Postcard from crazyland

Contrary to all appearances, this is completely real. O.O

Temereva_Tsitrin2013_3b

What you are looking at is the fluorescently stained muscles of a phoronid larva. (Phoronids are one of the zillion variations on “tentacled filter feeder sitting in a tube” nature has come up with. They are related to lamp shells.) I couldn’t care less about larval muscles – not even sure why I opened the paper – but that looks friggin’ awesome. And a bit psychedelic.

(Source: Temereva & Tsitrin (2013), BMC Developmental Biology 13:14)

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Shining a light on retinoic acid

I was planning to do more bioinformaticky stuff tonight, but then I saw Shimozono et al. (2013), and… SHINY!

I derive a particular joy from seeing neat methods, and what these guys did is pretty damn neat. They used genetic engineering and a clever trick with fluorescence to (almost) directly study an important but rather elusive molecule in vertebrate development.

Retinoic acid (RA) is related to vitamin A; in fact, it is synthesised from vitamin A by enzymes in our cells. It is what developmental biologists call a morphogen: a molecule that spreads through an embryo by diffusion, and influences development depending on its concentration. Among other things, RA is thought to be responsible for the subdivision of the embryonic body axis by Hox genes, and also the correct formation of somites, the basic repeating units that eventually form our spine.

So RA is pretty darn important, but it’s also a bit difficult to investigate. It’s a relatively small and simple molecule that isn’t encoded in the genome, so some of the popular tools for detecting important molecules don’t work on it. Its activity can be monitored indirectly, though. Retinoic acid works by binding to proteins called retinoic acid receptors (RARs), which then latch onto certain DNA sequences that regulate nearby genes. So you can, for example, construct a piece of DNA that responds to an activated RAR by producing a fluorescent protein. You can also examine the distribution of the enzymes that make and break down RA, the assumption being that this corresponds to the distribution of RA itself.

The Japanese team, however, created a modification of retinoic acid receptors that is basically a direct indicator of RA level. Their RARs have been engineered to glow in different ways depending on whether or not RA is bound to them. They were able to zap these miniature RA detectors into zebrafish embryos without affecting the little creatures’ development, creating a gentle way to monitor RA levels in live animals.

They exploited a fascinating phenomenon called fluorescence resonance energy transfer (FRET for short). FRET needs two fluorescent molecules that glow at different wavelengths, such that the wavelength one of them emits is the same that turns the other on. (Wikipedia tells me FRET is actually based on spooky quantum effects involving virtual particles rather than ordinary light travelling from molecule to molecule. Wow, I didn’t know that!)

If the two molecules are very, very close, the emissions of the first one can give the other enough energy to light up. You can detect this by shining the colour of light needed to excite the first molecule on your sample, but then also measuring the fluorescence from the second molecule. The ratio of Molecule 1 to Molecule 2 glow can tell you how much FRETting is going on.

What Shimozono et al. did was to add the code for a FRET-capable pair of fluorescent proteins to various RAR genes. RARs change shape when retinoic acid binds to them, and in these engineered versions this means that they bring their fluorescent tags close enough for FRET to work. (The above figure, from Carr and Hetherington [2000], illustrates the principle – just substitute “Ca2+” with “RA”.) The scientists calibrated their little RA detectors by measuring how much FRET happened at various controlled RA concentrations first; this allowed them to turn FRET intensities into accurate measurements of RA. They then tested whether the detectors were truly RA-specific (and not activated by, say, vitamin A) by using them in fish embryos with their RA-making enzymes crippled.

Of course, they also got round to looking at the behaviour of RA during development, which was, after all, the point of their new toys. They did a basic visualisation of RA concentration throughout developing embryos – and confirmed that the established method of looking for the enzymes involved in RA synthesis and degradation is actually a decent substitute for measuring RA itself.

They then interfered with the production of a protein called FGF8 that is thought to regulate RA synthesis, and found that this altered the RA gradient – as well as the expression of the main enzyme that produces RA. Basically – the technique seems to work, and what it shows agrees with what we’ve thought about RA signalling. Hooray!

And, of course, they got pretty pictures like the ones below, coloured according to the amount of FRET (red = high, green = low) they measured. These two compare a normal embryo (left) and an embryo of the same age whose fgf8 gene has been messed with (right). If you have normal colour vision*, it’s pretty clear how the control embryo has this massive band of redness halfway down its body, and how even nearer the head and tail ends it’s more yellow than the sad green of the treated baby.

(I spliced these together from panels in an overwhelmingly massive figure and labelled them for those of you who don’t look at fish embryos much. No copyright infringement and no financial gain is intended, of course ;))
Shimozono_etal_fgfEdit

I think this whole thing is waaaaay cool. I wish I could come up with something clever like that. Oh well, at least I get to work with fluorescent things and take pretty glowy pictures every now and then. When I’m not neck deep in protein sequences and ‘omics data. 🙂

***

*Being a red-green colour blind developmental biologist must be a hell of a lot of fun. I just realised that pretty much everything involving fluorescence in biology is red, green or both – and developmental biologists love sticking fluorescent tags on everything. By the way, this particular figure could have been presented in any old combination of colours – they’re illustrating abstract numbers, not the actual colours of the specimens, which in this case would have been glowing in cyan and yellow. Of course, there’s probably a colour vision deficiency for every combination you can think of, so, uh. I probably overthunk this?

***

References:
Carr K & Hetherington A (2000) Calcium dynamics in single plant cells. Genome Biology 1:reports024

Shimozono S et al. (2013) Visualization of an endogenous retinoic acid gradient across embryonic development. Nature 496:363-366

I believe I can fly…

I feel like I should let the interwebz know I still exist. I’ve been doing a little too much computer-based work to have any typing energy left for proper meanderings, but in the meantime, I’d like to present one of my favourite pieces of palaeo-related fun. Meet Alain Bénéteau’s Archie, the early bird who tried a little too hard 🙂

The entire series can be found on deviantART. (If you are into palaeoart, his entire gallery is well worth checking out. Dinosaurs and extinct sharks galore, all gorgeous and life-like.)

Specialisation is good for your soul

When I was little, I wanted to know everything. At age six or seven, I could whip out an explanation of why the sun shines, nuclear fusion and all, and by the time I hit my teens, I’d memorised the basic properties of a couple of hundred dinosaur genera, everything cetacean, and every planet in the solar system (back when that still included Pluto :-P) My family members are still a bit surprised if a science question comes up over the dinner table and I answer “I don’t know”.

During my undergrad years, specialisation was my nightmare. While I could, I took classes in maths, programming, geology and something vaguely philosophy of science-ish in addition to my compulsory credits in biology. My BSc is called evolutionary biology, but the actual subjects I studied for it range all the way from biochemistry to ecology.

But you know what?

After 2+ years of working on a single part of a single animal, I finally feel like I know something.

As an obsessive learner and insufferable know-it-all, the real world was bound to give me some big shocks. The first was venturing onto the internet, and getting a near-infinite pile of information dumped on me by Google. That experience might have been why I lost most of my interest in dinosaurs – there just seemed to be too much to learn. That’s a hard pill to swallow for a young know-it-all!

And then I went to university, and met the scientific literature. Even more than first googling dinosaurs, that made me realise that I knew nothing. Ever since then, I’ve never quite felt secure about my grasp of any field. There were always papers I hadn’t read, ideas I didn’t really understand, facts I hadn’t included in my reckoning. I often feel like I can’t form an opinion on anything, because there’s a part of a discussion I’ve simply missed or didn’t pay enough attention to.

No, I’m nowhere near satisfied with my current knowledge of my own area (now that I have an area I can call my own). I don’t think I’ll ever be, and if it happens it’s probably a good sign that I should read more. But when I look at my animals, when I have to tell others about my work, I feel… comfortable. This is my stuff, and while I may not know everything, I know some things in an intimate way only close study can give you. It is an immensely satisfying feeling. And it makes me think that perhaps, specialisation isn’t such a bad thing after all.