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.<....

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 wit....

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