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My name is Eric Wieschaus and I'm a HHMI investigator and professor
at Princeton University. For the previous two parts of this lecture we've
been talking about how pattern is established in the early Drosophila
embryo, and we focused on a molecule called bicoid that is deposited as an RNA
at the anterior end of the Drosophila egg during oogenesis in the mother.
And then is translated into a protein and forms a protein gradient in
the early embryo. And the model and then the reason why this protein,
this bicoid gradient is important is that it is thought to be the major
determinant in establishing the pattern of gene transcription in the embryo
such that different concentrations of the bicoid protein at different points
along the anterior posterior axis activate expression of particular genes
like the hunchback gene in green here, or the Krüppel gene in red.
Now, we've seen that if you examine this gradient in Drosophila embryos
or if we examine the expression of the downstream targets, we see that their
extraordinarily constant from one embryo to the next. And this is probably
what you want if you want to have a system which is establishing pattern
and controlling the behaviors of individual cells in the embryo. Now
in the last lecture we talked about some of the biophysical parameters
and cell biological parameters that might give rise to these constant
distributions of bicoid or constant transcription patterns.
Now one of the underlying assumptions for all of that work
is based on actually a fact. If you look at fly eggs, not only are the expression
patterns constant, but the actual sizes of the eggs are constant. And that's
an important idea because if you think about any of the mechanisms
that we think about when we talk about how one would establish a gradient,
they are very sensitive to the size of the egg. If you want to establish
hunchback expression up to 48% egg length, and you are doing that
by having a gradient of a molecule that diffuses with a particular
diffusive constant and establishes a particular constant shape
in its distribution as bicoid appears to do, then that mechanism for
patterning would be very sensitive to variation in egg size.
And if you look at Drosophila eggs in the particular way you raise them
in the laboratory, in the way that we measure them, in the stocks
we've measured them to establish these gradients and look at them.
What we see is that actually individual fly eggs are remarkably similar,
wild-type normal eggs are remarkably similar in their size, and so it's basically
consistent that such a mechanism could function to pattern
embryonic development in Drosophila melanogaster. The problem though
with that model arises if you go outside of Drosophila. If you go outside of
fruit flies and extend your observations to other kinds of embryos
from even other embryos of other insect species, even other fly species.
As we all know, from our own personal experience, flies come in different sizes.
There are the nice little small Drosophila fruit flies with red eyes
that we kind of raise in the lab and have such affectionate feelings for
and there are also disgusting flies like house flies and blow flies that
kind of fly around and invade our picnics and are less attractive.
The bigger flies are the uglier ones, the smaller flies are nicer and sweeter, generally.
Now what is also true though, not only is the adult flies that we see
are of different sizes, but also if you look at the eggs in the embryos
that these different fly species make, they are also different sizes.
So Drosophila melanogaster for example makes eggs that are about
500 microns long. There are even Drosophila species like
Drosophila busckii that make eggs that are even smaller than Drosophila,
but the big flies like Musca domestica, the house fly,
or Calliphora, blow flies or green bottle flies, Lucilia, that are big
obviously as adults much bigger than Drosophila, and the eggs
that they make are substantially bigger. Now all of these flies
these higher Diptera are closely related. And even though
bicoid as an RNA or a gene product was a newly evolved solution
to the problem of patterning in the embryo and arose during the evolution
of the Diptera, all of these insects here share the common feature
that their anterior/posterior patterning depends on bicoid.
And yet the bicoid gradients that are forming in these eggs are forming
in eggs which are very large or very small. Now, one of the interesting
features of all these eggs though is even though they are different sizes
if you actually look at the development of these embryos
you can see that the early development is remarkably similar in that if you
go back and remember how early development in Diptera starts with
fertilization followed by these nuclear cleavages in the syncytial embryo
and then a pause at 2.5 hours to form the syncytial blastoderm
that then transforms itself by the formation of cell membranes between
nuclei and into a cellular blastoderm and a gastrula, that process
that takes about 2-3 hours in Drosophila melanogaster
is also observed in all of these other insects and it also is observed
with exactly the same time, 2.5 hours, the same kinetics
and if you look at divisions of the nuclei, if you look at individual,
say this is an embryo, at the syncytial Drosophila embryo from
the big green bottle fly, Lucilia versus Drosophila melanogaster
or Drosophila busckii, the eggs have the same shape
and although you can probably barely make it out, you can barely see the nuclei
in the Lucilia and the nuclei in Drosophila melanogaster or busckii
are somewhat smaller, if you blow up the pictures of the individual eggs
and look at the nuclear distributions you can see that all these eggs
at 2.5 hours have the same number of nuclei. They all have about 100 nuclei
going from the anterior to the posterior end of the egg.
They're all being patterned over the same time constraints
and they're all being patterned by bicoid. If you look at transcription
the other remarkable similar thing between all these insects that
are all so closely related even though their sizes are different, is that
all of them activate transcription at this critical 2 hour period in response to bicoid
and if you look at the patterns of gene expression
if you look at say hunchback or giant, the two different gap genes
in Drosophila or in Musca or pair-rule genes like
paired or evenskipped, they show exactly comparable scaled patterns.
Even though the eggs are bigger and even though the cells are bigger
the patterns per cell are exactly the same. Now these are transcriptional
responses, they're genes which are transcribed at the blastoderm stage
directly or indirectly in response to the bicoid gradient. And so the question
that you'd like to ask, is how is it during the course of evolution,
as egg size changes, how does the embryo or the species adjust to using
a bicoid gradient to establish pattern. There are really two simple ways
that you can think about it. One way is that each of these genes,
like hunchback and any of the targets of bicoid activation
is going to have a control region which will respond to bicoid concentration.
And as you change the length of the egg, one strategy
would be to keep the bicoid gradient the same shape
and the concentration distribution the same, and yet change
the cis-acting control regions of each of these genes. Adjust them
during evolution and we know that that's generally what happens
during the course of evolution. Alternatively, during evolution
you could adjust with the size of the egg not by changing
the control responses of individual genes, but by somehow
changing the manner or changing the physical properties
that establish the bicoid gradient itself such that in bigger eggs
the gradient extends longer and in smaller eggs it is shorter.
This seemed to us initially a less likely alternative, partially because
many of the cases in evolution that we know about involve
changes in cis-acting control regions. But when we actually looked
at the bicoid distribution in these other insect species, what we observed
was that not only does bicoid form gradients in big eggs and the small eggs
but if you look at the big egg, if you compared the distribution to say
in melanogaster. In melanogaster the gradient falls exponentially
over an area like this, and if you look in Lucilia the distribution
of bicoid protein extends much longer, much farther
into the length and into the egg. In terms of microns, that is the bicoid gradient
in a bigger egg, is bigger, proportionately bigger, because if we then
replot the data, not in terms of absolute lengths as here
but in terms of relative lengths along the eggs you can see that
the bicoid gradients in the big eggs and the small eggs are
exactly equivalent scaled to the size of the egg. What that means then
in turn, is that somehow during the course of evolution of these insects
the bicoid gradient has changed. The properties that establish the gradient
have changed to allow this gradient to now span
a bigger or smaller egg and can provide positional information along
the whole length of the egg. So how does this happen? One simple strategy
would be to change bicoid as the species evolve they change bicoid,
they change the properties of the bicoid protein such that it moves faster,
such that it is degraded less rapidly for example, such that it ultimately
the gradient that you get out of this bicoid would extend farther
and thus establish gradients of comparable shape when
scaled back to the actual shape of the egg. To begin to test or
think about those models we've cloned the bicoid genes from these
different species and compared their structure to
that of Drosophila melanogaster bicoid, and if you look at that
bicoid is reasonably well conserved particularly in regions of the protein
which are functionally well defined. The homeodomain that binds DNA
or other regions that have been implicated at least suggestively
as being involved in protein stability. Most of the sequences are the same
but not surprisingly when you look at any particular sequence,
any particular region of these proteins, there are amino acids differences.
And so one possibility is that these different species specific bicoids
have evolved and the changes in their sequence, either the ones
that I've indicated here or changes in other regions of the protein
are actually responsible for adjusting the shape of the gradient
such that it can now function in larger eggs or smaller eggs.
So to test that possibility, you need to begin to hope to identify
the regions that have changed in the bicoid protein.
What Thomas Gregor and Alistair McGregor in the lab did was to
take these cloned bicoid genes from the other species,
tag them with EGFP and put them back into melanogaster to ask
what type of gradients that they make. And the surprising result here,
one that we hadn't anticipated was that if you compare,
if you take a bicoid protein from Calliphora for example
that will make a large gradient that extends more than a
millimeter through the entire Calliphora egg which is
one and a half millimeters long, and you put it into a Drosophila egg
which is only five hundred microns long, one possible result would have been
that this bicoid protein because of its changed properties, the protein
that it moves faster or that it degrades less, would make a Calliphora
sized gradient in a Drosophila egg and you can imagine
that would result in a catastrophe for development
for the Drosophila embryo that was depending on that gradient.
But what you actually see, the amazing thing is that these bicoid gradients
that are established in these eggs, actually using the Calliphora protein
are identical, surprisingly identical not to the gradients
that those same proteins would have made in Calliphora
but to the gradient that is made in Drosophila melanogaster.
So, for example in this figure here we can see the Calliphora bicoid
extending out in a visible sense to about 48% where we'd be activating
hunchback, and that's very similar to the distribution that you'd see
when you took the Drosophila EGFP and put it in the same egg.
And you can measure that and show that these distributions overlapped
each bicoid that regardless of the source of the bicoid protein put into a
Drosophila melanogaster egg that protein will make a gradient
of the same shape, the same size, as the melanogaster bicoid.
So we know that what that's telling us is that the fact that Dipteran bicoids
expressed in Drosophila make Drosophila sized gradients is that during
the course of evolution it's not bicoid that has changed to allow for the
adjustment of the egg shape and the egg size, but some other property
of the egg. So these proteins put in a melanogaster egg will produce
melanogaster type gradients. Now we've done the same kinds of experiments
using other tagged proteins, EGFP, we've altered the bicoid area,
just put straight, if you can imagine just taking EGFP or EGFP
with an NLS, a nuclear localization sequence, and localizing the RNA
to the anterior end of the egg and ask will any protein,
any GFP-tagged protein put into a Drosophila egg make a gradient
the shape of bicoid. And what we found is that's not true.
That each individual protein put into the egg makes a gradient of a particular
size and a particular distribution. But all of the bicoid proteins
put into the Drosophila egg at the anterior end make gradients
of a particular size. So the interesting conclusion then from these
experiments is that during the course of evolution, it's not that bicoid
has changed to adjust for the size of the eggs, but actually
bicoid has been conserved. What's been conserved in bicoid is
the property of the protein that allows it to build gradients of a
particular size when put into Drosophila eggs. And all the bicoid molecules,
but other proteins do not have that property.
What's actually diverged during evolution has been not the protein itself
but the environment that we put the protein in
and then that raises the kinds of questions that you'd like to answer now.
What are those properties? What could influence bicoid? What's the
features of bicoid, the bicoid protein itself, that allows it
to respond and make gradients and there are obvious experiments
that we're in the process of doing where you can identify the regions
of the bicoid protein that are essential for it to make
a gradient of a particular size and shape that's characteristic of bicoid.
And then the other question is what are the features that change
as you change egg size that change those distributions.
How is it that you are able to maintain the property of the protein
on the one hand and then change the size of the egg
and change the movement of the proteins. So those are
really essential questions for understanding how during the course
of evolution you are able to use the same system, the same protein,
over and over again. They will require different kinds of measurements
and being able to work with multiple species and multiple variants
both of being able to put bicoid proteins into melanogaster
but able to also put variant proteins into the bigger and smaller eggs.
But what they'll also require is to distinguish between these different models
to give it the underlying mechanisms that are controlling the distribution
is again the kind of quantitation that we talked about in the second lecture
and it's my own belief that the future of developmental biology
in general and the refined understanding of problems
in development will depend heavily on our ability to combine
both those quantitative visual techniques for analyzing distributions
of molecules with the powerful techniques of molecular biology
that allow you to manipulate the sequences and structures
of those proteins and also the other features of the egg.
So I'll stop there and thank you for your attendance.