Happy Galentines day and a late happy International Day of Women and Girls in Science – that was February 11, and it’s meant, in part, to draw attention and appreciation to underrepresented voices in the field of science. So I decided to honor the day by writing another new Wikipedia article on a female scientist – one you likely haven’t heard of, but whose work has been critical to understanding how the brain works – Eileen Southgate. She, along with John White and Nichol Thomson in the 1980s, under the guidance of Sydney Brenner, mapped (by hand from ~8,000 serial section electron micrograph prints) every single nerve cell in the worm Caenorhabditis elegans (C. elegans). This was the first time such a map of an animal’s “mind” had been made, and I want to make sure her story never will fade!
Eileen does get credited for her work – she hasn’t been “excluded” from the history books, but she’s not widely-known. And one reason Eileen isn’t more well known is probably that she was “just” a lab technician/assistant – a few months ago I did a Bri*fing on lab structure and how there are employees called lab technicians. These days they typically have bachelors degrees, sometimes masters, but not PhDs and they carry out a lot of the more “rote” lab work to help the other scientists. They often do the things that no one else wants to do, but which need to be done for a lab to run! Things like preparing stock solutions, maintaining cell cultures, helping with molecular cloning, etc., depending on the type of lab and its needs. They are crucial to science getting done, and done well, but they’re also often greatly under-appreciated. more here: http://bit.ly/2ojdm0m
These days it’s typical for lab techs to have at least a bachelor’s degree, but Southgate started lab work fresh out of high school. In fact, she was only 16 years old when she began working at the Medical Research Council (MRC)’s Laboratory of Molecular Biology (LMB) in Cambridge, England. In the beginning at least, it wasn’t that she had a burning desire to study biology. Instead, it was just the best choice offered to her when career counselors came to her school with a few options; working on “helping with medical research” sounded best.
The MRC is kinda special in the way it’s run. It had big research units that were overseen by group leaders and then there were individual labs within those larger groups, each led by a different scientist. Over the course of her career, Southgate worked for several labs within the LMB, in different areas of research. She thus did a lot of different types of tasks, ranging from growing plants to preparing proteins. But she’s best known for doing a task that not many people would be willing to do, let alone want to do – mapping – by hand – each of the nerves in the roundworm C. elegans. This required slicing the worms up into thousands of sections, taking electron micrograph “pictures” of them, and tracing the path of each nerve throughout the tiny (~1mm long) worm’s body.
One might ask, “How did she get tasked with this technically difficult, yet terribly tedious task?” The answer – she went to work for Sydney Brenner. And Brenner wanted to know how the nervous system developed and worked and how that related to those “gene” things that were becoming all the rage – this was around the time of the molecular biology revolution and scientists were just beginning to be able to interrogate genetics in simple systems.
Humans aren’t very simple but, thankfully, in biology there’s this concept of “model organisms” – basically it’s really hard (scientifically & ethically) to conduct research on humans – so to understand how key biological processes work we often turn to other animals (or plants, if that’s your jam) to figure out what’s going on. Thanks to evolutionary conservation, “what’s going on” is often really similar – when molecular mechanisms work really well, why change things, right?
At a more scientific level, evolution occurs because of random mutations, but only the helpful (or at least not harmful) ones provide a “fitness advantage” (or at least no disadvantage) so they stick around and get passed on to progeny. So if the “best” forms are figured out early on, before species diverge, those best forms are likely to be kept by all the split-off groups.
Some model organisms commonly used to study animals are mice, rats, fruit flies (Drosophila melanogaster) and the soil-dwelling nematode (roundworm) Caenorhabditis elegans (C. elegans). Different model organisms are good for different things. And one of the things that makes C. elegans a great model organism for certain things is that it has a precise, relatively small number of cells (exactly 959) and a fixed development cycle. They’re also transparent, easy to grow (just feed them some E.coli on a Petri dish) and reproduce quickly (albeit strangely – instead of male and female they have male and self-fertilizing hermaphrodites). So a lot is known about these worms. A LOT. Including, thanks in large part to Eileen Southgate, the position and interconnection of all of their nerve cells (neurons).
Sydney Brenner introduced C. elegans to the MRC-LMB in 1963 and set out to study how the worms developed and how their genes controlled things. He also had to convince the scientific community that it wasn’t a “joke model organism.”
He was studying a bunch of “mutant” worms – worms with genetic mutations that made them have abnormal features or quirks (we call such observable effects of genetic differences phenotypes). He’d isolated some worms with “uncoordinated” phenotypes – he called these jerky worms unc mutants. He knew that nerves control movement, so he wanted to be able to connect the genetic mutations with changes in the nerves. So he needed to know how the nerves were laid out.
Despite the worm’s small size, this was an astronomical task! And the team who carried it out included Southgate (who did most of the pic-taking & tracing), John White (a grad student then postdoc who helped her put the pieces together), Nichol Thomson (an electron microscopist who helped them prepare thousands of worm slices); and Sydney Brenner (who oversaw it all and provided the $).
One thing that made C. elegans a good candidate for nerve-mapping is that these roundworms are transparent, and if you slice them up into thin serial sections (think of a bread loaf but with super super thin slices) you can image those slices using electron microscopy (EM). Basically, EM is like a light microscope, but it uses waves of electrons instead of visible light waves (which are made up of energy packets called photons) because electrons have the shorter wavelengths needed to make out (resolve) tiny features. In order to make those features (like the membranes surrounding separate neurons) stand out, you can stain them with heavy metals like uranium and lead which help to provide contrast.
In order to get the images, they grew worms on E.coli-coated agar Petri plates (agar is a sugar that’s often used to form a gel matrix). Then they washed the plates and fixated the worms in osmium tetroxide, an oxidizing agent that promotes the formation of additional stabilizing bonds between neighboring molecules to keep things stuck where they are. Next, they spread the fixated worms onto agar and cut them in half. Then they added more agar on top of them to give them half-worms trapped in agar blocks. After hardening up the blocks by dehydrating them with alcohols, they transferred them to Araldite resin and used an ultratome (section slicer) with a diamond knife to cut transverse serial sections, which they stained and imaged. We’re talking super super thin slices – each slice was only 1/20 of a micron thick (and a micron is a millionth of a meter, or a thousandth of a millimeter)
After literally cranking out (the scope had a Rolodex-style plate loader) EM images & printing them as 12×16-inch prints, Southgate would take each print-out & label and follow each neuron and its “processes” – in each of ~8000 images. This task was even harder than it sounds, because neurons aren’t simple cells… Neurons have “big” main cell parts – the “cell bodies “and extended “arms” called processes. Groups of processes from different neurons travel in bundles or “tracts” which end in different places, like muscle cells or other neurons. Nerve processes can have also have branches that make tracing even more challenging, but thankfully C. elegans’ neurons are mostly unbranched.
Southgate worked backwards and forwards between the different images to make sure she knew which process belonged to which neuron cell body. The worms are really small, so instead of making a life-size, or even a scaled-down model, she blew things up – for some of the work she made an ~2ft-long 3-D recreation out of thin pieces of plywood which she painted. The work was painstakingly difficult and took around 13 years to complete.
Some of the unanticipated difficulty was finding a publisher willing to publish the ginormous (340-page-long) manuscript – but they eventually did and, in 1986, Philosophical Transactions of the Royal Society B published their landmark paper, “The structure of the nervous system of the nematode Caenorhabditis elegans.” The paper is affectionately known by its running title, “The Mind of a Worm,” the fun name the group had labeled the notebooks containing their manuscript to easily find it.
To help you really understand the enormity of the task, here’s how the work is described in their own words:
“Small groups of processes were given arbitrary labels, which were written onto the prints with Rotring drafting pens. These labels were carried through all the pictures in which the associated processes were present, and this procedure was repeated until all process profiles were labelled. Processes could then be joined to other processes where branches had occurred, or ultimately be assigned to particular neurons if their cell bodies were within the scope of the reconstruction. When all the labelling was completed, each process was individually followed through every section in which it appeared, and a list was compiled of all the synaptic contacts that it made. In this way all synaptic contacts were recorded twice, once for each member of an interacting pair of processes. This provided a useful check on synapse scoring as any synaptic contact that was only scored once was reappraised.”
Yowza! By “synaptic contact” they’re referring to places called synapses where the ends of neuron branches interact with other cells – those can be other neurons or muscle cells. Some neurons (sensory neurons) receive sensory input (signals from touch, smell, etc.) from the environment and transmit that info to other neurons. Some neurons (interneurons) receive messages from other neurons and pass them on to additional neurons. And a third main type of neuron (motor neurons) pass info from neurons to muscle cells. The nerve-to-muscle connections are called “neuromuscular junctions” and the nerve-to-nerve interactions include chemical synapses that communicate by passing chemical messages and gap junctions that communicate through electrical signals.
They identified close to 8,000 total synapses: ~2000 neuromuscular junctions, 5000 chemical synapses & 600 gap junctions. Additionally, they grouped the neurons into 118 classes (which is quite a lot considering that they only found 302 total neurons). They found that the layout and connections were virtually the same in genetically-identical worms, which was a good thing for them because their map of a “single worm” was actually the result of slices from many different worms (they couldn’t get great slices of all the parts of a worm from a single worm and they needed multiple slices from the same area to resolve ambiguities).
The work was groundbreaking – not only was it the first animal to have its nerves mapped out, but having that map allowed scientists to answer questions about the nervous system. They were able to identify and characterize nerves involved in various biological circuits like ones important for-egg laying. Once scientists knew what nerves were involved with what they could perform further experiments to see how those nerves responded to various manipulations and what the broader effects were. In short, it helped develop C. elegans into the invaluable “model organism” it is today.
I first came across the name “Eileen Southgate” when I was doing research on hemoglobin, the protein that carries oxygen throughout the bloodstream. It’s actually thanks in part to Southgate that we know what hemoglobin looks like. The first hemoglobin structure was solved (by x-ray crystallography) by Max Perutz, who was Southgate’s first “boss” at the MRC when she began working there in 1956.
X-ray crystallography is a technique in which you get molecules such as protein copies to organize into a lattice formation and then you beam x-rays at them. The molecules scatter the x-rays, the scattered x-rays interact, and, long story short, you get a pattern of spots called a diffraction pattern, showing where the scattered, interacted, rays hit a detector. Then you can work backwards from the spots to find the positions of the scattering atoms to see how the molecule is laid out. more here: http://bit.ly/2QASc8h It requires proteins to be really pure – and Eileen, among other jobs, was tasked with helping prepare hemoglobin & the related protein myoglobin, for crystallography.
I first brought up hemoglobin a few months back because of its connection to sickle cell disease, a genetic disease in which a mutation in hemoglobin causes it to clump up and block blood vessels. It’s caused by a single DNA letter change leading to a single protein letter change that alters hemoglobin’s shape. That protein letter change was discovered by a scientist named Vernon Ingram – and Eileen helped him with his work.
In 1962, she went to work with Tony Stretton. And it was here (after first working on the sugar break-down-er β-galactosidase) that she was introduced to worm work – but not C. elegans. Instead, she was investigating another, larger, nematode called Ascaris. Unlike the soil-living C. elegans, Ascaris lives in pigs’ intestines and they got the worms from a nearby slaughterhouse…
Working with Ascaris, she developed skills that would prove invaluable for her later work – they were trying to map out some of this worms’ nerves, but using light microscopy, not EM. When Tony left, moving to Wisconsin with his “big worm” project, she joined John White (who was then a PhD student) on his “small worm” project – reconstructing the C. elegans nervous system. She was working at a much smaller scale worm-size-wise – instead of ~10cm, C. elegans are ~1mm. And that’s the whole thing – she was looking at tiny parts of these tiny things.
I can’t even imagine how difficult that must have been, but apparently Eileen had the perfect qualities for it. John White described her this way: “she had many attributes that I lacked: she worked steadily and meticulously, she had a good visual memory, and she took careful notes of all that she did” http://bit.ly/38viP66 And, since thousands of nerves weren’t enough of a puzzle for her, she did jigsaw puzzles as a hobby! Eileen retired in 1993.
Here’s a link to The Mind of a Worm, from which I chose just a few of the >100 figures to include in the pics: http://bit.ly/2SrrDV2
If you want to know more about Wikipedia editing, here’s some info: http://bit.ly/37qejEU
You can read her Wikipedia article here: https://en.wikipedia.org/wiki/Eileen_Southgate (but this post is more funnly-worded.
And speaking of this post, it’s part of my weekly “broadcasts from the bench” for The International Union of Biochemistry and Molecular Biology. Be sure to follow them on social media if you’re interested in biochemistry! They’re a really great international organization for biochemistry.