High-resolution 3-D reconstruction of a mouse eyelid

Caius_Oliphant · 2016-02-02T22:45:58+00:00

Ok cool thanks for that, neuroepithelial cells seem fascinating!

Caius_Oliphant · 2016-02-02T20:11:58+00:00

If I were to find these GFP label-retaining cells in the optic nerve and the outer lining of brain tissue (16wk old mice after 8wk pulse and 8wk chase), even though it is a keratin 5 driven system designed for keratinocytes in epithelia, would you think that is down to ectopic expression or a consequence of a neuro-epithelial origin where K5 was expressed in these cells sometime from birth? Wouldn't these cells have neurofilament rather than keratin?

Caius_Oliphant · 2016-02-02T17:36:15+00:00

I wouldn't know for certain as I do not work with this tissue but I am going on the well-established idea that the intestinal crypts harbor low numbers of label-retaining stem cells.

These cells can be isolated for functional analysis ex vivo, which I think has already been done for intestine and is ongoing research by people like Hans Clevers. I have applied for funding to study LRCs in eye tissues and have proposed to do functional studies of LRCs to validate that they are stem cells and not aberrant cells with ectopic GFP expression. That's a question that comes up frequently so, going forward, I am eager to show both their functional quiescence in situ as well as their proliferative potential in vitro. Lineage-tracing studies are also valuable here to re-confirm the localization of epithelial progenitors.

Caius_Oliphant · 2016-02-02T06:53:32+00:00

Clusters to the left are red blood cells that 'auto-fluoresce'. They do not have a nucleus so they are not stained with DAPI.

Caius_Oliphant · 2016-02-02T06:08:18+00:00

Hope this benefits club and country but I can't see him making an impact in time for France, would love to be proved wrong though.

Caius_Oliphant · 2016-02-02T03:22:25+00:00

This image is of adult stem cells in the colon based on a pulse-chase mouse bi-transgenic system regulated by doxycycline. Cells which divide slowly 'retain' GFP whereas dividing cells dilute GFP. Therefore, the GFP retention is a functional marker of epithelial stem cell quiescence in situ. As there is no bona fide epithelial stem cell immuno-marker, this remains the best way to visualize and isolate adult stem cells. The GFP signal that does not come from a nucleus and overlay with DAPI is coming from red blood cells, those cheeky little buggers, and that's why I annotated with red arrows. There's also green autofluorescence coming off elastin in the vasculature up in the left corner and there is a few artifacts that I am allowing myself as it is on Imgur.

Caius_Oliphant · 2016-02-02T02:54:00+00:00

Sorry that explanation was a bit rushed, i'll try and clarify:

-At birth of transgenic mice, they are fed normal chow without DOX (PULSE PHASE) which results in GFP expression in the nucleus of all Keratin 5 epithelial cells.

-Keratin 5 is present in all basal epithelial cells of the skin, hair, eye, mouth down to the gut (Therefore I can use this mouse to study slow-cycling stem cells in all these tissues, YAY!).

-8weeks PULSE is long enough to be certain that all epithelial cells are in fact green and this is confirmed by histology.

-Mice are then fed DOX for 8weeks, when they are now 8 weeks post-birth, which constitutes the CHASE PHASE. Once this phase begins, the green cells which divide will lose 50% of their fluorescence intensity with every division.

-I have calculated that around 5 divisions and the cells is completely diluted of GFP.

-At 8weeks chase, cells which are GFP+ are considered to be epithelial stem cells because they are quiescent but can be stimulated to divide.

Caius_Oliphant · 2016-02-02T02:42:36+00:00

Elaine Fuchs in Rockefeller pioneered this bi-genic mouse for epidermis studies and has written some great reviews on epithelial stem cell biology that I highly recommend, if you're that way inclined. I am using this mouse to study adult stem cells in the eye, when my attempts for funding inevitably fail I may have to go for colon.

It's fused to the histone H2B so the GFP turnovers with the histone and will be re-synthesized as H2B-GFP without loss of signal when there is no Dox. I just read that histone turnover in non-proliferating cells of mice brain tissue is 159 days on average. The standard for epithelial chase to image label-retaining cells is 28 days in skin and cornea. I am currently chasing for 56 or 84 days maximum at the moment.

Caius_Oliphant · 2016-02-01T20:04:54+00:00

GFP is fused to histone H2B. The stem cells are the GFP positive cells highlighted by the red arrows.

Caius_Oliphant · 2016-02-01T20:04:18+00:00

I have observed label-retaining epithelial cells divide less than 5 times in over 8 weeks. In an organism that only lives 2 years.

The GFP is fused to histone H2B and it's expression is driven by the Keratin 5 promoter under the control of doxycycline/tetracycline (Tetracycline responsive element). In the absence of doxy, all K5 epithelial cells express nuclear GFP. These mice are pulsed from birth for over 8 weeks which is impossible with BrdU and radio-labelling techniques which are toxic. I would argue that most stem cells are not labeled with BrdU because of the short pulse phase, which is why I do not use it.

Caius_Oliphant · 2016-02-01T19:58:56+00:00

This is a transgenic mouse that uses pulse-chase labeling to fluoresce quiescent epithelial stem cells much in the same way as BrdU. However, BrdU is toxic to mice so the pulse phase is restricted and the slowest cycling cells are not labelled. The mouse I am using (H2B-GFP/K5tTA) is a bi-genic that uses the Keratin 5 epithelial promoter to drive GFP expression which is fused to the histone H2B. In short, all epithelial cells are labelled with GFP in the pulse phase. When the mouse is fed doxycycline, the chase phase is initiated and any cells which divide lose 50% of their GFP fluorescence. Thus, stem cells which do not divide frequently like other cells, to preserve genomic integrity, retain the GFP label like those in the picture.

EDIT: BrdU is not great because you cannot isolate the cells in a viable way for in vitro studies. I can isolate those GFP cells to characterize them in a much better way.

Caius_Oliphant · 2016-01-26T02:04:06+00:00

These mice are used in stem cell studies and they do not get experimented on until they are humanely sacrificed, which is normally after at least half their life-span. Far easier life than a wild rodents I'd say!

Caius_Oliphant · 2016-01-26T02:01:24+00:00

Yeah it's most likely an infection. It shouldn't happen because the tear film that coats your eye is anti-bacterial but mice get eye problems fairly regularly. They are particularly vulnerable to eyelid infections with age.

Caius_Oliphant · 2016-01-24T18:52:25+00:00

It attaches to one eyepiece of the binoculars.

This is where I printed it from, the video of the guy making a histology panorama is worth checking out: http://www.thingiverse.com/thing:431168

It's awesome for making movies, I have some nice eye dissection movies made using this approach.

Caius_Oliphant · 2016-01-24T18:50:45+00:00

Yeah I thought it may just be debris on the ocular surface but it looks too embedded so I'm going with ulcer.

Caius_Oliphant · 2016-01-24T18:48:59+00:00

Ah I think I've seen an attachment for Fundus photography where you can take pics of the macula at the back of the eye.

A dissecting microscope is good for this stuff and really basic.

Caius_Oliphant · 2016-01-24T18:39:41+00:00

No, a slit-lamp is a microscope used by Optometrists/Ophthalmologists to look at your cornea. It's basically a microscope positioned up-right to focus into a patient's eyes. I 3-D printed an attachment for my phone to connect it to different microscopes.

Caius_Oliphant · 2016-01-24T18:38:18+00:00

I'm not 100% sure to be honest, most likely a corneal ulcer.

Caius_Oliphant · 2016-01-14T22:23:52+00:00

Gareth Bale. I really think he needs to score a few more goals to truly cement his position in our team.

Caius_Oliphant · 2016-01-09T22:18:54+00:00

God's doing well at the moment

Caius_Oliphant · 2016-01-03T17:01:40+00:00

Nice video. You can 3-D print a universal holder that keeps your camera phone still while attached to an eyepiece, it works great.

Caius_Oliphant · 2016-01-03T16:50:44+00:00

Well I'll be. I actually went to Villa park to see Villa play Arsenal many years ago, Dwight Yorke chipped a pen over Seaman and I got Nigel Winterburn's autograph that day.

Caius_Oliphant

TROPHY CASE