Dr. Wes Thompson - Current Research

1- Neuromuscular junctions in the soleus muscle of a rat. Antibodies (to neurofilament, red) were used to visualize axons. Three axons (arrows) approach 3 different muscle fibers where each arborizes into a terminal (arrowheads). A second antibody (to an unknown component located in the muscle fiber underneath the nerve terminal, green) identifies the site of synaptic contact.

2- A living transgenic mouse pup (about 7 days old) expressing GFP. The mouse bears a transgene consisting of the S100 promoter (a promoter that drives expression in the lens of the eye and in derivatives of the neural crest) coupled to GFP. We find that it is easy to identify (genotype) these mice, because the ones bearing the transgene light up under the dissecting microscope.

3- Vital labeling of the components of a neuromuscular junction in the mouse sternomastoid muscle. This figure illustrates that it is possible not only to identify the Schwann cells, nerve and acetylcholine receptors in a living muscle, but that one can come back 30 days later and identify this same synapse and its components. The top row shows the images obtained one the first view; the bottom row shows the images obtained 30 days later. By using different filters in the fluorescence microscope so we illuminate with and detect different wavelengths of light, we can detect the red acetylcholine receptors (using rhodamine-conjugated bungarotoxin), the green GFP located in the Schwann cells, and the blue CFP located in the axons. The camera we use is black and white (to enhance sensitivity) but we can color these images and superimpose them, as shown on the far right. Note that we know the neuromuscular junction examined in the two views is the same synapse because of the unique “fingerprint” in the acetylcholine receptors created in each muscle fiber by the pattern of branching of the axon.

4- The strategy for conditional gene expression in Schwann cells (based upon work by Bujard and his colleagues). The S100 promoter, which we have previously shown drives expression in Schwann cells and other neural crest derivatives, is used to make a transgene in which this promoter drives expression of a transcription factor, rtTA (for “reverse tetracycline transactivator”). In a transgenic animal bearing such a transgene, this protein rtTA is constitutively produced. The rtTA protein can bind to and activate transcription of a second transgene bearing a TRE, tetracycline response element, but only in the presence of a tetracycline antibiotic, doxycycline. When the animal is given doxycycline, transcription of the gene under the control of TRE is initiated. One can therefore switch on or off the expression of this gene by giving (or withdrawing) doxycycline to the animal. The gene under the control of the TRE in this example is a reporter gene, beta-galactosidase or LacZ, a gene product that can be easily identified by a simple histochemical procedure.

5- Demonstration of the concept of conditional gene expression. A mouse embryo, approximately 3 days prior to birth, was labeled using a histochemical procedure for LacZ, a procedure that causes deposition of a blue-black product where the LacZ is expressed. This animal was given doxycycline (through the diet of its mother). The blue-black stain shows that expression of the reporter occurs in the Schwann cells along the peripheral nerves. Animals not given doxycycline show no such expression.