In some of my goi::gfp images I see GFP expression in each of two paired inverted stirrup-like regions located centrally within the body. Could anyone suggest what these might be? (I would attach an image but I get a ‘Cannot access attachments upload path!’ error)
Do you mean perhaps the spermathecas? Are you looking at L4s or young adults?
If this works…
I see similar GFP expression with both a ‘transcriptional’ construct driving GFP alone and a ‘translational’ version generating the protein::GFP fusion.
Could be VC4 and VC5 neurons? With no other images to go off of, that would be my guess.
More information could help:
- Is this a roller animal? (which it looks like it might be, and which I definitely don’t recommend, especially for this purpose)
- What do the nuclei of those cells look like by DIC (size, morphology)?
- Where are the cells in the animal: ventral? lateral? How deep within the animal?
- What’s next to the cells? For example, is the vulva right between them? Do they seem to be associated with or right next to the gonad, the gut, the ventral cord, etcetera?
Yes, we use rol-6 as a marker. What would be a ‘better’ one for expression analysis?
A couple more images plus DIC below. These were both 100x. I’m very much a novice on worm anatomy but these tissues/organs look quite distinctive. I’ll look at some more worms tomorrow at higher mag.
rol-6 doesn’t just cause the worm to move distinctively: it twists the whole worm. I’ve known some people who actually liked this, because in a twisted worm it might be easier to see structures normally on the side of the resting animal that had been rotated by the twisting into being on the top of the resting animal, but it makes the anatomy harder to understand, especially for the novice and for anybody looking at a picture.
I still haven’t been able to figure out what I’m looking at in your images. Even if I could, there’s a fair chance I wouldn’t know the answer - I’ve only worked on certain things, after all. Still, a couple of landmarks would be very important: where are you with respect to the vulva, and to the anterior/posterior and dorsal/ventral axes? If your cells are lateral, are they left-right symmetrical?
Nomarski morphology of nuclei can be very helpful. Different cell types have distinctive appearances that can be very informative, and knowing what cells are near your cells can help to identify them. There is an extensive chapter in Wormbook that can help you learn to look at the anatomy, full of advice and excellent reference images with labeled cells. If your GFP is visible before adulthood, you’re likely to find you’ll get much better Nomarski in larvae, especially before the uterus fills with eggs. If you’re not getting good enough Nomarski to see nuclear morphology at least as good as in the images at the link when looking through the scope (photographing Nomarski well is difficult), you’ll need to fiddle with your scope.
Many thanks - I’ll do what you suggest.
Although it can be difficult to judge small images, I may be able to shed some light on your green stirrups. One can see this type of pattern abutting the vulva when GFP is present in the cytoplasm of hyp7. Depending on what part of the worm you are examining, the thickness of hyp7 varies. The parts that are over the muscle quadrants are very thin, and one usually cannot see much if any GFP, but the part of hyp7 that is associated with the ventral cord is a little thicker, and one can sometimes see cytoplasmic GFP there.
The ventral part of hyp7 is joined to the vulva. Because, as others have mentioned, your worms are twisted, you sometimes can see the vulva head on–a dorsal ventral view–and this is the case with the images you have posted. The vulva has little or no expression of your GFP and is therefore a dark circle relative to the cytoplasmic GFP of hyp7. Your stirrup pattern is consistent with what is expected for the junction of the anterior part of the ventral hyp7 with the vulva and likewise for the posterior part. At each junction, hyp7 extends a little bit around the vulva, thereby producing the pattern that resembles stirrups. The ventral hyp7 extends anteriorly and posteriorly from the vulva, which are the faint lines that you see twisting away from the vulva.
Some of your images have additional evidence for expression of your construct in the cytoplasm of hyp7. In the head, one can see the pharynx in relief. It is black relative to extensive, somewhat diffuse GFP. This is the expected pattern of hyp7 for this region.
It has been difficult putting all this in words, but basically, I think you have a dorsal-ventral view of the vulva, which does not have much if any GFP, and GFP in the cytoplasm of hyp7.
The thickness of hyp7 in different parts of the body is nicely illustrated in cross section in John White’s anatomy chapter in the first Cold Spring Harbor C. elegans book, and there are scanning EM and other dorsal-ventral illustrations of the junction of the vulva and the ventral part of hyp7 in Hall and Altun’s C. elegans Atlas.