Hi,
I am currently working on a neuron expressing gene, bbs-5, which is expressed in all CSNs, while, as you may know, che-1 is specifically expressed in ASE neurons.
A tricky question I got is how to find if che-1 could affect bbs-5 expression in ASE. Given that there are a pile of neurons that are crowded in the amphid,
it is really hard to observe whether a specific neuron changed or not among these monsters. If I introduce another gene that is exclusively expressed in ASE,
it would another hell since there are three loci to moniter. This question really hits me. Could anybody help me out?
TIA.
Emily
One seemingly obvious option would be split-gfp: express half of GFP under the control of the bbs-5 promoter, and the other half in an overlapping set of cells that includes the ASE but not other chemosensory neurons. You should get reconstitution and fluorescence only in the ASE (unless the two promoters’ expression overlaps elsewhere), and can more easily look for effects of che-1; just remember that you’ll have to make sure that expression from your second promoter in the ASE isn’t strongly affected by loss of che-1.
I’d also add that while amphid sensory neuron identification sounds like a nightmare, it really isn’t terribly difficult. With practice, it is like identifying star constellations: for amphids, look for the laterally located arrow head…
Consider using DIC to definitively spot ASE, then switch to epi to see whether your GFP is in your reticle crosshairs. Without an NLS in your GFP I agree it would be difficult. With one NLS (and definitely 2XNLS), you’d be surprised at how easy it is, even in a pan-neural exressing line.
Even without NLS, you may be able to do it by DIC ID, then switching to epi: the absence of fluorescence in the ASE cell body amongst the crowd might be pretty obvious.
Anyway, you can do this with what you have in hand. Little extra effort beyond a few hours on the scope. You can even get a Pche-1::gfp animal to confirm your ASE identification. If it doesn’t work, then I agree with Hillel’s spit-GFP recommendation.