Here, we describe a method for reducing the size of zebrafish embryos without disrupting normal developmental processes. This technique enables the study of pattern scaling and developmental robustness against size change.
In the developmental process, embryos exhibit a remarkable ability to match their body pattern to their body size; their body proportion is maintained even in embryos that are larger or smaller, within certain limits. Although this phenomenon of scaling has attracted attention for over a century, understanding the underlying mechanisms has been limited, owing in part to a lack of quantitative description of developmental dynamics in embryos of varied sizes. To overcome this limitation, we developed a new technique to surgically reduce the size of zebrafish embryos, which have great advantages for in vivo live imaging. We demonstrate that after balanced removal of cells and yolk at the blastula stage in separate steps, embryos can quickly recover under the right conditions and develop into smaller but otherwise normal embryos. Since this technique does not require special equipment, it is easily adaptable, and can be used to study a wide range of scaling problems, including robustness of morphogen mediated patterning.
Scientists have long known that embryos have a remarkable ability to form constant body proportions although embryo size can vary greatly both under natural and experimental conditions1,2,3. Despite decades of theoretical and experimental studies, this robustness to size variation, termed scaling, and its underlying mechanisms remain unknown in many tissues and organs. In order to directly capture the dynamics of the developing system, we established a reproducible and simple size reduction technique in zebrafish4, which has the great advantage in in vivo live imaging5.
Zebrafish has served as a model vertebrate animal to study multiple disciplines of biology, including developmental biology. In particular, zebrafish is ideal for in vivo live imaging6 because 1) development can proceed normally outside the mother and the egg shell, and 2) the embryos are transparent. In addition, the embryos can withstand some temperature and environmental fluctuations, which allows them to be studied in laboratory conditions. Also, in addition to conventional gene expression perturbation by morpholino and mRNA injection7,8, recent advances in CRISPR/Cas9 technology has made reverse genetics in zebrafish highly efficient9. Furthermore, many classical techniques in embryology, such as cell transplantation or tissue surgery can be applied4,10,11.
Size reduction techniques were originally developed in amphibian and other non-vertebrate animals12. For example, in Xenopus laevis, another popular vertebrate animal model, bisection along the animal-vegetal axis at blastula stage can produce size-reduced embryos12,13. However, in our hands this one-step approach results in dorsalized or ventralized embryos in zebrafish, presumably because dorsal determinants are distributed unevenly and one cannot know their localization from the morphology of embryos. Here we demonstrate an alternative two-step chopping technique for zebrafish that produces normally developing but smaller embryos. With this technique, cells are first removed from the animal pole, a region of naïve cells lacking in organizer activity. To balance the amount of yolk and cells, which is important for epiboly and subsequent morphogenesis, yolk is then removed. Here, we detail this protocol and provide two examples of size invariance in pattern formation; somite formation and ventral neural tube patterning. Combined with quantitative imaging, we utilized the size reduction technique to examine the how the sizes of somites and neural tube are affected in size reduced embryos.
All fish-related procedures were carried out with the approval of the Institutional Animal Care and Use Committee (IACUC) at Harvard Medical School.
1. Tool and Reagent Preparation
2. Preparation of Zebrafish Embryos for Surgical Size Reduction
3. Surgical Size Reduction and Recovery
4. Live Imaging of Zebrafish Somitogenesis
5. Imaging of Neural Tube Patterning
Yolk volume reduction is important for normal morphology
As recently described in Almuedo-Castillo et al.17, size reduction of embryos can be achieved without reducing yolk volume. To compare with and without yolk volume reduction, we performed both two-step chopping (both blastula and yolk) and blastula-only chopping (Figure 2 and Supplemental Movie 1). Two-step chopped embryos showed seemingly normal overall morphology compared to the control (dechorionation only) embryos, other than size difference, throughout the developmental stages (see top and middle panels in Figure 2). On the other hand, blastula-only chopped embryos showed a peculiar morphology, especially at earlier stages. During epiboly, the embryos had a constricted and indented appearance (See bottom panel for 70% epiboly in Figure 2). At the following somite stage, midline structures were found to be flattened (i.e. DV length is relatively shorter than ML length) at many axial levels (See bottom panels for 8 and 20 somite in Figure 2). At later stages, the body structures adjacent to the yolk, such as the mid- and hindbrain, and the first ~10 somites, still showed a relatively flattened shape, possibly due to increased tension from the relatively larger yolk.
Somite size reduction in size reduced embryos
Somites are segmental structures that appear transiently during embryogenesis and give rise to vertebrae and skeletal muscle. From presomitic mesoderm (PSM), somites are formed one by one from the anterior to posterior direction in a periodic manner (e.g. 25 min for zebrafish, 2 h for mice) (Figure 3A). We performed time lapse imaging of somite formation both for control and chopped embryos and measured the size of most newly formed somites (Figure 3B). In both control and chopped embryos, the sizes of somites that were formed at later stages were found to be smaller compared to the ones from earlier stages. Also, throughout the somite formation stages, chopped embryos had smaller somites than the ones in control embryos (Figure 3C).
Neural tube heights are reduced following size reduction
To see the effect of embryo size reduction on neural tube size, we performed our two-step chopping technique on mem-mCherry injected embryos and imaged their spinal cords at 20 hpf using our confocal imaging system (Figure 4A,B). In this dataset, neural tube heights were reduced following size reduction by 12.4% ± 3.2%, as measured manually using custom image analysis code (Figure 4C). Taken together, these data show that size reduction reduces neural tube height. This technique can be used to measure the effects of size reduction on neural patterning.
Figure 1: Size reduction technique. Approximately 30%-40% of the cells were cut from the animal pole (top panels). The membrane surrounding the yolk was carefully wounded so that the yolk oozed out (middle panels). For the following few minutes, yolk oozed out and then the wounds on both blastoderm and yolk healed up (bottom panels). Scale bar = 200 μm. Please click here to view a larger version of this figure.
Figure 2: Comparison between two methods of size reduction. Control embryos (top panels, top embryos for 24 hpf and 30 hpf), size reduced embryos with two-step chopping (blastula and yolk, middle panels, middle embryos for 24 hpf and 30 hpf) and size reduced embryos with blastula-only chopping (bottom panels, bottom embryos for 24 hpf and 30 hpf) are compared along developmental stages. Note that in blastula-only chopped embryos, the blastoderm volume is much smaller compared to the yolk (at 70% epiboly). As a result, the embryo has a disproportionately flattened shape at somite stages (i.e., DV axis is relatively shorter compared to AP axis in blastula-only chopped embryos, compared to the control or a two-step chopped one). Scale ba = 200 μm. Please click here to view a larger version of this figure.
Figure 3: Size reduction reduces the length of somites. (A) Schematic illustration of somite formation. (B) Bright field images of control and chopped embryos over time. Yellow arrowheads indicate the most newly formed somite at each somite stage. (C) Somite length (in anterior-posterior axis) measurements over time for both control and chopped embryos. Error bars represent standard deviation. Please click here to view a larger version of this figure.
Figure 4: Size reduction reduces the height of the neural tube. (A-B) Example images of normal sized (A) and size reduced (B) tg(ptch2:kaede) embryos which were injected at the single cell stage with mem-mCherry mRNA. Scale bar = 20 μm. (C) Neural tube heights extracted from manual segmentation of the neural tube in each z-stack. Statistically significant differences are observed in average neural height when values are compared using an unpaired t-test (p = 0.0397). Please click here to view a larger version of this figure.
Supplemental Movie 1: Comparison between two-step chopping versus blastula-only chopping. Top row = control embryos, middle row = size reduced embryos with two step chopping, bottom row = size reduced embryos with blastula only chopping. Movies were taken every 3 min for 12 h. Scale bar = 1 mm. Please click here to view this video. (Right-click to download.)
Historically, among vertebrate animals, size reduction has been mainly performed using amphibian embryos, by bisecting the embryos along animal-vegetal axis at a blastula stage12. However, there are mainly two differences between frog and zebrafish embryos when we bisect embryos. First, at the stage when zebrafish embryos become tolerant of bisecting (blastula stage), the organizer is located in a restricted area of blastula margin18,19,20,21. Because one cannot tell the position of the organizer from the morphology of embryos, randomly cutting the embryo along the animal-vegetal axis produces dorsalized or ventralized embryos. Second, unlike frog embryos, zebrafish embryos go through a process called epiboly, where cells move towards the vegetal pole around a separated yolk until it is completely surrounded by cells. If only a portion of blastoderm is removed, fewer cells remain to engulf a yolk of relatively larger volume, and as a result, morphology appears affected after epiboly. Therefore, we employ two-step chopping in which we chop blastulae near the animal pole, to avoid cutting off the organizer, and wound the yolk membrane, to make the yolk size proportional to the blastula.
In addition to two-step chopping, we found the medium in which the size reduction surgery is performed is critical for recovery of embryos following the surgery. Among several media we tried (Egg water, Egg water + Albumin, Danieau buffer, L15, L15 + FBS, ⅓x Ringer, 1x Ringer), only ⅓x Ringer and 1x Ringer yielded high survival rates; in other media, embryos failed to recover from the wounds.
An important troubleshooting tip for low survival rate is to use healthy embryos from healthy and young parental fish. We noted that even when the control non-size reduced embryos show almost 100% survival rate, when size reduced, embryos from older fish tend to show lower survival rate. Also, note that the survival rate tends to decrease when the size reduction is combined with additional perturbations, such as morpholino injection.
The simplicity of the size reduction technique described here allows researchers to apply this technique without specialized equipment or intensive training. Further, since the size reduced embryos remain smaller until later stages of development (once they start eating, their size seems to catch up with the control fish), this technique can be applied to study scaling of many tissues and organs. Therefore, this technique makes it possible to combine size reduction and quantitative in vivo live imaging to study scaling and size control of various systems.
The authors have nothing to disclose.
The work was supported by the PRESTO program of the Japan Science and Technology Agency (JPMJPR11AA) and a National Institutes of Health grant (R01GM107733).
60 mm PYREX Petri dish | CORNING | 3160-60 | |
Agarose | affymetrix | 75817 | For making a mount for live imaging |
Agarose, low gelling temperature Type VII-A | SIGMA-ALDRICH | A0701-25G | |
CaCl2 | EMD | CX0130-1 | For 1/3 Ringer's solution |
CaSO4 | For egg water | ||
Cover slip (25 mm x 25 mm, Thickness 1) | CORNING | 2845-25 | |
Disposable Spatula | VWR | 80081-188 | |
Foam board | ELMER'S | 951300 | For microscope incubator |
Forcept (No 55) | FST | 11255-20 | |
Glass pipette | VWR | 14673-043 | |
HEPES | SIGMA Life Science | H4034 | For 1/3 Ringer's solution |
INCUKIT XL for Cabinet Incubators | INCUBATOR Warehouse.com | For microscope incubator | |
Instant sea salt | Instant Ocean | 138510 | For egg water |
KCl | SIGMA-ALDRICH | P4504 | For 1/3 Ringer's solution |
Methyl cellulose | SIGMA-ALDRICH | M0387-100G | |
NaCl | SIGMA-ALDRICH | S7653 | For 1/3 Ringer's solution |
Petri dish | Falcon | 351029 | For making a mount for live imaging |
Phenol red | SIGMA Life Science | P0290 | |
Pipette pump | BEL-ART PRODUCTS | F37898 | |
Pronase | EMD Millipore Corp | 53702-250KU | |
Tricaine-S (MS222) | WESTERN CHEMICAL INC | NC0135573 | |
Ultra thin bright annealed 316L dia. 0.035 mm Stainless Steel Weaving Wires | Sandra | The wire we used was obtained ~20 years ago and we could not find exactly the same one. This product has the same material and diameter as the one we use. |