Culture of Isolated Floor Plate Tissue and Production of Conditioned Medium to Assess Functional Properties of Floor Plate-released Signals

Summary

The floor plate is a crucial structure of the developing spinal cord, providing diffusible signals to progenitors and differentiating neurons. We describe a method to produce floor-plate conditioned medium, to apply it to fresh spinal cord tissue, and to assess the consequences on proteins of interest by biochemistry.

Abstract

During development, progenitors and post-mitotic neurons receive signals from adjacent territories that regulate their fate. The floor-plate is a group of glial cells lining the ependymal canal at ventral position. The floor-plate expresses key morphogens contributing to the patterning of cell lineages in the spinal cord. At later developmental stages, the floor-plate regulates the navigation of axons in the spinal cord, acting as a barrier to prevent the crossing of ipsilateral axons and controlling midline crossing by commissural axons¹. These functions are achieved through the secretion of various guidance cues. Some of these cues act as attractants and repellents for the growing axons while others regulate guidance receptors and downstream signaling to modulate the sensitivity of the axons to the local guidance cues^2,3. Here we describe a method that allows investigating the properties of floor-plate derived signals in a variety of developmental contexts, based on the production of Floor-Plate conditioned medium (FP^cm)^4-6. We then exemplify the use of this FP^cm in the context of axon guidance. First, the spinal cord is isolated from mouse embryo at E12.5 and the floor-plate is dissected out and cultivated in a plasma-thrombin matrix (Figure 1). Second two days later, commissural tissue are dissected out from E12.5 embryos, triturated and exposed to the FP^cm. Third, the tissue are processed for Western blot analysis of commissural markers.

Introduction

The floor-plate is a well-known patterning center of the developing spinal cord, playing key roles in the specification of progenitors and postmitotic cell lineages and controlling axon navigation^7,8. The experimental procedure described here to produce FP^cm allows investigators to assess the functional properties of floor plate-derived signals in a various contexts of developmental processes, from cell patterning and survival to cell and axon migration.

To illustrate the use of such FP^cm, dorsal spinal cord tissues containing commissural neurons are dissected, dilacerated and stimulated with the FP^cm. The tissues can then be processed for Western blot analysis. This allows investigating regulations of the axon guidance machinery by floor-plate released signals. The method of treatment of dilacerated fresh tissue holds the great advantage of preserving the microenvironment of cells within the tissue. Thus the consequences of the treatment by FP^cm or any types of treatment are assessed in a more physiological way than in cell and tissue culture conditions.

Protocol

DAY 1 1. Dissection of the Spinal Cord Floor Plate (FP) from E12.5 Mouse Embryos Note: The entire procedure requires the use of sterile conditions. It is preferable to perform the dissection under a dissection hood to avoid contamination. The surface of the hood should be cleaned with ethanol. All dissection instruments must be sterilized and kept in a sterile Petri dish. The liquids (medium, dissection medium) must be kept closed and put in an ice …

Representative Results

Commissural tissues were treated by the FPcm and the samples were processed for analysis of receptor levels in Western blot. Application of the FPcm was shown to increase the levels of a guidance receptor, PlexinA1, which mediates the sensitivity of commissural axons to the midline repellent Semaphorin3B after crossing (Figure 2). This revealed that signals released by the FP regulate PlexinA1 levels4,6. <i…

Discussion

The production of floor-plate conditioned medium provides an efficient and easy way for assessing the biological properties of floor plate released signals.

The plasma-thrombin matrix provides excellent condition for tissue survival. Nevertheless, this enriched environment might be a limitation for some types of experiments. Thus, the floor plate tissue can also be cultivated in agarose matrix. The quality of the floor plate conditioned medium can be assessed by the detection of known floor-pl…

Materials

Name of Reagent/Material	Company	Catalog Number	Comments
			REAGENTS REQUIRED
PBS	Invitrogen	14190-094
HBSS, Ca2+/Mg2+– free	Invitrogen	14170-088
Glucose	Sigma	G-7021
Neurobasal	Invitrogen	21103-049
Plasma	Sigma	P-3266	1mg/ml (Reconstitute in H2O and filtrate)
Thrombin	Sigma	T-4648	10mg/ml(Reconstitute in H2O and filtrate)
B27	Invitrogen	17504-044	50X
Hepes (260g/mol)	Sigma	H-7006
EDTA	Sigma	E-5513	0,5M pH8
MgCL2	Euromedex	2189	1M
Glycerol	VWR	24388.295
IGEPAL	Sigma	I-3021
Complete Protease inhibitor Cocktail Tablets	Roche	04 693 116 001
Sodium Orthovanadate (Na3VO4)	Sigma	S-6508
distilled H2O
			Table 1. Reagents required
			TOOLS AND MATERIAL
Surgical scissors	Fine Science Tools	14002-12
Adson forceps	Fine Science Tools	11027-12
Dumont #5 Fine Tips forceps	Fine Science Tools	11254-20
micro knives	Fine Science Tools	10136-14
			Table 2. Tools and material
			Dissection and FP culture Ethanol 70% dissection hood dissection microscope Petri dishes glass coverslips 18mm x 18mm Bunsen gas burner 24 well plate tissue culture incubator (37°C, 5% CO2, humidity controlled) Centrifuge RECIPES FP culture media Neurobasal B27 1/50e dilution from stock HBSS – Thrombin HBSS 1 ml Thrombin 10mg/ml 30 μl Lysis Buffer pH7,4 Hepes (260 g/mol) 25mM EDTA (pH8) 5mM MgCL2 1mM Glycerol 10% IGEPAL (NP40) 0.10% Protease Inhibitor Cocktail 1X Sodium Orthovanadate 0,2M H2O Laemmli Buffer Tris 0,5M, pH 6,8 375 mM SDS 6% Glycerol 60% DTT 0,6M Bromophenol blue H2O