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 specific tissues, such as neuroectoderm and somites, can be grafted between embryos to produce chimeric animals^1,2,4,5,6. For nearly a century, studies of such chimeric animals have provided crucial insights into regeneration, tissue differentiation, size control, and patterning^1,7,8.

In the last the decade, numerous transcriptional studies of regenerating tissues have produced insights into the genetic programs underlying salamander regeneration^{9,10,11,12,13}. These studies have added to an expanding list of candidate genes that, to date, are largely uncharacterized in the context of regeneration. Targeted mutagenesis techniques, such as CRISPR/Cas, now permit the investigation of such genes, and such genetic approaches are greatly facilitated by the recent sequencing and assembly of the large axolotl genome^14,15,16.

We sought to develop techniques that coupled classic developmental biology with new genetic technology for the purpose of dissecting the mechanisms of regeneration. Methods for generating haploid embryos of axolotls and other salamanders have been established for decades¹⁷. While these techniques have long been noted to be advantages of salamanders as genetic model organisms¹⁸, few subsequent genetic studies have incorporated haploid animals. We use in vitro activation in the axolotl to produce haploid embryos that serve as tissue donors for grafting¹⁹. Using embryos carrying fluorescent genetic markers, we have devised reliable methods for generating limbs derived almost entirely from donor tissues (Figure 1A). By combining these two techniques, we have bypassed the late embryonic lethality associated with haploidy, allowing for the production of fully developed, grafted haploid limbs (Figure 1B, Figure 1B', and Figure 2).

By conducting CRISPR/Cas-mediated mutagenesis in haploid embryos prior to grafting to create chimeric axolotls with mutant haploid limbs, we may investigate gene function specifically within the context of limb development and regeneration. This allows the rescue of limbs from potentially embryonic-lethal mutant phenotypes. While CRISPR/Cas microinjection can generate animals that are highly mutant, such animals are typically highly mosaic, with some degree of retention of wildtype alleles and a variety of distinct mutations at targeted sites^14,20. CRISPR-based mutagenesis in haploid cells increases the penetrance of single allele loss-of-function mutations, as they cannot be masked by retained wildtype alleles. For this reason, CRISPR-based screening in haploid cell lines is increasingly used to investigate the genetic basis of many cellular processes^21,22,23. By combining CRISPR-based lineage tracing with our haploid limb bud grafting protocols, the approach described here can serve as a platform for haploid genetic screens in living animals²⁰.

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 the eggs thoroughly with 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).
4. Place the eggs in fresh 40% Holtfreter's solution and store at 12 °C.
   NOTE: Diploid embryos should always be obtained before moving on to haploid embryo generation.

2. Haploid Embryo Generation

1. Female gamete donor preparation
   1. 48 h before conducting in vitro activation, anesthetize a sexually mature white or white/RFP female axolotl by immersion in 1 g/L HEPES-buffered MS-222 in 40% Holtfreter's solution²⁵.
   2. Ensure that the animal is fully anesthetized after approximately 30 min of immersion by firmly pinching its tail between the thumb and forefinger. Fully anesthetized animals will not physically respond to any pinch.
   3. Prepare a solution of human chorionic gonadotropin (HCG) containing 10,000 U/mL in sterile saline.
   4. Using a 30 G insulin syringe, inject 0.15 CC of HCG (1,500 units) into the musculature dorsal to the hind limb of the anesthetized female at 45° angle to the midline to avoid contact with the spinal cord.
   5. Return the female to fresh 40% Holtfreter's solution and place in an 8−12 °C refrigerator.
   6. After 48 h, return the female to room temperature. Place a few stones or plastic plants in her container as surfaces for egg laying.
      NOTE: Females injected with HCG will often deposit empty jelly cases for several hours before laying eggs.
   7. Remove the stones or plastic plants after the female has begun consistently laying eggs.
   8. Allow the female to sit in the empty tank for 30 min to 1 h.
      NOTE: Withholding materials for the animal to lay eggs on will allow tighter control of oocyte collection.
2. Male gamete collection
   1. While the female's egg laying is stalled, anesthetize a sexually mature GFP+ male axolotl to serve as a sperm donor, as in step 2.1.1.
   2. Place the fully anesthetized male on his back on damp paper towels under a dissecting microscope.
   3. If the experimenter is righthanded, position the tip of a P1000 pipette at the base of the cloaca with the right hand.
   4. Place the left forefinger and thumb of the left hand 2−3 cm rostral to the pelvis. Gently squeeze the animal while moving the fingers towards the hind legs to flush out spermic urine samples.
   5. Collect each that is flushed out of the cloaca into an individual microcentrifuge tube. Repeat this process to collect 6 to 10 samples.
   6. Pipette 5.0 µL from each sample onto a petri dish to inspect the quality of the sperm using an inverted microscope.
      NOTE: Concentrated sperm samples are a milky white color, typically range from 5 to 20 µL, and are usually retrieved after several, higher volume spermic urine samples are extracted. Sperm will collect at the bottom of the tubes in higher volume samples when left undisturbed. Concentrated samples of healthy sperm are highly motile and lose activity as their concentration decreases. Healthy males can produce up to 50 µL of concentrated sperm.
3. Female gamete collection
   1. After obtaining and confirming a healthy sperm sample, anesthetize the HCG-injected female axolotl as in step 2.1.1.
   2. Place the fully anesthetized female on damp paper towels on her back.
   3. Extract unfertilized eggs from the female using a hand motion similar to that in step 2.2.4.
   4. Collect the eggs using wet forceps and transfer them to a 10 cm petri dish. Treat eggs with irradiated sperm within 15 min of collection.
4. Male gamete preparation and in vitro activation
   1. Use a P10 or P20 pipette to pipette the sperm up and down, breaking apart the clumps to form a homogenous suspension.
   2. Approximate the percentage of motile sperm by placing a 0.5 µL drop of undiluted sperm suspension on a petri dish lid or glass slide and examining with an inverted microscope.
   3. Add 9.5 µL of 0.1x Marc's modified Ringer's solution (MMR; Table of Materials) to this sample to make a 20x sperm dilution. Gently pipette up and down to mix.
   4. With an inverted microscope, count the sperm in three 1.0 µL drops of the 20x diluted aliquot on a petri dish cover or hemocytometer to estimate the sperm concentration of the undiluted suspension.
   5. Prepare the sperm for irradiation by diluting an aliquot of the original sperm sample to about 80,000 motile cells/mL in sterile 0.1x MMR.
   6. Count the number of eggs obtained within the past 15 min. Add 0.5 µL of the freshly diluted sperm from step 2.4.5 per egg to a petri dish. Use the pipette tip to spread this suspension into a 1 mm thick layer.
   7. Using a plastic lift, place the sample 4 cm from the bulbs of a 254 nm crosslinker. Genetically inactivate the sperm by irradiating the sample with 800,000 uJ/mm².
   8. Using a P10 pipette, pipette the suspension onto the unfertilized eggs, coating each egg with 0.25−0.5 µL of irradiated sperm. Allow the eggs to sit at room temperature for 30 min.
   9. After 30 min, flood the eggs with 0.1x MMR. Immediately place the eggs in an 8−10 °C incubator for injections the next day or at 18 °C for injections within 7 hours post activation (hpa).
   10. Dejelly haploids using sharp forceps 30 min after hydration. Immediately place the eggs in an 8−10 °C incubator for injections the next day or at 18 °C for injections the same day.

3. Haploid Mutagenesis and Maintenance

1. CRISPR/Cas9 microinjections
   1. Design sgRNAs using CRISPRscan and synthesize^26,27.
   2. For multiplex mutagenesis, prepare a stock of 5 sgRNAs (10 ng/µL for each sgRNA) and Cas9 protein (1 µg/µL). Prepare a 100-fold dilution of this stock (0.1 ng/µL per sgRNA, 10 ng/µL Cas9) for injection. For single gene, high mutation frequency mutagenesis, follow the protocol outlined previously²⁸.
   3. Inject haploid embryos at the single cell stage 7 hpa if stored at 18 °C or inject embryos the next day at the 2−8 cell stage if they are stored at 8−10 °C.
   4. Transfer the embryos to 1.0x MMR with 20% polysucrose 400.
   5. If single cell, inject each embryo with a 5 nL drop of the injection solution (approximately ¼ radius of the egg) containing a total mass of 0.5 pg/sgRNA and 50 pg Cas9 protein. If multicellular, distribute this mass by injecting smaller volumes into multiple cells.
   6. Allow the embryos to heal in the polysucrose 400 for a minimum of 4 h and a maximum of 18 h at 18 °C.
2. Haploid embryo housing
   1. Transfer the embryos to 0.1x MMR with antibiotic-antimycotic.
   2. House each embryo in an individual well of a 24-well plate, as some will die or develop abnormally. Maintain at 16−18 °C. Lower temperatures can be used to prolong development, if necessary.
   3. Replace the media with fresh 0.1x MMR with antibiotic-antimycotic every other day.

4. Diploid Host Embryo Preparation

1. Maintain embryos in the jelly coating at 12 to 16 °C until they reach stage 22 to 26.
2. Collect the embryos that are ready for grafting, place them within a sieve (4 mm mesh size), and gently rinse them with 40% Holtfreter's solution.
3. Transfer the embryos into filter-sterilized 0.1x MMR containing 1.5% bleach for up to 2 min. Completely submerge the embryos in the bleach solution and swirl them gently to ensure that the jelly coating makes full contact with the bleach solution to kill the microbes present on the embryos.
4. After 2 min, dilute the bleach solution containing the embryos with an equal volume of filter-sterilized 0.1x MMR.
5. Gently pour the embryos into a bleach-sanitized sieve (4 mm mesh size) and rinse the embryos five times with filter-sterilized 0.1x MMR.
6. Place the embryos into sterile 10 cm petri dishes with 0.1x MMR with antibiotics for dejellying (penicillin 100 units/mL, streptomycin 100 µg/µL, 0.25 µg/mL, gentamicin 25 µg/mL).
7. Under a fluorescent stereomicroscope, remove the jelly coats and vitelline membranes from GFP+ embryos using sharp forceps (tip dimensions 0.05 x 0.01 mm²).
8. Transfer the GFP+ embryos to a new petri dish. Minimize the amount of liquid transferred from the petri dish where they were dejellied.
9. Rinse the GFP+ host embryos with sterile 0.1x MMR with antibiotics four to six times in order to remove contaminants.
10. Place the clean embryos at 4 °C overnight before grafting.
    NOTE: Cooling the embryos makes them more rigid and clean separation of the mesoderm from the endoderm feasible.

5. Haploid-diploid Chimera Generation

1. Surgical dish and media preparation
   1. Prepare sterile surgical operating dishes by pouring autoclaved 2% agarose in 0.1x MMR into sterile 35 mm easy-grip petri dishes. Fill the petri dishes halfway with agarose.
   2. After the agarose cools, use a sterile scalpel to cut a 25 mm long, slanted trough in the agarose to hold the embryos in place.
   3. Fill the dish with sterile surgical media (0.1x MMR with anti-mycoplasma 2.5 µg/mL, amphotericin B 0.25 µg/mL, and ciprofloxacin 10.0 µg/mL) and refrigerate at 4 °C.
2. Embryo grafting procedure
   1. Place one healthy haploid donor with one or two stage-matched GFP+ diploid host embryos inside the trough of the prechilled operating dish containing surgical media (Figure 3).
      NOTE: Perform the procedure on a cooling stage (10 °C or lower) if possible.
   2. Use two ultra-fine, autoclaved forceps (straight tip, tip dimensions 0.05 x 0.02 mm²) to remove the ectoderm and mesoderm layers from the host with the limb bud near the center (Figure 4).
      NOTE: The rectangular tissue graft region encompasses the limb bud and extend from the ninth somite to the posterior half of the gill bulge, about 2 mm, along the anterior posterior axis. Along the dorsoventral axis, the grafted region spans approximately 1.5 mm, including the somites to just beyond the ventral edge of the gill bulge. The grafted region includes all lateral plate mesoderm and the lateral halves of the somites, without disturbing underlying endoderm. See Figure 4 and the accompanying video for details.
   3. Set aside the host tissue and remove an equivalently sized tissue sheet from the haploid donor using the same methods.
   4. Place the haploid donor tissue sheet onto the corresponding region of the donor embryo.
   5. Secure the tissue by covering it with an autoclaved, rectangular glass shard from a crushed microscope cover glass and gently pressing it into the host embryo body.
   6. Flip the haploid donor embryo onto its other side to harvest the limb bud for the second host embryo. Repeat steps 5.2.2 through 5.2.5.
   7. Carefully remove the remaining excess host tissues and the donor embryo from the dish.
   8. Leave the grafts with the glass shard anchors in place for 60−75 min, checking every 20 min to ensure that the glass has not slipped off.
   9. After the tissue grafts fully adhere, use the forceps to slowly peel off the glass shard anchors.
3. Chimera maintenance
   1. Transfer the engrafted embryos to fresh surgical media and maintain them at 8−12 °C overnight to heal. Individually house engrafted embryos in 12 or 24-well plates.
   2. After 36−48 h, transfer the engrafted embryos to sterile 0.1x MMR antibiotic-antimycotic. The strong antibiotics in the surgical media cause toxicity in engrafted embryos after 3 days.
   3. Replace the media with fresh 0.1x MMR and antibiotics every 2−3 days.
   4. Maintain the engrafted embryos at 18 °C until they are able to feed²⁸.
   5. After 1 to 2 months of development and care, haploid limbs can be scored for purity of the graft using a fluorescent dissection microscope.
      NOTE: The presence of non-neural or non-blood host-derived GFP-tissue in limbs is an indicator of impure grafting and is often associated with abnormal limb development. These animals should be excluded from further analysis.

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