I am trying to visualize NSM cells in the pharynx. I have a tph-1: RFP strain. I know there is an NSM pair in the anterior bulb of the pharynx.
However, sometimes I just see one. Sometimes I do see two; however, most are not across from one another, one appears in the middle, while the
other appears towards the side (ventral).
Can I be confident that it is true that I am seeing both cells? and that the differences in positions is attributed the worm’s loading plane on the agar pad?
When I see only one, does that mean that the other simply doesn’t exist? I tried to scan the literature, some of their images showed only one, but they didn’t
explain, instead just labelled.
I am new in using the microscope, and therefore I am not comfortable explaining what I see/don’t see. There seems to be no black/white answer,
but rather a grey range of “guesses” at this point associated with different focuses ???.
Thank you in advance for reading/replying to my post. I hope I can get some ideas to try.
Obviously it depends on what you’re doing, and there are applications where the marker will be absolutely necessary, but you shouldn’t need a marker to find the NSMs on a good Nomarski scope. The triangular arrangement of the NSM, I2, and MC nuclei in each of the two ventral planes should be easy to spot. Remember that the two ventral planes are not parallel to each other; if one is flat with respect to the plane of focus, the other will be rotated out of that plane. This can also explain why one of the NSMs you see is rotated “towards the middle” rather than being entirely ventral.
As to why your marker doesn’t reliably detect two NSMs: I assume you’re looking at a high enough magnification that you can tell two from one (i.e. a dissecting scope may not suffice)? I assume you’re using an integrated transgene, and you’re not just seeing mitotic loss of an extrachromosomal transgene from some of the NSMs? Do you ever see animals with no NSMs?
In any case, I recommend that you practice finding both NSMs by Nomarski. This should also tell you a lot about how good your reporter is - and whether it might be affecting the positions or morphology of the NSMs. I don’t think tph-1::rfp should do this, but there might be another defect in the strain, an effect of the integration or some such.
I am using Zeiss Axio Observer (inverted microscope). I believe it has enough magnification. I will try to count the cells in wild type again, though it is not so clear
in this microscope.
We don’t have a Nomarski, but perhaps I can try to see if there is in another lab.
I think I understand more now that you mentioned that the ventral sides are not parallel.
, the NSMs being in the subventral part of the picture
attached.
That might be why perhaps I see it close to the middle in many cases.
As for the single versus NSM pair. In about 90% of the worms counted (~180 worms), I was able to see 2 cells. The remaining 10% had only 1. But none had
an absent NSM.
The strain I am using is LX993 from CGC.
hmm the idea of mitotic loss of the transgene didn’t cross my mind. But it is very worth taking it into consideration.
I will definitely be trying the suggestions. Thank you very much for your reply!
LX993 is apparently lin-15AB(n765) vsIs108, so the transgene is integrated into the genome and should not be subject to mitotic loss - which is a shame, because that would be a simple explanation. You might look at heterozygotes (in case a mutation that’s homozygous in the reporter strain is recessively causing NSM loss); this is a little unlikely, as the transgene has been outcrossed a few times, but there could be a mutation at or near to the integration site. You might also request some of the many other integrated tph-1 reporters described on the Reagents panel of the gene’s WormBase page (though mostly they’re gfp transgenes, if that makes a difference), either from the CGC or from the originating lab.