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.
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,…
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. |