My name is Richard Harland.
I'm at UC Berkeley, and I'm a developmental biologist.
I'm interested in how one goes from a fertilized egg, a simple organized cell, to a complex organism.
And we study the early phases of that, where the egg is making its initial axes,
making the anterior and posterior, the front and back, as well as the back and the belly,
the dorsal and ventral organization.
And in addition to those general overall axes, the particular tissues that are laid down
during early development.
So, we study that in the Frog, Xenopus.
And I'm going to start this lecture by discussing why Xenopus.
I'm going to talk about some of the experimental advantages from the biology.
And then talk more about some of the experiments that have been done, and how the animal becomes
organized.
So, why do we use amphibians and why Xenopus in particular?
As we all know, when we go out and see these eggs in a pond, there are very many of them.
So, the amphibian lays a lot of eggs.
They're outside the mother, so they're easy to access.
You can easily find them and... and manipulate them.
They're very big, so they're easy to manipulate.
And that's important for both injection of materials into the egg as well as,
in the embryo, we can do microsurgery.
We can cut out pieces of the embryo and study them in isolation, or we can cut and paste:
we can transplant tissues around the embryo.
So, there are all of these advantages, but of course an additional feature is that
these are vertebrate embryos.
So, just like us, vertebrates.
And here is a picture of the standard vertebrate embryo.
I'll just briefly allude to some of the features that make us similar to frogs.
So, first of all, the notochord.
So, you see this red stripe that runs down the back of the animal.
It's a stiff rod that enables swimming in the larval vertebrates that actually do swim.
But also, in the placental mammals like us, it's an important signaling center.
So, we're very interested in how the notochord forms and what it does.
Secondly, we all share this dorsal hollow nerve cord.
So, we have, running down our spine and in the brain, ventricles,
hollows in the nervous system.
And we all have this feature.
Thirdly, although we may not realize it, we all have the residue of gill slits and
branchial apparatus.
The branchial apparatus is often used for feeding in fishes and tadpoles,
but in us it's an important developmental component in the transition from making the simple embryo
to the face of the mature animal.
And finally, not very glamorously, perhaps, the post-anal tail.
Many simpler animals have an anus right at the back of the animal, but, here,
one of the unusual features of the vertebrate is having a tail, often used for swimming,
behind the anus.
So, all of these are common features to the vertebrates that the frogs have in common
with us.
So, let's talk a little bit about these large and abundant eggs, and how they're used.
So, I'm going to give a couple of Nobel Prize-winning experiments as examples.
The first one is from John Gurdon.
And what he was able to do, starting in the 1950s, was to take specialized nuclei
from differentiated cells, and, because the eggs are so large, he could transplant those nuclei
physically into a new egg, and study what kind of development they could support.
And at the time there were very obvious different models.
So, for example, one model would be that in order to make a red blood cell you might
throw away all the genes that are needed to make a muscle cell or a nerve cell.
Instead, what Gurdon found was when you transplant a nucleus into an enucleated egg,
that specialized nucleus can be reprogrammed and make all of the tissues of the tadpole.
And that showed very clearly that these nuclei did not get to be specialized by throwing away genes,
but rather those genes are selectively used in normal development to achieve their
differentiation.
So, the first experiments were done in a species called Rana, but they really brought to their
most successful experiments, where the differentiated cells were shown to be developmentally totipotent,
as it's called, in Xenopus.
I want to remind you that although the fine-maned John Gurdon that we see nowadays is
an older gentleman, when he did this work, as is common with most of the Nobel laureates,
they did the work when they were young people.
So, I just show this picture of Gurdon when he was doing this kind of work.
So, that's the advantage of a big egg -- easy to inject.
Also, the microsurgery and transplants on development... developing embryos.
Let me give a couple of examples of that.
So first of all, let's watch an egg developing.
So, in this time-lapse movie made by Huw Williams, we can see that the initial big, yolky egg
is cleaved completely into separate cells, or blastomeres.
These big cells are called blastomeres.
And so we've gone through these synchronous cleavages and those synchronous cleavages
lasts up to the 4000-cell stage, the mid-blastula transition, which I'll come back to.
So, here's the cleavage.
And that makes it useful because these eggs are cleaving up and packaging the yolk into
each progeny cell.
So, here we see the consequence of that complete cleavage.
And we're looking at two stages here: the hollow blastula stage at about 4000 cells...
we're gonna to come back to this stage later because that's when gene expression
really starts; then we're going to be talking about this later gastrula stage, where the
movements of gastrulation bring all of this stuff inside and cause the gastrula...
the primitive gut, to form.
And the whole embryo is enclosed by this top, this ectoderm.
The point I want to make here is that that complete cleavage encloses each... each cell
encloses yolk.
It encloses food.
And so this gives us the property that we can cut out pieces of the blastula and
put them in isolation.
They have their own food, so it's... even in simple buffered salts,
they'll continue to develop and display their normal characteristics
that they would have done in the whole embryo.
So, we can do that from the blastula.
And we'll also be exploiting this from the gastrula, particularly explanting regions
from this dorsal region to look at the cell behaviors that occur there.
So if we zoom in on this, you will... you'll see then that, indeed, these cells are
packaging up a lot of yolk.
The yolk is segregated into all the blastomeres, and you can see these little hollow ovals
here in this fixed section.
Each of these hollow ovals is a package of yolk that's been taken up from the bloodstream
during oogenesis, the formation of the egg, and is... is taken up and then processed and
crystallized into these little food sources that are used later.
So, that property was really exploited a lot at the beginning of the last century to study
what different parts of the embryos thought they should make, as well as, if you confront
different parts of the embryo, what happens as a result?
And this of course is the famous culmination of that in the organizer experiments
of Spemann and Mangold.
What they discovered was there are certain regions of the embryos that have very special
signaling properties and can tell their surrounding neighbors what to do.
In order to be able to infer from the experiments that that was the case, they used newts of
different pigmentation.
So, these were naturally breeding newts out there in Germany, and there are
some that are pale, like this one on the left up here, Triturus cristatus,
and then there are darker species as well.
And this provided a natural label so that after they did the graft -- in this case,
at the gastrula stage -- they could follow the fate.
So, you can see here, there's a little stripe of white tissue here that has come from
the graft.
And the spectacular result here was... this is two sides of the same embryo up here.
This is the belly side, and this is the dorsal side.
You can see that the results of that graft is it's making... made a second dorsal side.
So, this graft from the dorsal lip -- this region over here, this special lip here --
when that's grafted onto the ventral side, the prospective belly side, it induces
a whole secondary set of structures.
And especially it is... it is recruiting those and telling them what to do.
You can see from this picture here, this white strip has recruited surrounding black cells
to make the neural folds of the animal.
And in her sections, this was also shown to be the case.
In other experiments, just pure explants we used, rather than transplants.
And many of the cell behaviors that we understand today come from initial observations
from Johannes Holtfreter, a German embryologist who trained with Spemann but emigrated to
the United States during the... before the Holocaust.
So, Johannes Holtfreter did experiments to study how explants behaved in culture.
He developed methods to culture these explants so that they would undergo their normal behaviors.
And he could take the embryo apart and see how different behaviors of cells could
sum together to give the overall behavior, the shape change, or morphogenesis
of the whole embryo.
He was an entertaining character who was also an active cartoonist, as you can see here.
Okay, so here's a couple of experiments that we can show.
There's the possibility of cutting out the dorsal side.
We'll talk about this more in detail later, but this is a simple macroscopic view of
how one can cut out a piece of the embryo, put it in isolation, and it will undergo behaviors
that it would normally do in the normal context, though now in a dish.
So, one can take apart the movements and understand the cellular behaviors.
This is a case where we can study the behavior of cells in... in an explant at high resolution
using microscopy.
And I'll come back to that later.
Okay, so there all these advantages of being able to do experimental embryology with these
amphibians.
But which one do we choose?
Here we see different amphibian eggs cleaving up.
We can see that they have different sizes.
So, for instance, this axolotl is very big, so perhaps very easy to manipulate.
But Xenopus is the animal we've mostly alighted on.
It cleaves completely.
And we use it because, in the wild, Xenopus develops in seasonal rains.
And it has to be ready to lay eggs at any time of the year when it rains.
And so as a consequence, we can get the eggs at any time of the year.
In contrast, almost all of the northern hemisphere amphibians are very seasonal and
only make eggs in the spring.
And of course, we want to do experiments at any time of the year.
And so Xenopus has that advantage.
There are different species of Xenopus.
And down here we can see two different species in the same dish: Xenopus laevis, the big ones;
and Xenopus tropicalis, the little ones.
The little ones are not so easy to manipulate, but they have the advantage that
that particular species is a true diploid, which is good for genetic manipulations.
In contrast, Xenopus laevis, the workhorse Xenopus, is a tetraploid, or correctly a paleotetraploid.
And so that makes its genome a little more complicated for experiments.
Nonetheless, we can use either one essentially interchangeably, and for injection purposes,
for example, they're essentially the same in how they behave.
So, again, why Xenopus?
It is interesting that Lancelot Hogben discovered, in using Xenopus from South Africa,
that it could respond to simple hormones and lay eggs.
And ultimately, what he found was that these animals can respond to conserved gonadotropins,
which are made by a variety of animals.
And most usefully, they're made by pregnant women and secreted into the urine.
And so, in fact, what was found is if you want to inject a preparation of sort of crystallized urine
from a pregnant woman or another kind of pregnant mammal, the injection of that material,
that human chorionic gonadotropin, will induce ovulation.
So, it's very convenient to be able to use that.
In fact, it's so convenient that this was the original pregnancy test.
And so hospitals throughout the U.S. and Europe maintained large colonies of Xenopus.
And when one did the pregnancy test, would take the urine from a putatively pregnant woman
and inject it in the frogs and ask, do they lay eggs?
Of course, now we've moved on from that, and we buy a box from a pharmacy and pee on a stick,
which is much simpler.
But of course, in... back in those days, it didn't escape people's notice that there were
all these animals that could lay eggs, and they would be, in principle, a very good
experimental organism.
And so, as people like Michail Fischberg, who recognized that and started using Xenopus
actively in the lab, and most notably convinced John Gurdon as a graduate student
to study them.
And it was those experiments that John Gurdon did, using Xenopus, that led to his Nobel Prize.
So, some other good things about Xenopus.
We can get the eggs and put them in a dish, fertilize them with sperm, and get a
large number of synchronously developing animals.
So, for time courses, that's very handy.
And of course we can get these... these large embryos... there's a lot of biochemical materials.
So, we can harvest a lot of material for study.
There's also a consistent fate of different parts of the animal.
As you see here, as they develop, we're going to stop the movie at the four-cell stage.
You see different appearances of these animals.
And if we take this one, for example, we see there's a pale side and a dark side.
And we know, because of the rearrangements of pigment in the first cell cycle that determined
the dorsal-ventral -- the back-to-belly-- axis, that the top here, the pale part,
is going to make the dorsal side, and the darker part will make the ventral side.
And so, if we want to target manipulations to particular territories of the embryo,
we can choose embryos like this and inject into particular cells and study the consequence.
That's shown in this case, where we're doing the very simple manipulation of injecting
the two-cell stage.
And if we choose the right embryos, we can inject into that... that two-cell stage.
And the embryo will cleave up such that that blastomere populates just one side
of the animal.
So, we have this left-right difference between the manipulated and unmanipulated side.
Here, we just show it in a case where we're using the now very fashionable CRISPR/Cas9,
and using that to mutagenize the pigment gene.
Now, you see this tadpole here.
It's been injected in one side with the reagents that will mutagenize a pigmentation gene.
And as a result, it has an unpigmented eye on this side.
This same tadpole was also simultaneously injected with a lineage tracer.
This is a neutral molecule that is not going to affect development, but which you can
see by virtue, in this case, of its fluorescence.
And it's not going to pass between cells because it's attached to a big sugar molecule.
So, we inject that, and it's a cell-autonomous tracer for the ultimate fate.
And so here on the right, you see the exploitation of that lineage tracer confirming that
only the right side of the animal has been injected.
So, this fate mapping goes on, of course.
Here's the early 32-cell stage, and we can see the different germ layers now developing,
or schematized at least.
So in blue, these... using the classical colors of American embryologists, the blue is the outside,
the ectoderm of the animal, which includes the epidermis and the central nervous system.
This marginal zone around the equator is colored red, and it's the mesoderm, the meat of
the animal.
And down here the fatty endoderm, which will make the gut.
And so, at the 32-cell stage in this schematic, we can inject different parts of the embryo
and see how they develop.
Because the embryo is dynamic, and so the fate map changes as the embryo changes its shape.
But ultimately, we can develop this into a tadpole.
And so, for instance, if we inject one of the blastomeres over here... sorry, over here,
the red notochord territory, for example, we can look and see that population later.
So, here's the tadpole and the section through it.
And you can see in this picture of the section, we've got the standard vertebrate organization
with the outside epidermis in pale blue; the nervous system with its hollow nerve cord
in dark blue; and here's that red stiff rod, the notochord; muscle on either side;
and then the rest of the territory, the mesoderm, including things like the kidneys and the blood.
So, in the next slide I'll show a schematic... schematic of how this can be exploited
in particular.
So, if we want to know about the notochord, and manipulate just the notochord,
as was shown by Les Dale and Jonathan Slack, we can inject this C1 blastomere over here
-- this is a dorsal tier-three blastomere -- and ask what it normally makes.
And in the animal, all of the notochord, essentially, comes from this part of the embryo.
You can see from the anterior end, here, where the notochord is, all the way out to the tail,
the notochord is populated by the progeny of this blastomere.
So, if we want to manipulate just the notochord, we can do that by injecting that blastomere.
This also illustrates an important point, that during early development we call this
the dorsal and that the ventral side, and there's been some discussion about how
we label these axes, but if we focus on the notochord itself, we can see that this dorsal side
is indeed dorsal in the animal.
It makes the most dorsal mesodermal component.
And it stretches all the way from the anterior to the posterior of the animal.
So indeed, this region of the animal gives rise to the dorsal animal, all the way
from the head to the... the head to the tail.
It's more complex in the rest of the animal, but we'll leave that to a later discussion.
Okay, so here we're showing that embryo parts do become different.
And here we're showing a simple RNA expression experiment, where all of these genes
are turning on at a particular phase, about the 4000-cell stage.
And just to give a couple of examples, here's one.
This is a signaling molecule that has initially turned on around the margin.
You can see, turned up around the margin.
And if we look up on the vegetal pole, we see it's expressed all the way around
in a ring.
So, that shows that this region of the embryo is special at that stage.
It's making the prospective marginal zone and turning on special genes.
Let's look... just look at this one.
This is a gene marking the endoderm.
And you can see, again, that that is different in expressing just this gene at this stage.
Then finally, dorsal-ventrally, we can see that this goosecoid gene is expressed
just on the dorsal side, down here.
In Particular, you can see it in just a little patch of the prospective dorsal mesoderm.
So already at this stage, different regions of the animal are special.
I'll just emphasize that point here.
This is looking up on the vegetal pole.
We get genes expressed initially in a mutually exclusive way, and I'll be talking about that
more in the third lecture.
Here's another case that illustrates the dynamic behavior of gene expression, a dorsal and
a rest-of-the-embryo expression of the blue and the brown gene, nodal and wnt8.
And you can see how that changes very quickly.
Over the course of about an hour, you get this change in the territory of expression
of the blue nodal3 gene and a restriction of expression of wnt8.
So although initial expression may be quite simple, it very quickly becomes dynamic and
more complex.
Let's discuss some of the movements of gastrulation.
So in this movie, we see... in this movie from Dave Shook and Ray Keller,
the whole progress of the formation of the gastrula through formation of the neural plate and
the tadpole.
Let's see that again.
So here we're seeing that... we're gonna stop this movie right here.
And we're going to see that in the initial phase of gastrulation -- this is the stage
at which all of that yolky endoderm has to be brought inside the embryo
and the blue ectoderm has to cover up the entire embryo -- we're seeing the first stage of that.
So, let's just play that again.
So, we see cells moving inside the embryo.
And so this starts with the behavior of these cells on... at least the visible behavior
starts with these cells, which start to constrict and dive inside the embryo.
And they lead the movement of these cells... this is the prospective dorsal mesoderm up here...
these cells are going to go inside and come to lie against the inside of that
hollow blastocoel.
Okay, so there they go.
You can see them turning the corner and going over the edge.
And that same behavior occurs all the way around, as this blastopore spreads around
the entire animal.
So, here's the rest of gastrulation.
There's not much tissue moving inside anymore, but what does happen is that
the overlying cells come and cover the entire animal.
So, let's watch that one... one more time.
So, you see these cells now stretching, covering up the yolk, so that all of the
prospective gut is inside.
And now we move into neurulation, the formation of the neural tube, which occurs by this folding
movement of the dorsal part of the animal.
The animal's opaque, so we can't readily see inside, we're using a special technology.
This is magnetic resonance imaging from the Jacobs and Fraser labs.
Using a very high-resolution MRI, we can watch the behavior of these cells.
So, we see here the early cleavages of the egg into the blastula and then on through
gastrulation.
And I'm gonna focus in on the first part of this.
This is the cleavage stage, where you saw the blastomeres dividing up.
And we...
I should make here the important point that the egg is not growing at all during
this phase of development.
The egg starts with a certain mass and it's subdivided into smaller and smaller cells.
So, during early development, in contrast of the mammals, there's really no net gain
in dry weight, no net gain in size.
And so in many ways this simplifies the analysis of early development, because we don't have
to worry about cell division.
Instead, we can concentrate on the movements of the cells.
So, here we have this hollow blastomere... sorry, hollow blastocoel.
And here, down in the black region, this is the increased contrast of the yolky endoderm,
which is quite a handy marker here.
So, now we're going to see the movements of gastrulation, as these cells move inside.
And we'll go through that a couple of times.
The other thing is you'll see this black yolky material has to spread at the level of the
blastomere... blastocoel floor, and all of this ends up inside.
So again, we'll watch that, as this black material is all engulfed in the rest of
the embryo.
So, using these kinds of techniques, we can study this whole process not only from
the outside, but we can see the behaviors from the inside.
And then, in conjunction with the explants, we can take the embryo apart and study
the individual behavior of different parts of the embryo.
I show this picture just to show that not all cases of neural tube formation are identical.
In Xenopus, it's not particularly spectacular, but here is an axolotl that's neuralating,
and you can see this really dramatic behavior of the neural folds as they come up and close
over the top.
So finally, we're going to see in the schematic what we'll be discussing in the second talk,
for those that are interested in morphogenesis.
Here, we've got the colors going in this great movie from an undergraduate at Berkeley,
Mengsha Gong, who worked with Ray Keller and John Gerhart to animate this whole process.
We see the enclosure of the entire embryo by the blue ectoderm; the stretching,
the elongation of the neural plate, over here, going from initially a stack of about 30 cells high.
The cells come in from the rest of the embryo.
It becomes extremely long, about a millimeter long.
We see the cells going inside the embryo, and we'll talk more about the behavior of those.
You saw those pigmented cells, these green cells that are special cells that
undergo an apical constriction, so we'll discuss those.
So, although gastrulation as a whole appears horrendously complicated, by taking
these movements apart and studying pieces in isolation, we can get a good understanding of all
the individual types of cell behaviors that contribute to the overall process.
So, that's it for the first talk.
In the next, Part 2, I'm going to talk more about gastrulation and take those behaviors apart
and consider them in isolation.
Then in Talk 3, I'll discuss the signaling events and the experiments that have led to
our current understanding of how the embryo becomes different in its different parts.
So, thanks very much.
