1Department of Biological Sciences, University of Southern California, 2Neuroscience Graduate Program, University of Southern California
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Hazen, V. M., Phan, K., Yamauchi, K., Butler, S. J. Assaying the Ability of Diffusible Signaling Molecules to Reorient Embryonic Spinal Commissural Axons. J. Vis. Exp. (37), e1853, doi:10.3791/1853 (2010).
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 plate3,4 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 axons3,4 as well the necessity for this signal to direct the commissural trajectory9.
Part 1: Preparation of Aggregates of Transfected COS Cells using Hanging Drops
Part 2: Preparation of Dorsal Spinal Cord Explants
Part 3: Priming Collagen
Note: The exact amount of NaHCO3 will need to be titrated for each batch of collagen. Start low (11μl) and then add 0.5-1μl stepwise until the solution is a slight shade of orange. The "right" amount is usually 1ul less than the smallest amount of NaHCO3 that would turn the collagen pink. The amount of NaHCO3 does not scale linearly up or down.
Part 4: Dissection of Dorsal Spinal Cord Explants and Positioning of Them with COS Cell Aggregates in Collagen Matrix
Part 5: Visualization of Commissural Axons by Immunohistochemistry
Part 6: Representative Images of Explants
In a successful experiment, the explant and COS aggregate will remain adjacent to one another and not drift apart as the collagen sets. There will be minimal overgrowth of axons; significant axonal overgrowth indicates that the dorsal spinal explant was cultured for too long. There will be no blebs growing from the explant; blebbing occurs if the tissue comes into direct contact with the culture medium.
The ability of the signaling molecule to reorient axons is assessed using confocal microscopy. The extent of axon reorientation can be quantified by measuring the average angle of reorientation of the axons closest to the COS cell aggregate3. As shown in Figure 1, COS cells expressing myc-tagged BMP7 (blue) reorient Tag1+ commissural axons (green) away from the source of BMP7 (Figure 1B) similar to the manner to which an explant of roof plate repels commissural axons (Figure 1A). COS cells expressing a control vector have no effect on the orientation of commissural axons3,4. These explants were also labeled with antibodies against the transcription factor Isl1/2 (red), which decorates motor neurons and dorsal interneurons. This result was the first indication that a member of the BMP family mediates the activity of the roof plate chemorepellent3.
Figure 1: Please click here to see a larger version of figure 1.
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.
This protocol was developed in the laboratories of Jane Dodd, Thomas Jessell and Andrew Lumsden. Many people, including Konrad Basler, Ann Calof, Thomas Edlund, Phil Hamilton, Domna Karagogeos, Ariel Ruiz i Altaba and Toshiya Yamada determined how to culture explants of spinal cord in collagen. Marysia Placzek and Marc Tessier-Lavigne pioneered the technique of using axon growth from in vitro explants as a means of identifying axon guidance molecules. Work in the Butler laboratory is supported by grants from the March of Dimes and R01 NS063999 from NIH/NINDS.
|Type 1 rat tail collagen||BD Biosciences||354236|
|10x Minimal Essential Medium||Invitrogen||11430-030|
|Fetal bovine serum||Mediatech, Inc.||35-016-CV|
|Cell dissociation solution||EMD Millipore||S-004-C|
|Trypsin 0.25% EDTA||Invitrogen||2520-0056|
|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|
|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|