Generation of Chimeric Axolotls with Mutant Haploid Limbs Through Embryonic Grafting

Summary

This goal of this protocol is to produce chimeric axolotls with haploid forelimbs derived from Cas9-mutagenized donor tissue using embryonic tissue grafting techniques.

Abstract

A growing set of genetic techniques and resources enable researchers to probe the molecular origins of the ability of some species of salamanders, such as axolotls, to regenerate entire limbs as adults. Here, we outline techniques used to generate chimeric axolotls with Cas9-mutagenized haploid forelimbs that can be used for exploring gene function and the fidelity of limb regeneration. We combine several embryological and genetic techniques, including haploid generation via in vitro activation, CRISPR/Cas9 mutagenesis, and tissue grafting into one protocol to produce a unique system for haploid genetic screening in a model organism of regeneration. This strategy reduces the number of animals, space, and time required for the functional analysis of genes in limb regeneration. This also permits the investigation of regeneration-specific functions of genes that may be required for other essential processes, such as organogenesis, tissue morphogenesis, and other essential embryonic processes. The method described here is a unique platform for conducting haploid genetic screening in a vertebrate model system.

Introduction

Historically, embryonic tissue grafting in amphibians has been an important technique for exploring fundamental mechanisms of developmental biology and regeneration. The axolotl, a species of salamander, possesses an impressive ability to regenerate tissues and complex structures such as limbs and organs after injury or amputation. Similarly impressively, they can receive, without rejection, tissue grafts from other individuals at embryonic, juvenile, and adult stages^1,2,3. Regions of embryos that produce whole structures such as limbs, tails, eyes, and heads, and more specif....

Protocol

Experimental procedures used in this protocol were approved by the Yale University Institutional Animal Care and Use Committee (IACUC, 2017–10557) and were in accordance with all federal policies and guidelines governing the use of vertebrate animals. All animal experiments were carried out on Ambystoma mexicanum (axolotls) in facilities at Yale University.

1. Diploid Embryo Generation

1. Obtain GFP+ diploid embryos to serve as graft hosts through natural mating using one or two gfp parents²⁴.
2. Collect freshly laid diploid eggs and place them in a metal sieve.
3. Rinse....

Results

Developing haploid embryos can be distinguished from diploid embryos by their 'haploid syndrome' phenotype²⁹. At graft-stage, haploid embryos exhibit reduced curvature along the anterior-posterior axis and incomplete enclosure of the yolk plug (Figure 3A). A fluorescent microscope can be used to ensure that haploid embryos are free of paternally derived GFP expression (Figure 3B

Discussion

There are a few critical steps in our protocol for generating haploid-diploid chimeras that the operating technician should consider for consistent grafting results.

The most likely reason for haploid generation to fail is due to poor in vitro activation conditions. The proper quantities of motile sperm must be used to activate eggs. To prolong motility, sperm samples should always be maintained at 4 °C. Before applying any sperm sample to eggs, check the viability of the sperm using an i.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
#55 Dumont Forceps	Fine Science Tools	11295-1	Only use Dumostar material (can be autoclaved)
Amphotericin B	Sigma Aldrich	A2942-20ML	20 mL
Antibiotic-Antimycotic 100x	Thermo Fisher	15240062
Ciprofloxacin	Sigma Aldrich	17850-5G-F
Ficoll 400 (polysucrose 400)	bioworld	40600032-3	Ficoll 400
Gentamicin	Sigma Aldrich	G1914-250MG
Heating/Cooling Incubator	RevSci	RS-IF-233
Human Chorionic Gonadotropin	Merk		Chorulon
Megascript T7 Transcription Kit	Thermo Fisher	AM1334	40 reactions
Miroscope Cooling Stage	Brook Industries	Custom	Custom
NLS Cas9 Protein	PNAbio	CP01-200	4 vials of 50 µg protein each
Plasmocin	Invivogen	ant-mpt-1	Treatment level
Recipes
1.0x Marc's modified Ringer's solution (MMR)			0.1 M NaCl, 2 mM KCl, 1 mM MgSO4, 2 mM CaCl2, 0.1 mM EDTA, 5 mM HEPES (pH 7.8), ph 7.4
40% Holtfreter's solution			20 mM NaCl, 0.2 mM KCl, 0.8 mM NaHCO3, 0.2 mM CaCl2, 4 mM MgSO4, pH to 7.4