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.
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 cues2,3. 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 (FPcm)4-6. We then exemplify the use of this FPcm 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 FPcm. Third, the tissue are processed for Western blot analysis of commissural markers.
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 navigation7,8. The experimental procedure described here to produce FPcm 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 FPcm, dorsal spinal cord tissues containing commissural neurons are dissected, dilacerated and stimulated with the FPcm. 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 FPcm or any types of treatment are assessed in a more physiological way than in cell and tissue culture conditions.
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…
The authors have nothing to disclose.
We thank Karine Kindbeiter, Muriel Bozon, and Florie Reynaud for their help. The work is supported by CNRS, ANR YODA program and Labex DevWeCan.
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 | |||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
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