I decided to do a little electronic housekeeping with my Gmail account this morning; archiving all of my old mail to save on space.  While I was scanning some subject lines from the Cambridge days and I came across this little gem.

One of the down-sides to working with living biological material is that you’re subject to their rhythms; a slave to the organism, if you will.  This was the case whenever working with a developing mouse embryo.  I was trying to understand the first fate decision the embryo makes: make placenta or make embryo.

A quick (if not slightly surreal) embryology primer

In mammals, embryos implant to some extent inside the mother’s uterus.  The connection between mother and embryo is mediated by a placenta, and this organ is embryonic in origin, and the cells that make it up become specified in development quiet early.  Think of it this way, if you put eight marbles in a small bag and started adding marbles, eventually some of those marbles would have to be on the inside of the mass-of-marbles, surrounded by the remaining outer marbles.  Similarly in the embryo, once the 8-cell embryo divides to make 16-cells, two groups of cells emerge.  The outer group of cells will go on to form the placental tissues, and the inner cells will go on to form the embryo itself.  Our group at Cambridge was trying to understand these early fate decisions in the mouse embryo: why do some cells decide to make one type of tissue and others make others?  In developmental biology, this is referred to as differentiation, when an unspecialised (stem) cell adopts a more specialised function.  Often, this happens because some cells, like say your muscle cells, utilise certain genes in your genome that others, say the cells in your brain, do not. So if the genome was a magazine that cells could subscribe to, then every cell in the embryo has the exact same subscription.  However for some reason neighbouring cells have differing reading habits. One cell decides to only read the articles in the odd-numbered issues whose authors’ surname begins with the letters A-M, while the one right beside it keeps to the articles whose titles contain less than ten words.  There will be some overlap, but ultimately cells read from differing sets of instructions, leading to different shapes and functions.

So at any rate, if the goal is to understand what’s going on from 8-to-16 cells, then all experimental manipulations have to happen beforehand.  In the case of the day the universe smiled on me, I was having to work with 4-cell embryos.  The goal was to modify one or two of the four cells by injecting them with fluorescent markers or something to alter their genetic circuitry.  By observing how the embryos develop and comparing the relative contributions of inner or outer cells between modified and non-modified cells, we can start to understand how those genetic circuits control this decision process.

Here are some stills from time-lapse imaging of one of the first embryos I ever modified. In this case the embryo was injected with two genes, one that coded for a fluorescent marker in the cell membrane (RED) so that the individual cells were visible, and a fluorescently tagged tubulin (GREEN), part of the network of proteins that makes the spindle that pulls copied chromosomes into daughter cells at cell division.  What you’re seeing here are sections through the centre of the cells.  The black spots in the green are the nuclei, where the genome is housed.  The orientation of the spindle helps guide cells to either the inside or outside position in the embryo. What orients the spindle is still very much an open question.

Meanwhile back at the lab, those mouse embryos would arrive at the late-4-cell stage somewhere between 2-4 AM.  Factoring in arrival time and prep, etc, my night-before was pretty much reduced to dinner, some down-time then back to the lab. [sigh] It took me a good few months to figure out the perfect dosage of coffee necessary to produce adequate alertness without getting so jittery that I would lose all fine motor skills.  Microinjection involves using a glass needle, pulled to a point that is less that 1 micron or a one-millionth of a metre, and injecting it under a microscope using (quasi) robotic manipulators into a cell that’s about 10-15 microns in diameter, without running it though with the needle, or poking the nucleus which usually results in the needle coming back covered in stringy DNA (also not good). Delicate to say the least. [hand-tremour]

So after injecting, it’s about six-ish AM. I was feeling good; it had been a good injection session. The Strokes were playing on my MP3-player. I put the embryos on a warming plate, while I started preparing a new dish for the embryos.  While still looking down the oculars of the microscope, I flipped over the dish containing my little embryos, my experiment (read: about four days worth of planning and work). I watched everything in that dish ooze out all over the work bench.  The whole situation happened out of my view in a fraction of a second, but I imagined the whole thing in slow-mo, like in the movies with the voices slowed down to that demonic drone.  What followed were the 4-STEPS of grief experienced by all researchers who watch days of their work flash before their eyes.  1. An impressive barrage of vulgarity and profanity. 2. Denial: No, I didn’t just knock over [the dish with my experiment], it was [that OTHER dish]. 3. More vulgarity and profanity.  4. Existential Crisis:  Why didn’t I go into [insert profession], I bet THEY never have to deal with this [profanity]!!!

But then the universe smiled down on me in those wee hours of the morning.  In a last-ditch, semi-defeated attempt to reclaim the previous four days and six-ish hours of lost sleep, I put the dish, now empty save for an oil slick with microscopic droplets of the medium we culture embryos in (we keep them under oil, hence the slick).  The miracle that is the physico-chemical relationship between water (well, media+embryos actually) and oil came through for me! In one of those tiny droplets, there were all of my embryos; my masters. Sigh. Existential what?!  Five or so hours later was a brand new day.

AND NOW LADIES AND GENTLEMEN…HEEEEEEEEEERE’S GRACE!!

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