The first few days working with dii staining I had a few issues with the entire worm being fluorescent making it impossible to actually see the amphid neurons. This was just caused by the dye clumping at the bottom of the centrifuge tube and was easily solved by making sure I mixed the solution better. The other difficulty came with identifying exactly which neuron was which. This was difficult because almost every worm I looked at was oriented differently on the slide and sometimes all the neurons looked too clustered to accurately identify.
I have looked at a decent amount of these worms now and from what I’ve observed there is not a difference in expression between males and hermaphrodites.
I have also still been working on getting a successful cross between my him-5:dmd-6::GFP and him-5:AWA::mCherry worms. I’ve tried numerous times and still haven’t had any success. I’m observing a few more crosses today that I set up so hopefully I’ll have some luck.
Using liquid culture for the dauer assays has proven to be a little more tricky than expected. For whatever reason, we still haven’t been able to induce dauer even after drastically reducing the amount of food in the culture and doubling the ascaroside concentration found in the literature. Time to go back to the drawing board!
The leaving assays have been going well! Based on the data collected over the past couple weeks, we are going to hone in on a few of the sex reversal strains that looked promising. Over the final few weeks I’ll be working on collecting some more data and crunching numbers with Renee.
I can’t believe how this summer has flown by! In two short weeks we’ll be presenting our projects at a poster session- it seems unreal. I’m trying not to get sentimental just yet but as excited as I am to watch things come together over this final stretch, I can’t help wishing this experience wasn’t ending so soon.
Until next time! 🙂
Things have been going pretty well the past two weeks! I’ve been scoring my prab-3::dmd-6 worms as well as my ptdc-1::dmd-6 worms. Just yesterday I learned the technique of dii staining. The actual process of the dii staining is very easy; you essentially just let the worms soak in fluorescent dye that is mixed with water. Then after an hour and a half or so you pipette that liquid dye containing the worms onto a clean plate. The more difficult part is actually looking at and identifying the stained cells on a microscope. It stains the amphid neurons in the worm and some of these neurons are clustered, so depending on what angle you’re looking at the worm from it can be challenging to tell exactly what each neuron is. Once I observe more worms in the next few days though I’m sure it’ll become significantly easier to identify these neurons!
Looks like our liquid culture protocol still needs some adjusting! First, the concentration of worms in the culture needs to be significantly increased to make plating them easier, so I’m hoping reducing the volume of the culture and increasing the amount of eggs added will solve this problem. Second, even with the small number of worms I was able to look at, there were hardly any dauer. Hannah thinks reducing the bacteria in the culture might help by preventing the response to the ascarosides from being reduced by the high availability of food. We really only need enough bacteria to keep the worms from arresting at L1. I’m going to give it a try and see if we get better results!
In the meantime I’ll be doing some trials for one of Renee’s experiments while she’s on vacation. Earlier on, did some mating assays for her which looked to see if male response to hermaphrodites or location of the vulva was impaired by any of the neuronal mutations she was looking at. This time around, I’ll be doing leaving assays which are similar in design. I’ll be working with tra-2 mutant males, which means they have pan-neural feminization. Any sex-specific roles of the neurons should be reversed, which may impact/reverse some sex-specific behaviors.When placed on a food source, males are known to be more likely to take excursions from the food or even leave it completely (presumably looking for a mate), where hermaphrodites are more likely to remain happily on the food. Tra-2 mutant males are known to have the leaving behavior of a hermaphrodite. I’ll be observing the leaving behavior of a few worms with additional mutations to see if any of these mutants will behave more like wild type males. This will help us take a look at which genes are involved in sex-specific leaving behavior.
Until next time! 🙂