Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Summary

A modified protocol for ploidy manipulation uses a heat shock to induce a one-cycle stall in cytokinesis in the early embryo. This protocol is demonstrated in the zebrafish but may be applicable to other species.

Abstract

Manipulation of ploidy allows for useful transformations, such as diploids to tetraploids, or haploids to diploids. In the zebrafish Danio rerio, specifically the generation of homozygous gynogenetic diploids is useful in genetic analysis because it allows the direct production of homozygotes from a single heterozygous mother. This article describes a modified protocol for ploidy duplication based on a heat pulse during the first cell cycle, Heat Shock 2 (HS2). Through inhibition of centriole duplication, this method results in a precise cell division stall during the second cell cycle. The precise one-cycle division stall, coupled to unaffected DNA duplication, results in whole genome duplication. Protocols associated with this method include egg and sperm collection, UV treatment of sperm, in vitro fertilization and heat pulse to cause a one-cell cycle division delay and ploidy duplication. A modified version of this protocol could be applied to induce ploidy changes in other animal species.

Introduction

This protocol allows the manipulation of ploidy in zebrafish embryos, such as in the generation of homozygous gynogenetic diploids from gynogenetic haploids (Figure 1) or the production of tetraploids. This is achieved by inducing a delay in cytokinesis corresponding to precisely one cell cycle (Figure 2A, 2B). The key one-cycle delay in cytokinesis is achieved by treatment with heat shock. The standard protocol of Heat Shock (HS) as originally described by Streisinger and colleagues involved a temperature pulse during the period 13-15 mpf, resulting in a one-cycle cell division stall during the first cell cycle ^....

Protocol

All animal experiments were conducted according to University of Wisconsin – Madison and Institutional Animal Care and Use Committee (IACUC) guidelines (University of Wisconsin – Madison Assurance number A3368-01).

1. Selecting Females for Egg Collection via Interrupted Mating

NOTE: IVF-based protocols rely on the extrusion of mature eggs from females through manual pressure ⁹. Previous protocols have used females directly from tanks or in pair matings without the females undergoing egg release behavior, but only a small fraction of these females (about 20% or less, depending on the zebrafish line) yield extruded and competent eggs u....

Results

In spite of the one-cell cycle cytokinesis stall, DNA replication occurs normally in such embryos, resulting in the duplication of the DNA content of the embryo (Figure 1). The Streisinger Heat Shock protocol (standard HS) involves a heat pulse during the period 13-15 minutes post fertilization (mpf) and induces primarily cytokinesis arrest during the first embryonic cell division at 35 mpf ^1,2, whereas the derived method described here, referred to as Heat Sho.......

Discussion

Critical steps

It is critical to work under conditions of effective in vitro fertilization. To insure a good supply of mature eggs (step 1), females set up for mating should not have been set up in mating crosses for at least 5 days and should appear gravid. During interruption of breeding, an observer can monitor 15-30 tanks adequately for the first appearance of natural egg extrusion. Interruption of mating should occur as soon as possible when the first eggs are released through natural mating.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Zebrafish mating boxes	Aqua Schwarz	SpawningBox1
NaCl	Sigma	S5886
KCl	Sigma	P5405
Na2HPO4	Sigma	S3264
KH2PO4	Sigma	P9791
CaCl2	Sigma	C7902
MgSO4-7H2O	Sigma	63138
NaHCO3	Sigma	S5761
Tricaine	Western Chemical	Tricaine-D (MS 222)	FDA approved (ANADA 200-226)
Tris base	Sigma	77-86-1	to prepare 1 M Tris pH 9.0
HCl	Sigma	920-1	to prepare 1 M Tris pH 9.0
Fish net (fine mesh) (4-5 in)	PennPlax	(ThatFishThatPlace # 212370)	available in ThatFishThatPlace
Plastic spoon			available in most standard stores
Dissecting scissors	Fine Science Tools	14091-09
Dissecting forceps	Dumont	SS	available from Fine Science Tools
Dissecting stereoscope (with transmitted light source)	Nikon	SMZ645	or equivalent
Reflective light source (LED arms)	Fostec	KL1600 LED	or equivalent
Petri plates 10 cm diameter			any maker
Eppendorf tubes 1.5 ml			any maker
Ice bucket			any maker
Pipetteman P-1000			any maker
Pipette tips 1,000 µl			any maker
Narrow spatula	Fisher	14-374
Depression glass plate	Corning Inc	722085 (Fisher cat. No 13-748B)	available from Fisher Scientific
UV lamp	UVP	Model XX-15 (cat No. UVP18006201)	available from Fisher Scientific. Although not observed by us with this model, some UV sources have been observed to experience a decrease of intensity over time (if this is the case, see Modifications and Troubleshooting)
UV glasses			any maker
Paper towels			any maker
Kimwipes	Kimberly-Clark	06-666-11	available from Fisher Scientific
Timer stop watch			any maker
Wash bottle	Thermo Scientific	24020500	available from Fisher Scientific
Tea strainer			available in kitchen stores
beakers, 250 ml (2)	Corning Inc.	1000250	available from Fisher Scientific
water bath (2)			any maker, with accurary to 0.1 C (e.g. Shel Lab H2O Bath Series)
Hanks’ Solution 1	see above	see above	8.0 g NaCl, 0.4 g KCl in 100 ml ddH2O. Store at 4°C.
Hanks’ Solution 2	see above	see above	0.358 g Anhydrous Na2HPO4, 0.6 g KH2PO4 in 100 ml ddH2O. Store at 4 °C.
Hanks’ Solution 4	see above	see above	0.72 g CaCl2 in 50 ml ddH2O. Store at 4 °C.
Hank's Premix	see above	see above	add, in the following order: 10.0 ml Solution 1;  1.0 ml Solution 2;  1.0 ml Solution 4;  86.0 ml ddH2O;  1.0 ml Solution 5. Store at 4 °C
Hanks’ Solution 6	see above	see above	0.33 g NaHCO3 in 10 ml ddH2O. Prepare fresh the morning of the IVF procedure.
Hank's Solution (final solution)	see above	see above	Combine 990 μl of Hank’s Premix and 10 μl of freshly made Solution 6 (NaHCO3 solution)