Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons

Summary

This assay assesses the ability of a signaling molecule, here Bone Morphogenetic Protein 7 (BMP7), to reorient commissural axons. An explant of embryonic dorsal spinal cord is cultured adjacent to an aggregate of COS cells secreting the candidate growth factors. Reoriented commissural axons growing within the explant are visualized by immunohistochemistry.

Abstract

Dorsal commissural axons in the vertebrate spinal cord¹ have been an invaluable model system in which to identify axon guidance signals. Here, we describe an in vitro assay, "the reorientation assay", that has been used extensively to study the effect of extrinsic and intrinsic signals on the orientation of commissural axons². This assay was developed by numerous people in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden (see acknowledgements for more details) and versions of this assay were used to demonstrate the reorientation activities of key axon guidance molecules, including the BMP chemorepellent in the roof plate^3,4 and the chemoattractive activities of Netrin1⁵ and Sonic Hedgehog (Shh)⁶ in the floor plate in the spinal cord.

Explants comprising 2-3 segments of the dorsal two-thirds of spinal cord are dissected from embryonic day (E) 11 rats and cultured in three dimensional collagen gels⁷. E11 dorsal spinal explants contain newly born commissural neurons, which can be identified by their axonal expression of the glycoprotein, Tag1⁸. Over the course of 30-40 hours in culture, the commissural axon trajectory is recapitulated in these dorsal explants with a time course similar to that seen in vivo. This axonal trajectory can be challenged by placing either test tissues or a COS cell aggregate expressing a candidate signaling molecule in contact with one of the lateral edges of the dorsal explant. Commissural axons extending in the vicinity of the appended tissue will grow under the influence of both the endogenous roof plate and signals from the ectopic lateral tissue. The degree to which commissural axons are reoriented under these circumstances can be quantified. Using this assay, it is possible both to examine the sufficiency of a particular signal to reorient commissural axons^3,4 as well the necessity for this signal to direct the commissural trajectory⁹.

Protocol

Part 1: Preparation of Aggregates of Transfected COS Cells using Hanging Drops

1. Seed COS-7 (COS) cells in 35mm culture dishes. When they reach 80% confluency, transfect 1μg of the expression plasmid into the cells using Lipofectamine2000 according to the manufacturer s protocol.
2. To prepare hanging drops, aspirate the transfection medium and rinse the transfected COS cells with 1ml of 1x Phosphate Buffered Solution (PBS). Treat cells with 0.5ml of enzyme-free cell dissociation medium for 15 minutes. Add 1ml of a solution of Opti-MEM + 1x Penicillin/Streptomycin/Glutamine (P/S/G) + 10% Fetal Bovine Serum (FBS) to stop the reaction.

Discussion

The critical factors that determine success in performing this assay are first, the tissue should not be treated with dispase for too prolonged a period, such treatment will result in the tissue becoming very sticky and having decreased viability. Two, the collagen must be perfectly primed and kept on ice as much as possible. It will become increasingly difficult to handle if it starts to set, i.e. turn pink. Three, the tungsten needle must always be kept very sharp.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Type 1 rat tail collagen	BD Biosciences	354236
COS-7 cells	ATCC	CRL-1651
10x Minimal Essential Medium	Invitrogen	11430-030
Opti-MEM	Invitrogen	51985-034
L15	Invitrogen	11415-064
Lipofectamine 2000	Invitrogen	1166-8019
Fetal bovine serum	Mediatech, Inc.	35-016-CV
Cell dissociation solution	EMD Millipore	S-004-C
Trypsin 0.25% EDTA	Invitrogen	2520-0056
Sodium Bicarbonate	Sigma-Aldrich	S6297
Triton X-100	Sigma-Aldrich	X100
Pen/Strep/Glutamate	Invitrogen	10378-016
Paraformaldhyde	Baker/VWR	JTS898-7
Dispase	Roche Group	10241750001
mouse anti Tag1 IgM (4D7)	Developmental Studies Hybridoma Bank
mouse anti Myc IgG (9E10)	Santa Cruz Biotechnology, Inc.	sc-40
goat anti mouse IgM FITC	Jackson	115-095-075
goat anti mouse Fcy Cy3	Jackson	115-165-071
Goat serum	Invitrogen	16210064
Vectashield	Vector Laboratories	H-1000
4 well Nunclon dish	Nalge Nunc international	62407-068
3 well depression slide	VWR international	48339-009
48 well dish	Falcon BD	62406-195
Aspirator tuve assembly	FHC, Inc.	30-32-0
#55 Dumont forceps	Fine Science Tools	11252-20
#5 Dumont forcept	Fine Science Tools	11255-20
Needle Holder	Fine Science Tools	26016-12