Centre for Regenerative Medicine, Department of Biology and Biochemistry, University of Bath
Porazinski, S. R., Wang, H., Furutani-Seiki, M. Dechorionation of Medaka Embryos and Cell Transplantation for the Generation of Chimeras. J. Vis. Exp. (46), e2055, doi:10.3791/2055 (2010).
Medaka is a small egg-laying freshwater fish that allows both genetic and embryological analyses and is one of the three vertebrate model organisms in which genome-wide phenotype-driven mutant screens were carried out 1. Divergence of functional overlap of related genes between medaka and zebrafish allows identification of novel phenotypes that are unidentifiable in a single species 2, thus medaka and zebrafish are complementary for genetic dissection of the vertebrate genome functions. Manipulation of medaka embryos, such as dechorionation, mounting embryos for imaging and cell transplantation, are key procedures to work on both medaka and zebrafish in a laboratory. Cell transplantation examines cell autonomy of medaka mutations. Chimeras are generated by transplanting labeled cells from donor embryos into unlabeled recipient embryos. Donor cells can be transplanted to specific areas of the recipient embryos based on the fate maps 3 so that clones from transplanted cells can be integrated in the tissue of interest during development. Due to the hard chorion and soft embryos, manipulation of medaka embryos is more involved than in zebrafish. In this video, we show detailed procedures to manipulate medaka embryos.
1. Development of the embryos
2. Removing the chorion
The chorion of medaka consists of two protective layers with a hard inner layer and a soft outer surface. Thus, a two-step protease treatment employing pronase and hatching enzyme is necessary to remove this chorion.
Once dechorionated, embryos should be kept in 1X BSS. Semi-sterile conditions will enhance the successful culture of dechorionated embryos, especially when longer periods of observation are required. These include using sterile solutions (e.g. sterilized 1X BSS with antibiotics) and tools sterilized with 70% ethanol followed by rinsing with 1X BSS.
As dechorionated medaka embryos are softer and more fragile than dechorionated zebrafish embryos, extra care must be taken to ensure they do not contact air or bubbles in the pipette, as this will cause immediate collapse. To ensure minimal damage to embryos, a wide-mouthed heat polished glass pipette with pipette pump should be used for transferring embryos and a hair loop should be utilized to orientate embryos for observation. Non-adherent Petri dishes should be used to prevent embryos from attaching to surfaces.
Figure 1. Comparison of rolled and unrolled embryos during dechorionation. Observe how the rolled embryos in panel B lack the hairs seen on the unrolled embryos in panel A.
3. Mounting dechorionated embryos
Agarose embedding is useful for longer periods of imaging (e.g. time-lapse imaging) for live embryos as well as for detailed observations of fixed embryos. During gastrulation and early organogenesis (stage 14 to stage 28), medaka embryos exhibit waves of rhythmic contractile movements across the periderm, a tissue layer covering both the developing embryo and the yolk 5. Embryos are treated with 3.5mM 1-heptanol to stop contractile movements 6.
To stop movement of embryos after stage 28 (64hpf), embryos are anaesthetized by adding drops of Tricaine mesilate (TMS) before embedding. TMS is also added to the agarose (add only a minimal amount, this dose needs to be optimized, approximately a 1:25 dilution).
4. Cell transplantation in medaka embryos
The goal of this procedure is to determine whether the gene of interest acts cell-autonomously (within a cell) or non cell-autonomously (between cells).
Donor cells can be labeled with a tracer dye such as rhodamine-dextran prior to transplantation (as outlined in the accompanying JoVE protocol 'Microinjection Of Medaka Embryos') or a transgenic strain with GFP expression may be utilized, allowing transplantation assessment. A combination of both labeling techniques is often useful to overcome auto-fluorescence that can be encountered. For time-lapse studies following transplantation, GFP expression is particularly useful.
Use a wide-mouthed glass pipette with pipette pump throughout this procedure and sterilize all tools (including slides) beforehand with 70% EtOH followed by thorough rinsing with sterile 1X BSS. Recipient embryos are usually developed to around stage 12 as this allows discrimination of the ventral and dorsal poles when carrying out transplantation.
If necessary, genotype donor embryo(s) by transferring to PCR tube(s) containing 25μl of 20mg/ml proteinase K. Incubate at 55°C for 4 hours followed by 10 minutes at 94°C and carry out PCR.
5. Representative results
Figure 2. Example position of an agarose-embedded embryo. Anterior is shown to the right and view is dorsal. Panel B: endothelial cells can be seen on either side of the embryo body and are labeled using GFP driven by the fli promoter.
Figure 3. Images of post-transplantation embryos. Image A shows a donor and recipient embryo immediately post-transplantation. The donor embryo is shown on the right with a completely red blastoderm (arrow). The recipient embryo is shown on the left and is easily identified by the small mass of red transplanted cells visible in the blastoderm (arrowhead). Image B shows two recipient embryos approximately 7 hours post-transplantation (incubated at 27°C). Notice the transplanted cells have migrated from the site of transplantation and are now dispersed across the blastoderm (arrows). Image C shows a recipient embryo at st.29 (approximately 74hpf). Note the pigmentation in the eye and the presence of melanophores in the trunk and brain region (arrows). The rhodamine-labelled cells can be seen to be colonising many of the embryonic structures indicating the successful production of a chimera.
In this video we demonstrate how to dechorionate and carry out cell transplanataion on medaka embryos. This is a powerful technique to produce chimeric embryos for the study of gene/protein function during development, as well as for elucidating the autonomy of the gene/protein in question. Medaka are a useful vertebrate model system for this technique due to well established fate maps and their transparency lending them well to in vivo real-time imaging. Transplantation can be combined with microinjection (as outlined in the accompanying JoVE protocol 'Microinjection Of Medaka Embryos') so that the behaviour of transplanted cells can be easily observed.
No conflicts of interest declared.
This work is supported by a grant from the MRC to M. F-S.
|Embryo medium||Reagent||Made in-house||N/A||200ml 50X stock solution, 1ml 1%methyleneblue in H2O/10 liter RO water (50X stock solution: NaCl 14.7g, KCl 0.6g, CaCl2 2H2O 2.4g, MgSO4 7H2O 4.0g/1 litre RO water.)|
|Micro-dissecting forceps||Tools||55 INOX A.DUMONT&FILS||N/A||Very sharp fine tips for removal of chorion.|
|Fine waterproof sandpaper - p2000 grit size||Tools||Hermes Abrasives Ltd.||N/A||Very fine grit size for rolling/cleaning embryos with chorions.|
|3% methylcellulose||Reagent||Sigma||M 0512||High viscosity for holding embryos in place during transplantation.|
|1X BSS||Reagent||Made in-house||N/A||20XBSS: 130g NaCl, 8g KCl, 4g MgSO4.7H2O, 4g CaCl2.2H2O, and 10mg Phenol Red in 1L MilliQ water and autoclave; 500mM Hepes in MilliQ water autoclaved. Add 25ml 20XBSS and 15ml 500ml Hepes pH7.0, fill up to 500ml and filter sterilize before use.|
|Penicillin-streptomycin||Reagent||Gibco||15140-122||Added to BSS when incubating dechorionated embryos.|
|Hatching enzyme||Reagent||Made in-house||N/A||For dechorionation.|
|Tricaine mesilate (TMS)||Reagent||Sigma||A-5040||400mg tricaine in 97.9ml distilled water and 2.1ml 1M Tris, pH adjusted to 7; 4.2ml of this solution in 100ml clean tank water.|
|3% ultra-low gelling temp. agarose||Reagent||Sigma||A-2576||Type IX-A. Used during embedding of embryos for observation.|
|Petri dish culture chamber||Tool||Iwaki, Asahi Techno Glass Corp.||11-004-008||Iwaki glass base dish 35mm with 12mm window. Used during embedding of embryos for observation.|
|Micromanipulator||Equipment||Narishige||N/A||Model MN151. For removal and insertion of cells during transplantation.|
|Borosilicate glass capillaries||Tool||Harvard Apparatus||GC100-10T||For making transplantation needles.|
|Flaming/Brown micropipette puller||Equipment||Sutter Instrument Co.||Model P-97||For making transplantation needles.|