Dissection of Xenopus laevis Neural Crest for in vitro Explant Culture or in vivo Transplantation

Summary

This protocol describes how to dissect premigratory cranial neural crest (NC) from Xenopus laevis neurulas. These explants can be plated on fibronectin and cultured in vitro, or grafted back into host embryos. This technique allows studying the mechanisms of NC epithelium-to-mesenchyme transition, migration, and differentiation.

Abstract

The neural crest (NC) is a transient dorsal neural tube cell population that undergoes an epithelium-to-mesenchyme transition (EMT) at the end of neurulation, migrates extensively towards various organs, and differentiates into many types of derivatives (neurons, glia, cartilage and bone, pigmented and endocrine cells). In this protocol, we describe how to dissect the premigratory cranial NC from Xenopus laevis embryos, in order to study NC development in vivo and in vitro. The frog model offers many advantages to study early development; abundant batches are available, embryos develop rapidly, in vivo gain and loss of function strategies allow manipulation of gene expression prior to NC dissection in donor and/or host embryos. The NC explants can be plated on fibronectin and used for in vitro studies. They can be cultured for several days in a serum-free defined medium. We also describe how to graft NC explants back into host embryos for studying NC migration and differentiation in vivo.

Introduction

The neural crest (NC) is a transient embryonic cell population that emerges from the neural tube at the end of neurulation in vertebrate embryos. The signaling and genetic events that control NC specification start as early as gastrulation. The NC is specified at the border between the neural and non-neural ectoderm by signals from surrounding dorsal tissues. At the end of neurulation, NC cells undergo an epithelium-to-mesenchyme transition (EMT) and migrate extensively in the embryo following stereotyped routes by responding to surrounding guiding cues. Once they have reached their final destination, they differentiate into a vast array of derivatives, e.g. neurons, glia, bone, cartilage, and pigmented cells^1-5. Because of their contribution to many cell types and embryonic tissues, defects at any step of NC cells development, from induction to final differentiation, can cause congenital syndromes named neurocristopathies⁶. Experimental manipulation of the developing NC at different stages - specification, EMT, migration, and differentiation - will improve our understanding of neurocristopathies and allow design of potential therapeutic strategies.

The Xenopus laevis embryo is a model of choice to study NC development. Large numbers of embryos are easy to obtain, and external fertilization gives access to the very first steps of development. Many tools are available to experimentally manipulate X. laevis embryonic development. Gene gain-of-function and knockdown are easy to perform by microinjecting individual cells of early blastulas. Embryonic tissues can be cut for in vitro reassociation ^7-11 or back-grafting assays^12,13.

In this protocol, we describe how to dissect out premigratory cranial NC in X. laevis late neurulas, prior to migration. These explants can be cultured on fibronectin-coated plates to study migration and differentiation in controlled in vitro experimental conditions. NC explants can also be grafted into normal or manipulated host embryos to study their migration and differentiation in vivo.

Protocol

Experiments comply with National and European regulation on the protection of animals used for scientific purposes and with internationally established principles of replacement, reduction and refinement.

1. Preparation of Fibronectin-coated Dishes¹⁴

1. Pipette 500 ml of 10 mg/ml culture grade fibronectin diluted in 1x Phosphate Buffered Saline (PBS) into a standard plastic Petri dish (Ø 40 x 11 mm). Incubate at 37 °C for 1 hr. Note: if using glass dishes or glass coverslips for subsequent imaging, use a 100 mg/ml fibronectin solution.
2. Rinse three times with 1x PBS.
3. Replace PBS with 500 ml of 1x PBS/0.1% Bovine Serum Albumin (BSA). Incubate at 37 °C for 30 min.
4. Rinse three times with 1x PBS.
5. Fill up with 1x PBS and keep at 4 °C until use (usually for 24-48 hr).

2. Dissection of the NC Explants

1. Prepare 3/4 Normal Amphibian Medium (NAM) (110 mM NaCl, 2 mM KCl, 1 mM Ca(NO₃)₂, 1 mM MgSO₄, 0.1 mM EDTA, 1 mM NaHCO₃, 0.2x PBS, 50 mg/ml gentamycin^10,15). Do not keep more than 1 week for dissection, store at 4 °C. Older NAM can be used for preparing agarose-coated dissection dishes (below).
2. Prepare 60 mm Petri dishes for dissection, coated with 10 ml of 2% agarose in 3/4 NAM.
3. Collect dissection tools: plastic Pasteur pipettes, a finest insect pin mounted on pin holder (see materials table for reference), an "eyebrow knife" (made with eyebrow hair embedded in paraffin at the tip of a glass Pasteur pipette¹⁶), two fine dissection forceps, P1000 pipette and tips, a binocular stereoscope with magnification 8-40X to 50, a bright light source with optic fibers guides. All dissecting tools should be kept clean; after each experiment, rinse them twice with distilled water, once with 100% ethanol and let dry. Store away from dust.
4. Fill in the dissection dish with fresh NAM almost to the top.
5. Obtain X. laevis embryos according to standard procedures¹⁰ and age them until they reach neurula stage 16 (according to Nieuwkoop and Faber developmental table¹⁷).
6. Place stage 16 embryos into the dissection dish using the plastic Pasteur pipette.
7. Carry out all subsequent steps under the binocular scope. Remove the vitelline membrane with two pairs of forceps. Be careful not to damage the dorsal part of the embryos.
8. Wait until the embryos have reached stage 17 to start the dissection. Stage 17 is appropriate since the neural crest is clearly set apart from the neural tube, in contrast to earlier stages. After stage 17, NC starts migrating and intermingling with the surrounding mesenchyme.
9. Dig a small hole in the agarose with forceps to make a niche for the embryos in order to maintain them during the dissection. Place one embryo dorsal side up. Note: modeling clay can be used to maintain the embryos instead digging a hole into the agarose.
10. Make an incision in the lateral part of the anterior neural fold with the eyebrow knife and the insect pin. Be careful not to go too deep into the embryo: aim to cut through 2-3 cell layers (never reach as deep as the archenteron!). During the whole dissection process, never allow the dissected tissues to enter in contact with the air-liquid interface since X. laevis embryonic tissues are lysed by surface tension.
11. Make a second parallel incision in the more dorsal part of the neural fold using the same instruments.
12. If needed, turn the dish around 90°. Make a third perpendicular incision with the eyebrow knife and the insect pin, at the posterior side of the first two incisions. The NC is a thick mass of cells underneath the pigmented outer layer of the ectoderm, lateral to the neural tube.
13. Use the tip of the eyebrow knife to detach the pigmented ectoderm layer from the underlying NC, starting from the third incision edge.
14. Use the side of the eyebrow knife to detach the NC tissue starting from the third incision. The NC tissue is rather greyish, while the underlying cephalic mesoderm is white. Carefully separate the NC from the mesoderm.
15. Detach the NC tissue anteriorly from the optic vesicle, and cut at this level to free the explant. As a control for first experiments, checking snail2 expression (a canonical NC marker) in some explants by in situ hybridization is recommended.

3. Plating Explants on Fibronectin

1. Fill-in the fibronectin-coated dish with Modified Danilchick Medium (MDM, NaCl 53 mM, Na2CO₃ 5 mM, K Gluconate 4.5 mM, Na Gluconate 32 mM, MgSO₄ 1 mM, CaCl₂ 1 mM, BSA 0.1 %, adjust pH to 8.3 with 1 M Bicine¹⁸).
2. Transfer the explants into the fibronectin-coated dish using a P1000 pipette. Never allow the explants to touch the air-liquid interface. First, pipette some liquid into the tip, then the explants, then some liquid again. Do not allow any air into the tip while transferring the explants into the fibronectin-coated dish. Once the tip is inside the liquid of the fibronectin-coated dish, let the explants slowly go down by gravity, or push very gently. Avoid ejecting the explants too fast.
3. Place 10-30 explants in the dish using the eyebrow knife. Avoid the sides of the dish, which are not adequate for subsequent imaging.
4. Try not to move the dish until the explants have attached on the fibronectin (it takes about 15-30 min). If needed, move the dish very carefully.
5. Place the dish in a refrigerated incubator (between 15-20 °C, for temperature chart see¹⁷). When placed at 15 °C, cells are usually migrating efficiently and scattering after 6 hr of incubation (this timing may vary between experiments).

4. Grafting Explants into Host Embryos

1. Cut a fine glass coverslip into very small pieces using coarse forceps. The size of the pieces should be approximately 1.5 mm². Immerse the pieces into a Petri dish containing 3/4 NAM or PBS using forceps.
2. Preparation of the host embryo: dissect out the host NC in 3/4 NAM as described in 3) with special care not to damage the host embryo while removing the vitelline membrane and dissecting the NC. The tissue tension tends to increase the size of the ablation: let the host embryo heal partially while dissecting the donor embryo.
3. In another agarose-coated dish or in another part of the same dish, dissect out the NC explant from the donor embryo as described in Protocol 3. Transfer the NC graft into the dish containing the host with care, as described in Protocol 3.
4. Immediately place the NC explant onto the host ablated area using forceps. The explant should attach partially. Put a piece of glass coverslip onto the grafted embryo to maintain the graft in place. Choose a piece of glass bigger than the embryo to avoid damaging it. The embryo will be a little bit flattened.
5. Wait for at least 15-20 min, then gently remove the coverslip. Let the embryo recover for another 10 min and carefully pipette it into a clean dish filled with 3/4 NAM, using the plastic Pasteur pipette.

Results

When plated on fibronectin, the neural crest explants attach rapidly (15-30 min) and the explant spreads flat within 2 hr (Figure 1A). After 3-6 hr cells start to scatter. At 24 hr at 15 °C, many cells have started to migrate away from the explant (Figures 1B and 1C). Yolk makes cells very bright under phase contrast (Figure 1B). Cell protrusions (filopodes and lamellipodes) are clearly visible after phalloidin staining of the actin cytoskeleton (

Discussion

This protocol describes an easy technique to explant premigratory cranial NC in X. laevis embryos. The embryos used for such experiments need to be robust and heal well. Discard any batch of unhealthy embryos. In addition, grow embryos at various temperatures (from 12 to 18-20 °C), in order to excise neural crest at stage 17 and not later. After stage 17, neural crest may be mixed with cephalic mesoderm and cannot be removed completely. Xenopus tissues heal quickly and then loose their adh...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Fibronectin from bovine plasma	Sigma	F4759-1MG
Bovine Serum Albumin, Fraction V	Euromedex	04-100-811-C	Lower quality grade may  be suitable for this application
Stainless Steel Insect Pins, Size 000	FST	26001
Dumont #5 forceps 0.05 mm x 0.02 mm	FST	11252-20