Visualization of Axonal Projection Patterns of Embryonic Motor Neurons in Drosophila

Begin with a tube containing late-stage-16 Drosophila embryos in a mounting medium.

The motor neurons of Drosophila are immunolabeled.

Transfer an embryo to a glass slide and remove it from the mounting medium.

Then, place the embryo ventral side up and dissect it.

Orient the anterior section containing the motor neurons with the dorsal side up.

Using a tungsten needle, cut along the dorsal midline and spread the body wall. Move the embryo into the mounting medium. Then, remove the internal organs to expose the labeled

neurons.

Transfer the embryo to a new slide, then mount and seal it.

Observe under a differential interference contrast microscope as light passes through the embryo, generating a high-resolution image.

The labeled neurons with axonal projection patterns appear dark brown on a light background.

Motor neurons branch into two major nerves: the intersegmental and segmental nerves, and a minor branch, the transverse nerve, that innervates the muscles.

To fillet the embryos, stage them on a glass slide

First, move them into a drop of glycerol ventral side up. Next, under a dissection microscope, cut off the anterior part at 1 quarter of the body length. And remove the most posterior region, which is free of motor axons.

Now, using a needle probe, move the preparation out of the glycerol, oriented dorsal side up, and position it horizontally in the field of view. The next step requires a fine tungsten needle that has been inserted into the hollow region of a needle probe and then bent at the end. Using the tungsten needle, make a small cut at the posterior end of the embryo, and continue to cut down the dorsal midline.

Make sure the tip of the tungsten needle does not touch the surface of the glass slide during the dissection, because the needle will easily bend against the glass.

Then with the probe, return preparation back to the glycerol drop, and position it dorsal side up. There, detach the gut from the body wall by unfolding each body wall in a ventral lateral direction. The two body walls should come apart. Now using the probe, carefully move the body wall flaps onto a glass slide. On the slide, use a tungsten needle to remove the internal organs by pushing them laterally.

To stabilize the tungsten needle and keep it from being damaged, keep the hollow needle probes that hold the tungsten needle braced against the surface of the glass slide while manipulating the tungsten needle.

Now mount the preparations and 8 microliters of mounting solution on a new glass slide. Attach a coverslip, and seal the edges down with regular nail polish. Then, capture images at high resolution using DIC optics.