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Developmental Biology

Ploidy Manipulation of Zebrafish Embryos with Heat Shock 2 Treatment

Published: December 16th, 2016

DOI:

10.3791/54492

1Laboratory of Genetics, University of Wisconsin, 2Department of Neurobiology, University of Massachusetts Medical School, 3Interdisciplinary Biomedical Graduate Program, University of Pittsburgh School of Medicine
* These authors contributed equally

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.

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.

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 ....

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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.......

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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.......

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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.......

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This work was supported by NIH grants R21 HD068949-01 and RO1 GM065303.

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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 1000 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 8.0g NaCl, 0.4g KCl in 100ml ddH2O. Store at 4°C.
Hanks’ Solution 2 see above see above 0.358g Anhydrous Na2HPO4, 0.6g KH2PO4 in 100ml ddH2O. Store at 4°C.
Hanks’ Solution 4 see above see above 0.72g CaCl2 in 50ml ddH2O. Store at 4°C.
Hanks’ Solution 5 see above see above 1.23g MgSO4 ∙ 7H2O in 50ml 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 0.33g NaHCO3 in 10ml ddH2O. Prepare fresh the morning of the IVF procedure.
Hank's Solution (final solution) see above see above Combine 990ul of Hank’s Premix and 10ul of freshly made Solution 6 (NaHCO3 solution)

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