Neural Explant Cultures from Xenopus laevis

Summary

Culturing neural explants from dissected Xenopus laevis embryos that express fluorescent fusion proteins allows for imaging of growth cone cytoskeletal dynamics.

Abstract

The complex process of axon guidance is largely driven by the growth cone, which is the dynamic motile structure at the tip of the growing axon. During axon outgrowth, the growth cone must integrate multiple sources of guidance cue information to modulate its cytoskeleton in order to propel the growth cone forward and accurately navigate to find its specific targets¹. How this integration occurs at the cytoskeletal level is still emerging, and examination of cytoskeletal protein and effector dynamics within the growth cone can allow the elucidation of these mechanisms. Xenopus laevis growth cones are large enough (10-30 microns in diameter) to perform high-resolution live imaging of cytoskeletal dynamics (e.g.^2-4 ) and are easy to isolate and manipulate in a lab setting compared to other vertebrates. The frog is a classic model system for developmental neurobiology studies, and important early insights into growth cone microtubule dynamics were initially found using this system^5-7 . In this method⁸, eggs are collected and fertilized in vitro, injected with RNA encoding fluorescently tagged cytoskeletal fusion proteins or other constructs to manipulate gene expression, and then allowed to develop to the neural tube stage. Neural tubes are isolated by dissection and then are cultured, and growth cones on outgrowing neurites are imaged. In this article, we describe how to perform this method, the goal of which is to culture Xenopus laevis growth cones for subsequent high-resolution image analysis. While we provide the example of +TIP fusion protein EB1-GFP, this method can be applied to any number of proteins to elucidate their behaviors within the growth cone.

Protocol

Note: We describe the steps in the first two sections only in brief, as excellent protocols with detailed information have been published elsewhere that focus more specifically on these steps (e.g.^8-12). Additionally, the general protocol of working with Xenopus spinal neurons in live cell culture has been previously published in a detailed methods article⁸. We highly recommend reviewing that article as a complement to this video, although here we do provide enough information to successfully perform and troubleshoot the neural tube dissection and plating protocol in step 3.

1. In Vitro Fertili....

Discussion

Xenopus laevis neural explants send out neurites in a very robust manner by 24 hr after plating on the laminin/poly-lysine substrate if conditions are appropriate. With this substrate, growth cones are highly motile and can achieve axon lengths of up to 1 mm, extending in all directions outward from the explant, although typical lengths are 100 μm or more. If neurites do not grow out, there are a limited number of reasons for this to be the case. One possibility is that the neural explants did not properly a.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Chorionic Gonadotropin	Argent Labs	C-HCG-ON-10
Cysteine	Sigma-Aldrich	52-90-4
mMessage mMachine kit	Ambion	AM1340
Capillary Borosil Needles 1.2 mm (OD) x 0.9 mm (ID)	FHC, Inc.	30-31-0
Ficoll	Sigma-Aldrich	F2637
Dumont #5 Biologie Inox Forceps	Fine Science Tools	11252-20
Collagenase	Sigma-Aldrich	9001-12-1
Mattek dishes	Mat Tek Corporation	P35G-1.5-14-C
L-15	Invitrogen	21083-027
Poly-l-lysine	Sigma-Aldrich	P-1399
Laminin	Sigma-Aldrich	L2020
NT3	Sigma-Aldrich	N1905
BDNF	Sigma-Aldrich	B3795