Fruit fly chromosomes

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Wes
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Fruit fly chromosomes

#1 Post by Wes » Wed Jul 31, 2019 8:19 pm

Drosophila larvae grow to a large extent due to a process called endoreduplication, where the genome will double a few times but the cell would not divide. One consequence of this process is that cells become rather large. Another, more interesting effect of endoreduplication is the formation of giant polytene chrosomomes where multiple DNA strands associate along their length maintaining gene co-linearity with a single nucleus carrying up to 1024-2048 copies of the fruit fly genome! Polytene chromosomes have a characteristic banding pattern where inactive genes form condensed bands whereas active genes form less dense expanded structures (sometimes referred to as puffs).

Here I sacrificed a Drosophila larva by dissecting it in an isotonic salt solution and making a temporary microscope slide. Below is a 2-image stack of a giant nucleus carrying polytene chromosomes. There were several different kinds of tissues carrying such giant chromosomes so I'm not quite sure what kind exactly this is but it might be salivary gland tissue. You can clearly see the nuclear membrane surrounding the clear nucleoplasm inside of which is a bundle of elongated structures and those are the chromosomes. There are protocols that result in nuclear membrane rupture where the chromosomes nicely spread out and you can even map the individual genomic regions based on the banding pattern.
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Drosophila polytene chromosomes, 40/0.95 DIC
Drosophila polytene chromosomes, 40/0.95 DIC
polytene_chromosome.jpg (371.09 KiB) Viewed 6994 times
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MichaelG.
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Re: Fruit fly chromosomes

#2 Post by MichaelG. » Wed Jul 31, 2019 8:39 pm

Supeb image, and useful information, Wes
... Many thanks

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75RR
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Re: Fruit fly chromosomes

#3 Post by 75RR » Wed Jul 31, 2019 9:50 pm

Hoping to better understand what I was looking at of looked up endoreduplication in Wiki. Wish I hadn't ;)

Endoreduplication (also referred to as endoreplication or endocycling) is replication of the nuclear genome in the absence of mitosis, which leads to elevated nuclear gene content and polyploidy. Endoreplication can be understood simply as a variant form of the mitotic cell cycle (G1-S-G2-M) in which mitosis is circumvented entirely, due to modulation of cyclin-dependent kinase (CDK) activity.[1][2][3][4] Examples of endoreplication characterized in arthropod, mammalian, and plant species suggest that it is a universal developmental mechanism responsible for the differentiation and morphogenesis of cell types that fulfill an array of biological functions.[1][2] While endoreplication is often limited to specific cell types in animals, it is considerably more widespread in plants, such that polyploidy can be detected in the majority of plant tissues.[5]

Beautiful image by the way!
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mrsonchus
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Re: Fruit fly chromosomes

#4 Post by mrsonchus » Wed Jul 31, 2019 10:01 pm

Lovely image - I wish I could image plant chromosomes like that!

I do see hints of banding in my slides but not to any useful degree, or of course suitable spread. But it's quite satisfying even to see that they exist on my slides for me!

Excellent image, thanks for posting.
John B

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Wes
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Re: Fruit fly chromosomes

#5 Post by Wes » Wed Jul 31, 2019 10:46 pm

Many thanks MichaelG., 75RR and mrsonchus for the positive feedback. I find an amazing array of cells and tissues in both the larvae and adult flies along with all sorts of internal intricacies from doughnut-shaped nucleoli to sausage-like chromosome.
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tgss
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Re: Fruit fly chromosomes

#6 Post by tgss » Wed Jul 31, 2019 10:55 pm

Hi Wes
I must concur with the other responders - wonderful image! Can you provide an idea of scale - width of the imaged field or something similar?
Tom

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Wes
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Re: Fruit fly chromosomes

#7 Post by Wes » Wed Jul 31, 2019 11:02 pm

Thanks tgss, I will get a measurement. I keep forgetting to take an image of my stage micrometer but I will do it first thing in the morning.
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MichaelG.
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Re: Fruit fly chromosomes

#8 Post by MichaelG. » Wed Jul 31, 2019 11:17 pm

75RR wrote:Hoping to better understand what I was looking at of looked up endoreduplication in Wiki. Wish I hadn't ;)
:shock: I see what you mean

This one is rather more comprehensible:
https://en.m.wikipedia.org/wiki/Polytene_chromosome
... unless of course you really feel a need for the technical jargon

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apochronaut
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Re: Fruit fly chromosomes

#9 Post by apochronaut » Wed Jul 31, 2019 11:51 pm

Quite remarkable, although if you hadn't explained what it was, I would have thought it was an old coin depicting a Rugger scrum.

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75RR
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Re: Fruit fly chromosomes

#10 Post by 75RR » Thu Aug 01, 2019 5:53 am

MichaelG. wrote:
75RR wrote:Hoping to better understand what I was looking at I looked up endoreduplication in Wiki. Wish I hadn't ;)
I see what you mean

This one is rather more comprehensible:

https://en.m.wikipedia.org/wiki/Polytene_chromosome
Indeed it is! Many thanks
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MicroBob
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Re: Fruit fly chromosomes

#11 Post by MicroBob » Thu Aug 01, 2019 12:07 pm

Hi Wes,

great image! I would be interested in a closer description of the preparation. You dissected the fly in an isotonic solution - and then, did you squash the sample under the cover slip?

Bob

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Wes
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Re: Fruit fly chromosomes

#12 Post by Wes » Thu Aug 01, 2019 2:58 pm

Thanks Bob. I place the larva in a drop of saline and then grab it on both ends with two fine tweezers. I then pull it apart so the digestive tract comes out completely or partially. Using the tweezers I then tear the remaining tissue into smaller pieces and once there are no large chunks I squish it with the coverslip. Moments like these make me wish I had a stereomicroscope because then I could isolate individual organs and systems.
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Sauerkraut
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Re: Fruit fly chromosomes

#13 Post by Sauerkraut » Thu Aug 01, 2019 3:33 pm

Wes wrote:Moments like these make me wish I had a stereomicroscope because then I could isolate individual organs and systems.
What?! You did this without a stereomicroscope??? That's impressive. Very nice work. I find myself hoping you get a stereomicroscope so we can see isolated organs and system slides.

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Wes
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Re: Fruit fly chromosomes

#14 Post by Wes » Sat Aug 03, 2019 10:23 am

tgss wrote:Can you provide an idea of scale - width of the imaged field or something similar?
I measured the pixel distance corresponding to 10 µm on a stage micrometer photographed under the same conditions and then set this as a global scale in imageJ which I used to generate the image below. The nucleus is 42 µm in diameter whereas the chromosomes are about 2-3 µm thick.
Sauerkraut wrote:
Wes wrote:Moments like these make me wish I had a stereomicroscope because then I could isolate individual organs and systems.
What?! You did this without a stereomicroscope??? That's impressive. Very nice work. I find myself hoping you get a stereomicroscope so we can see isolated organs and system slides.
Its not a very precise procedure to be honest, more of a microscale butchering hoping to get something interesting on the slide.
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polytene_scalebar.jpg
polytene_scalebar.jpg (244.27 KiB) Viewed 6780 times
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tgss
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Re: Fruit fly chromosomes

#15 Post by tgss » Sat Aug 03, 2019 2:41 pm

tgss wrote:Can you provide an idea of scale - width of the imaged field or something similar?
Wes wrote:I measured the pixel distance corresponding to 10 µm on a stage micrometer photographed under the same conditions and then set this as a global scale in imageJ which I used to generate the image below. The nucleus is 42 µm in diameter whereas the chromosomes are about 2-3 µm thick.
Wow! Thank you for going to all that trouble Wes. But, to me, a very interesting result. That is huge! Based on your post I am now checking out polytene chromosomes for myself - quite a fascinating subject. I also came across this paper, which you may find interesting, if you are not already familiar with it:

"Preparation of Drosophila Polytene Chromosome Squashes for Antibody Labeling"
which is here:
https://www.ncbi.nlm.nih.gov/pmc/articles/PMC2818712/

Thanks again for sharing.
Tom

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Re: Fruit fly chromosomes

#16 Post by MicroBob » Sat Aug 03, 2019 6:33 pm

Wes wrote:more of a microscale butchering hoping to get something interesting on the slide.
Hi Wes,
this is about what we do in our microscopy group. We cover widely spread topics so usually only a few people have experience in this area. Where experience is lacking we depend on our enthusiasm :lol: and it is always a joy to view and take part.
I have saved this topic as a possible future topic as it would fit in our time time frame and is very interesting.
We once had a topic about chromosomes and genetic manipulation were we looked for chromosomes in onion root tips and were showed a PCR, a power bank sized genetic analyser and a method to extract the DNA of a kiwi with household methods. This was a very impressive topic that I will remeber a long time.
We don't have a lot of members who could do the dissecting with the bare eye - but we have stereo microscopes and can help each other.

Bob

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Wes
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Re: Fruit fly chromosomes

#17 Post by Wes » Sat Aug 03, 2019 8:01 pm

MicroBob wrote: extract the DNA of a kiwi with household methods.
Hi Bob, If you ever get to repeat the extraction, save the DNA and throw it under a polarizing microscope. If its concentrated enough and with the right salts you may observe liquid DNA crystals.

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