You know, a lot of times, when you--as a scientist--think back to experiments that you've done
even really successful experiments, you realize that at the time that you were doing them,
you didn't really fully understand the science involved or the science behind them
or the science which was going to affect whether you were going to get results or not,
and you probably also didn't really appreciate how lucky you were.
Today, we're going to talk a little bit about one experiment--a wonderful experiment--
that I did many years ago with Christianne Nüsslein-Volhard
at a time when we were both starting out our scientific careers.
We, like many people at the time (this was the late 1970s)
were interested in the problem of embryonic development,
but at a very basic level.
We knew that genes controlled things that you could see happening in the embryos.
We wanted to identify what those genes were and what they did.
And there were a lot of different molecular strategies--conceptual strategies--
that you could use to do this.
We decided that we would try a genetic approach and what that meant for us
was that to build on our lack of knowledge and to just randomly mutagenize all genes--
knock them all out and see what happens,
and get some estimate at least or some global picture of what it was the genes were doing during development,
how gene activities were organized in patterns and in sequences
to produce these extraordinary phenomena that you see in development.
We knew that there were lots of genes,
and we knew that understanding the process would probably require that we identify most of them,
so we had set up in our minds at least this goal that we wanted to identify everything.
We wanted to identify all the genes, and this meant a really big experiment.
And for those of you who have any sense or experience working with Drosophila,
what a big experiment looking for lots of genes means is lots of tubes,
growing lots of flies, and setting up crosses.
Basically, our plan was to feed flies mutagens, establish inbred lines from single males
in the first generation and then carry it through an appropriate number of generations
to produce inbred heterozygous lines that we could collect homozygous embryos from.
And we thought about it, and our estimates of what we needed to do was something like
in the range of maybe 40,000 lines established through many generations.
And for us to do that, we realized
(and this is actually the first real secret and the unappreciated aspect of this experiment
is that we were starting our jobs in Heidelberg)
and we knew that we had to set up lots of tubes, and we would identify all these genes
and if it was going to be effective, we would have to have ways of almost a mechanized inbreeding of flies.
We couldn't sit and set up crosses manually by hand.
We would have to have selective procedures that allowed us
to avoid collecting individual flies and set up crosses.
The first two years of our stay in Heidelberg was involved in this.
Setting up and designing genetic crosses that allowed us simply to put a fly in a tube.
Wait two weeks, there would be lots of flies... Put two flies in a tube, actually.
Wait a couple of weeks for the next generation,
and by raising it at a different temperature, or with some other selective technique,
we could just shake the surviving flies over and go through and gradually inbreed the lines
such that, at the end of the cross down here at the bottom, after three generations,
we had balanced, inbred lines so that we could collect homozygous embryos.
Now, it took us two years to set that up and to get crossing schemes that would actually work.
We set out to do the experiment, and the experiment actually, itself only took us...
well, two rounds of experiments, each of which were less than two months in advance.
So, I guess the first lesson is the idea that if you have a vision and the vision's clear enough
you may still have to spend a lot of work just getting the assays and getting the procedures right.
But then the next thing, and this is the thing that I really wanted to talk about
We did actually start out with trying to make 40,000 lines.
As you'll see, we only really carried about 27,000 of them through to the third generation
But, by the time we got to that generation and began to get our first results...
We'd gotten our first important result.
We'd used enough mutagenesis that, in the lines we were looking at,
we had about, in these 27,000 lines, we had about 18,000 different mutations.
So that meant that our mutagenesis procedures worked--everything was working fine
Our interest, though...these mutations kill the fly at any time during its life cycle,
and what we were really interested in were genes that controlled the way embryos developed.
And, this is the point, actually, where I think we were extraordinarily lucky.
Because, what we had to do was now, from all these 18,000 different lines
we had to collect mutant embryos, look at the embryos that die,
and decide whether the gene that was mutated in those flies
actually killed the embryos and killed them in an interesting way.
And the great result--the thing we couldn't have anticipated
was that, of the 18,000 different lethal mutations, only a tiny, tiny fraction
actually changed homozygous embryos in a way that was really meaningful.
Most of the time, the homozygous mutant embryos hatched and crawled away.
It didn't have to be that way, and what would have been really, really horrible for us, in thinking back to it
is if every one of those 18,000 lines, we picked up and looked at embryos and there was some phenotype...
something had gone wrong.
We would never have actually been able to, I think, since there was really only the two of us doing these experiments
to sort things out and come up with a coherent picture.
So, we were really lucky that what had actually happened was that, in a way that we didn't fully anticipate,
the screen was very selective...
selective for mutations that caused phenotypes in homozygous embryos.
That is to say, we were selecting for genes that had to, themselves, be transcriptionally active in embryos,
and that, in flies, that number is very small.
And so, the number of mutations was about 586,
and actually the number of genes, if you do complementation tests among those mutations,
there are only about... we only found 139 different genes.
So that's a really small number that reduced the problem
of thinking about development down to 139 components--139 genes,
each of which were producing different phenotypes
and could be arranged in pathways and sequences that made, for me at least,
the whole process of development almost understandable or structural for the first time.
That understanding came from the fact that the numbers are small.
And the numbers are small, not because we planned them that way.
The numbers are small because of the peculiar biological features... the way flies develop.
It turns out that flies' embryonic development is very rapid, the mothers puts everything the embryo needs...
[everything] that she can possibly put into the egg is put in by the mother,
and the transcriptional requirements early during these patterning stages are very minimal.
So, the fly has stripped down its transcriptional requirements to the rare genes that can't be supplied
by the mother--that must be supplied by transcription in this cell and not in this cell.
There's genes...the embryo is using transcription to control patterns, to control transitions in time.
So, we did a genetic experiment that was oriented towards identifying phenotypes in homozygous embryos
and because of the biology of flies--the way they work--
we ended up identifying the central set of core regulatory genes
whose activity temporally and spatially control the development of pattern.
And if I think back to what both Janni [Nüsslein-Volhard] and I, at the time...
We thought a lot about this experiment.
As I said, we planned it for more than two years, we actually spent two years trying to get the thing set up,
and yet we didn't really fully understand how the system worked--
how the fly embryos developed until we actually had the numbers
and had the results.
And I think that's actually a great lesson in biology...
On the one hand, you don't understand the experiment really that you're doing
until you get the result.
And it's really only when you get the result that you potentially understand
what biology and the whole system that you work with is actually giving you.
That's true, and I guess it's pretty much the nature of science.
It's really great to do experiments where you don't know the outcome.