Wednesday, December 4, 2013

[Wormpaper outtakes and bloopers] elt-2 RNA level dynamics after heat shock in wild-type and HS::end-1 strains

- Gautham

Second in a series of outtakes and bloopers related to our paper on the relationship of gene expression dynamics and cell division times in the early C. elegans embryo.

elt-2 RNA level dynamics after heat shock in wild-type and HS::end-1 strains

This one is a blooper.

In our paper we perturbed cell divisions using mutants and asked if gene expression would track with those cell divisions. Conversely, it would have been great to modify the levels of transcriptional activators and see if gene expression could start before the cell divisions that normally precede them.

Strains with end-1 under the control of a heat shock promoter are available. end-1 is a well-known activator of elt-2. So we went ahead.

Methods: We obtained strain RJ663bc3 from Joel Rothman's group with the kind help of Yewubdar Argaw. We isolated embryos from a synchronized batch culture and aliquoted them at 25C. We heat shocked each aliquot at a different time, for 5 min. at 34C, and returned them to 25C. These brief but accurate heat-shocks were accomplished by centrifuging the embryos and resuspending them in M9 at the appropriate temperature. All samples were fixed simultaneously. The different aliquots spent between 0 and 40 min. at 25C after heat shock and before fixation.

Results: We did RNA-FISH on all the samples for end-1, end-3, and elt-2. We analyzed the data for each aliquot and obtained the following very interesting-looking result for elt-2 expression after end-1 over-expression by heat-shock:

Each panel shows embryos that were heat shocked within a particular window of their development as indicated in the grey shaded region. The red dots are the elt-2 expression levels for these heat-shocked embryos after they've been returned to 25C. The blue dots are the expression trajectory in wild-type without heat-shock for reference and the dashed vertical lines are E lineage divisions. It was super interesting to see elt-2 starting before the 2E divisions, and that it didn't appear to be sensitive if the heat shock came too early.

Blooper: This result is not in the paper because we see qualitatively similar behavior if we heat-shock wild-type worms, with no end-1 overexpressing transgene. Todd Lamitina alerted me to the critical need of doing that control experiment, which in hindsight I should have done first.

Perspective: What was much worse is that I had reason to know that the experiment would be a failure if I had paused to think about all I'd done. Well before doing this experiment I had looked at wild-type embryos that had been left at 30C (not a viable growth temperature for C. elegans) for an hour and found precocious elt-2 expression in these embryos. I disregarded that result as due to any number of things that could go wrong when you try to grow worms under lethal conditions. Without this confounding problem, the overexpression experiment would have been a valuable addition to the paper no matter what the outcome.

Why we were even looking at gene expression trajectories of wild-type worms under non-viable temperatures is a separate story, and another example of Perhaps there will be something interesting.

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