Surgical Size Reduction of Zebrafish for the Study of Embryonic Pattern Scaling

Summary

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.

Abstract

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. 

Introduction

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 conditions^1,2,3. 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 injection^7,8, 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 applied^4,10,11.

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 embryos^12,13. 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.

Protocol

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 Make a wire loop to chop embryos Take 20 cm of stainless steel wire that is stiff and non-corrosive with a diameter of 40 μm. Loop the wire through into glass capillary (1.0 mm outer diameter, 0.5 mm inner diameter, no filament), making a small loop at t…

Representative Results

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 compare…

Discussion

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 margin^18,…

Materials

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.