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

Explants of dorsal spinal cord are dissected from rat embryos and cultured in a three-dimensional collagen. Gel in opposition to cause cells expressing a candidate signaling molecule. The ability of the signaling molecule to reorient axons growing within the dorsal spinal explan is assessed using immunohistochemistry.

Hi, I'm Virginia Hazen from the laboratory of Samantha Butler in the Department of Biological Sciences at the University of Southern California. I'm Keith Fun also from the Butler lab. And I'm Ken Ya Mai from the Butler lab.

Today we will show you a procedure for culturing dorsal spinal implants and aggregates of transfected cost cells in a collagen matrix. We use this procedure in our laboratory to assay the ability of candidates, ling molecules to orientate comm spinal neurons. In this experiment we'll be examining the ability of bone morphogenetic protein or BMP seven to act as a chemo repellent.

Let's get started. Prepare co cell aggregates used to challenge the trajectory of comm choral axons from co cells. Transfected with the expression plasmid for the molecule of interest here, bone morphogenetic protein or BMP seven.

Aspirate the medium from the dish and rinse the cells with one milliliter of one XPBS. Remove the PBS and treat the cells with half a milliliter of enzyme free cell dissociation medium for 15 minutes. Stop the reaction by adding one milliliter of medium with fetal bovine serum.

Using a 1000 microliter pipette tip, iterate the cells until none remain on the surface of the culture dish. Transfer the cells to a 15 milliliter conical tube and pellet the cells for two minutes. At 2000 RPM, remove the supernatant and pipette up and down to resuspend.

In 100 microliters of medium spot multiple 20 microliter drops of the cell solution on the inside of a 35 millimeter culture dish lid. Close the lid over the dish and place in the incubator for several hours to allow the cells to aggregate. To prepare dorsal spinal cord implants, keep freshly harvested.

E 11 rat embryos on ice. In L 15 medium with a sharpened tungsten needle, remove a section of four to six mite segments from the trunk region immediately below the forelimb bud of each embryo. Using a plastic pipette, collect the Tissue in one well of a four.

Well no dish on ice. When all of the segments have been dissected, use the pipette to transfer the pieces into a second well of the no dish containing L 15 medium plus disc paste. Incubate the tissue at room temperature for no more than five minutes.

To prevent cell death, do not over digest the tissue. Add one milliliter of fresh L 15 medium with 5%heat inactivated goat serum to the third well of the no dish. And transfer the tissue pieces to this well.

At the end of the dis space incubation, allow the tissue to rest on ice for an hour. To make the subsequent dissection easier in a micro centrifuge tube, add 40 microliters of 10 x minimal essential medium to 360 microliters of collagen. And vortex briefly to mix spin down in a pico fuge.

The solution will be yellow and should be kept on ice as much as possible to prevent the collagen from setting for each batch of collagen titrate with the amount of 0.8 molar sodium bicarbonate that turns the collagen solution slightly orange. Mixing the solution after each edition. When the collagen solution is this color, it will remain liquid if kept on ice, but is primed to set when brought to room temperature if the solution turns pink after mixing, start over with a fresh tube of collagen with medium spot 20 microliters of collagen in each well of a clean four well no dish.

Using the tip of the pipette, spread out the collagen to form a small pad. Let the collagen set at room temperature for about 10 minutes. Under the dissection microscope, use a freshly sharpened tungsten needle and a pair of fine forceps to remove the mesoderm surrounding the spinal cord from one embryo with the tungsten needle, remove the ventral portion of the spinal cord, which can be distinguished by the presence of the floor plate.

At the ventral most edge, the floor plate is slightly thicker than the roof plate at the dorsal edge. In this example, the spinal cord is ventral side down. Carefully cut the tissue working parallel to the floor plate and discard the ventral fifth of the spinal cord.

Use the needle to bisect the dorsal spinal cord explan vertically. Make sure the edges are as cleanly cut as possible and to retrim them if necessary. Use a mouth pipette fitted with a 0.5 millimeter diameter glass capillary tube to transfer each x explan onto a separate collagen pad.

With the tungsten needle, cut the aggregate of transfected co cells into squares approximately the same width as the lateral edge of the dorsal spinal cord.Explan. Use a mouth pipette to transfer one of the squares onto the collagen pad with the x explan. Use the mouth pipette to aspirate off any excess liquid.

Do not over aspirate since a small amount of liquid will aid in the subsequent application of collagen, apply four microliters of primed collagen onto the pad. Use the tungsten needle to move the collagen over the implants without directly touching the tissue. By moving the needle in the collagen orient each x explan such that the co cell aggregate is adjacent to its lateral edge.

Allow the collagen to set for 15 to 20 minutes. Repeat the application process with another four microliters of collagen to ensure that the x explan is completely encased in collagen before moving on to the next embryo in a tissue culture hood at 0.5 milliliters of medium and culture for 30 to 40 hours In an incubator in a fume hood, fix the explants with 0.5 milliliters of 4%Para formaldehyde solution in PBS for 45 minutes. Keeping the tissue on ice wash twice with 0.5 milliliters of the blocking and permeation solution.PBTN.

Incubate the x explants in blocking solution at four degrees Celsius, preferably overnight. Use forceps to cut the X explan out of the four. Well plate and place each one in one well of a 48 well dish.

Add 200 microliters of primary antibody to each well. To visualize comm choral axons, use mouse anti ttag antibody and to determine whether cross cell transfection was successful, include an antibody against the molecule being tested. Incubate overnight at four degrees Celsius.

Wash each sample in PBTN blocking solution five times for one hour each wash at four degrees Celsius. Remove the PBTN and add 200 microliters of secondary antibody to each. Well cover the edition foil to prevent bleaching and incubate overnight at four degrees Celsius.

Wash the samples five times in PBTN as before. Transfer each explan to a three well depression slide. Remove excess liquid mount invecta shield medium and cover with a cover slip.

Store the slides in a light safe folder or box at either four or minus 20 degrees Celsius until ready to visualize on the microscope. Now we'll show some representative images of x explants after they've been cultured, fixed, stained and imaged by confocal microscopy panel A shows that an X explan of roof plate outlined in white can reorient the tag one positive choral axons away from it, demonstrating that the roof plate is the source of an axon chemo repellent. Panel B shows that co cells secreting the M tact signaling molecule, bone morphogenetic protein or BMP seven can repel carceral axons in a similar manner to the roof plate.

Axons co cells secreting only the expression plasmid have no effect on the orientation of axons. We've just shown you how to dissect, orient and culture X explan of spinal cord and cause cell aggregates to allow you to determine whether a candidate singling molecule has axon reorienting activity. When doing this procedure, it's important to keep the collagen on ice as much as possible, completely encase the explan in collagen so that it does not come in direct contact with the cultural media.

And finally, culture only as long as it takes for the axons to extend to the ventral edge of the x explan. So that's it. Thanks for watching and good luck with your experiments.I.