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March & April 2023
March & April 2023
After successfully heat-shocking a sample of eya-enhanced flies in February, I was unable to collect data before breaking for Minicourse. Frustratingly, I ran into the same timing issue in March when I had to restart the process but didn't have enough time to collect data on the heat-shocked, crossed flies before spring break. I hope that over senior project, when I can be in the lab every day, I will be able to run through the whole process – fly cross, heat shock, anesthetization, amputation, imaging –i n the first 2 weeks.
February 2023
February 2023
I ran a successful heat shock test run on a small sample of eya-enhanced larvae. The larvae survived the heat shock trial and matured into adult flies, which is exactly what I need to happen to perform the rest of my experiments and collect data. Hooray!
I also dedicated time to working on my manuscript. I have drafted an introduction and methods section so far. It is exciting to be putting together a paper because it feels like a tangible, summative product of all the work I have been doing the past year and a half.
January 2023
January 2023
I am preparing to collect data! The first step is setting up a cross between the heat shock strain and the UAS-eya strain to generate flies that can overexpress the eya gene. Currently, I am working on cultivating healthy fly stocks.
Last week, during my mentor meeting, we discussed possible publication routes. Depending on the results of my data (assuming a binary of YES regeneration or NO regeneration) I have a few options. If I do NOT see any leg regrowth, I will reach out to the authors of the agreeing bioRxiv preprint to see if they are interested in my data/discussing. If I DO see leg regrowth, I will work on a manuscript, either full or partial, that I could submit to a journal. In the likely event I am not able to produce an entire paper, I will submit to the microPublication of biology.
December 2022
December 2022
Big month! I carried out the heat shock and staining protocols with success! I stained two groups of larvae – a heat shocked group and a control. The tissues of the heat-shocked group appear bright blue after staining while the control group appears yellow/colorless. It seems that the heat shock works to induce the expression of the gene of interest.
control (no heat shock) tissue
heat shock tissue
November 2022
November 2022
11/10
So far, I've progressed by finishing all of the solutions necessary for my heat shock and staining protocols. Because it took a little while to make everything, I was unable to use the larvae from my first cross attempt as they grew into adult flies. Today I initiated a new cross so that I will have larvae for my experiments next week (now that I am fully ready to begin!)
October 2022
October 2022
10/29
I spent this week making solutions for my heat shock and LacZ staining protocols! So far, I've whipped up Poel's Salt Solution and glycerol solution.
10/6
As I wait on the first generation of fly cross offspring to develop, I am ironing out my protocols and reagants for the heat shock and lacZ staining. A GIANT thank you to Jennifer for helping me gather everything I need and concoct all necessary solutions.
September 2022
September 2022
This month, I successfully initiated a cross between my heat shock and lacZ fly stocks. The process requires some serious vigilance and care, so it's huge that my first attempt went smoothly. Since fly crosses can only use females who have never mated before (in order to ensure the genome of offspring) I had to watch my collection vials closely, being sure to separate out female flies within 8 hours after they hatch. After gathering enough flies, I set up cross vials with males and females from the different strains and waited for larvae to appear. Once the new generation of adults emerge, I will take on the heat shock and staining protocols.
Looking Ahead
Looking Ahead
Fans of https://sites.google.com/ma.org/regeneration have lots to look forward to in the coming months. Starting in September, I will attempt my first cross and heat shock experiments (!!) I will use a "test" stock of flies unrelated to the regeneration question (described in July section below) and assess if the gene of interest (in this case, lacZ, in the eventual cross, eya) is prominently expressed. While not technically complex, the drosophila cross procedure requires vigilance, precise timekeeping, and little margin for error. Stay tuned to hear how this goes.
Heading into my final year in MARC is exciting but also scary. I have huge ambitions for a tight and limited schedule, but I can't wait to see where my project will take me. I'm most eager for collecting data and analyzing results. After so much planning and preparation, my project will really take flight this semester.
August in the Lab
August in the Lab
August in the lab begins out of the lab at the 2022 International Limb Conference on Development, Regeneration, and Evolution!!! Over 4 jam-packed days, I learned about model systems I didn't know existed and listened to keynotes where I understood (at best) every third word. Still, my experience was mindblowing, enrapturing, and unforgettable. Some of my personal highlights were hearing about the accidental generation of an 8-legged frog from Dr. Beck and from the Tabin Lab, hijacking/rerouting cell fate determination (effectively reprogramming non-limb cells to behave as limb progenitors)
I focused the rest of the month on taking over more and more MARC lab countertop space with my growing fly colony (now including 4 separate stocks), studying cross protocols, tracking down some elusive reagents for said protocls, and occasionally amputating a leg or two. I have the photos to prove it. Not currently, as they're on a digital camera and not my computer. But soon. You'll see. Just you wait.
example of imaginal disc LacZ staining via Tomizawa et. al
July in the Lab
July in the Lab
This month's major excitement:
receiving CO2 regulator and all required components for my anesthetization protocol
met with Jessica and discussed protocols for a "practice" cross with heat shock flies (hs-GAL4) and UAS-LacZ flies. Once I've successfully crossed these stocks, I can fix and plate the offspring, apply some staining and other reagents, and hopefully see a dark blue precipitate that indicates LacZ presence (and therefore successful protocols all around)
June in the Lab
June in the Lab
6/27-7/1
I have made great progress this week with imaging. Previously, I was only able to generate blurry, poorly-focused images by trying to aim my phone camera through the eyepiece by hand.
But this week, thanks to Jon's help, I was able to set up a phone stand for the microscope that has enhanced the ease of the process, as well as the focus magnification of the images.
old photo
new photo
6/20-6/24
From the Kumar Lab, I received a copy of their UAS-eya stock which I will eventually cross to a hs-GAL4 line to generate flies with the capacity for eya upregulation
Kumar shipment arrives
6/13-6/17
While I wait on the arrival of the CO2 monitor I need for anesthetization, I have been working on maintaining /growing the fly colony, practicing microscopy and imaging, and studying the protocols I will use later on.
May 2022
May 2022
5/22/22
After another fabulous and productive meeting with Jessica on 5/17, I have nailed down my experimental plan! Here is the recap of our meeting:
We ran into some initial difficulties with being unable to find an existing line of hs-eya flies from any online stock center. We reached out to 2 scientists involved with the original 1993 Bonini paper that created this line of flies to see if they had any in their labs. While neither scientist had the stock we were looking for, Dr. Justin Kumar graciously offered to send me 3 stocks with which I will be able to create my desired genotype by setting up a cross.
As for summer plans, I am still planning on reaching out to Dr. Hariharan regarding potential lab space this summer.
Jessica and I also discussed my attending the Limb Conference (of which she is an organizer) and the ISSCR Conference in San Francisco
Unfortunately the Flowbuddy Amy found online fell through and I am waiting for confirmation that Jessica will be able to purchase and send one to me as she so generously offered
5/9/22
I am planning to meet with Jessica on 5/17 (stay tuned for updates)
On the agenda is finalizing my experimental plan as well as discussing summer plans i.e. reaching out to Dr. Hariharan
Thanks to Amy, the Flowbuddy has been ordered and is expected to arrive relatively soon
Meanwhile, I am maintaining my rapidly growing fly colony!
April 2022
April 2022
I have obtained and have begun to maintain a small number of wild type Canton-S flies that will serve as the control animals in my experiments.
I have developed 3d printing plans for a flypad for CO2 anesthetization
Acquiring a flowbuddy machine for CO2 regulation is a top priority
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