transplants within the zebrafish forebrain. A) A frontal view of wild type host embryos with transplanted tg[gfap:gfp] cells in the diencephalons and telencephalon of the zebrafish forebrain. Axons are labeled in red (α-Acetylated Tubulin antibody) and transplanted cells are labeled in green (endogenous GFP expression). Embryos are 30 hpf. Inset shows radial glial morphology of GFP+ cells. B) Dorsal view of the telencephalon of a live tg[gfap:nuc-GFP] host embryo with Rhodamine fluorescing transplanted cells (red). Nuclei of host embryos are labeled in green (GFP). Dashed line outlines the anterior surface of the forebrain. Solid line indicates the ventricular zone. Scale bar is 10μm.
Movie 1. The relationship of ectopically labeled radial glial cells amongst forebrain commissures. Cells from a gastrula staged tg[gfap:GFP] embryo were transplanted into a non-GFP transgenic wild type line. A Laser Scanning Confocal Z-Stack was collected and then processed for 3-D rendering using Volocity software. Initially, the image is a frontal view of the zebrafish forebrain at 30hpf, and it has been flipped horizontally as compared to Figure 2B. Axons (a-Acetylated Tubulin, red) within the anterior commissure (AC, top) and post-optic commissure (POC, bottom) are seen. The green cells are the GFP+ transplanted cells that took root in the forebrain and generated clear radial glial morphology. As the movie progresses it focuses in on two radial glial cells possessing end fee that contact the POC. Click here to Download Movie 1.
Movie 2. Timelapse imaging of transplanted Alexa 594 Labeled Cells in the forebrain. Cells from a gastrula previously injected with Alexa 594 were transplanted into tg[gfap:nuc-gfp] transgenic embryos. This movie represents a 3-D projection of a 2h timeseries, in which Z-stacks were taken every 5 minutes. Z-Stacks were collected on a laser scanning confocal microscope, and 4-D renderings completed using Volocity software. Transplanted cells are labeled with Alexa 594 (red), and GFP+ nuclei are green. As this timelapse runs, the 4D image starts with a dorsal view of the forebrain at 30hpf and will rotate about the X-axis, as clear movement is detected in the cell membranes of all transplanted cells and in the GFP+ nuclei as well. Click here to Download Movie 2.
Generating chimeric embryos is a powerful tool that addresses many research questions within several model systems, namely the fruit fly (D.melanogaster), worm (C.elegans), zebrafish (D.rerio) and mouse (M.musculus). We argue that the zebrafish model system is uniquely suited for generating chimeras in a relatively easy, fast and versatile way. The zebrafish is a vertebrate that develops outside the mother making it significantly more accessible as compared to the mouse model. Zebrafish are also significantly larger than flies or worms, which simplifies embryological manipulations such as cell transplantation. In addition, the ability to combine cell transplantation procedures with transgenic techniques enables a large array of possible experiments in which gene function can be manipulated in a localized tissue-specific manner. For instance, small clones of GFP+ or Rhodamine labeled cells enable the characterization of full cell morphologies that are often lost in a homozygous transgenic donor or are impossible to visualize due to differential subcellular labeling with common antibodies. Furthermore, by using transgenically tagged or fluorescently filled cells, we can track donor cells in the live zebrafish embryo over time. Timelapse imaging of individual cells provides a novel analysis of cellular behavior in the context of the whole organism.
Our lab also combines the use of heat shock inducible transgenic lines with cell transplantations such that certain axon guidance cues can be misexpressed in donor cells following an elevation in incubation temperature (data not show). This technique is an extremely powerful and direct approach to locally misexpress a protein of interest in a spatial and temporal-specific manner, which allows us to see how the specific protein affects development. In our case, this technique can extend our knowledge behind the function of Slit-Robo guidance cues in the positioning of commissures and glial cells at the midline 5.
Other approaches to locally misexpressing genes such as focal UV uncaging and localized heatshock tools (laser or soldering iron), do offer alternative ways to manipulate the expression of genes and markers in a small cluster of cells 7-9; however, cell transplantation establishes a final environment of analysis that is free of any trauma induced by UV, laser or heat making cell transplantation a more controlled experimental approach. In conclusion, we have found cell transplantation procedures to be a crucial part of our neurodevelopmental biology research. Generating clones of cells with different properties is a critical tool for the developmental biologist in addressing fundamental questions of cell behaviors, gene function, and cell autonomy.
The authors have nothing to disclose.
We would like to thank members of the Barresi Lab for their support and helpful comments on this manuscript. We thank Alexander Workman for his constant technical assistance as well as the animal care staff for their help in maintaining the Smith College Zebrafish colony. We also thank Mike Hallacy, Rudi Rottenfusser, and Carl Zeiss Microimaging for lending some microscopy equipment for the filmin of this protocol. This work was supported by an N.S.F. funded research grant, 0615594.
|Petri dishes||Tool||Fisher Scientific||08-757-13||100 x 15 mm|
|Glass bottom culture plates||Tool||MatTek Corp.||P35G-1.5-2.0-C||35 x 15mm, No 1.5, Uncoated, Gamma-irradiated|
|Wide Bore Glass Pasteur pipets||Tool||Fisher Scientific||63A-53-WT|
|Glass filament capillaries for trough molds (without filament) and injection needles (with filament)||Tool||World Precision Instruments, Inc.||1B150F-4||4 in. (100 mm), 1.5 / 0.84 OD/ID (mm)|
|Transplant Capillaries (TransferTip)||Tool||Eppendorf||83000122-8||Type IV, sterile, Int. 15μm; 20 degree bend|
|Transplant mold||Tool||Adaptive Science Tools||PT-1||150 triangular divots to hold individual embryos|
|Agarose, Type I||Reagent||Sigma-Aldrich||A6013-250G||1.5% agarose made in EM|
|(Antibiotic) Embryo Medium||Reagent||See Westerfield, 2007|
|Phosphate Buffer||Reagent||See Westerfield, 2007|
|Watchman Forceps||Tool||Fine Science Tools||100+mm tip (dull), 50mm tip|
|Rhodamine B Dextran||Lineage tracer dye||Molecular Probes, Life Technologies||D1824||MW 10,000 Da, lysine-fixable, red (590nm) fluorescent dye excited with green (570nm) light|
|Dissecting Microscope||Microscope||Olympus Corporation||SZX7|
|Fluorescent Stereo Microscope||Microscope||Carl Zeiss, Inc.||Lumar||Fully Automated Joystick Controlled (Sycop)|
|Automated Micromanipulator||Tool||Eppendorf||TransferMan NK2||Joystick Controlled|
|Micromanipulator||Tool||Applied Scientific Instrumentation||00-42-101-0000||MM33 Right|
|Microinjector with Back Pressure Unit||Tool||Applied Scientific Instrumentation||MPPI-2 BPU|
|Flamming/Brown Micropipet Puller||Tool||Sutter Instrument Co.||Model P97||Program: Heat 550;
Pull force 120.