Okay, so we're coming to the third part,
the last part,
on how the chordate got its chord.
Let me just...
this is sort of a 'just so' story, you know,
where we're gonna tell you all the little features
that have arose in the animals
in the course of evolution
that we couldn't normally see,
but are revealed by homologies
in the molecular details.
In the first part,
I told you essentially that
the anterior-posterior axis of the hemichordate,
which is a sister phylum to the chordates,
was very, very similar
to the chordate anterior-posterior axis.
In the second lecture,
I showed you that the basic patterning mechanism
of the dorsal-ventral axis
was the same,
but the details were very different.
And I argued, based on a lot of evidence,
that the idea that Geoffroy Saint-Hilaire had,
and Eddie De Robertis,
much more recently in the late 20th century, had,
of an inversion was basically correct.
That is, there has been an inversion of the nervous system
and the mouth
relative to a conserved body plan.
And now, in this lecture,
I wanna deal with the fundamental patterning mechanism
of chordate development,
that is, the Spemann organizer
and the way in which
the chordate body is generated.
And one of the primary structures
which is generated there
is an anatomical structure
called the notochord,
which also signals many other structures
and it organizes them in the course of development,
and there's no, or didn't seem to be,
any obvious precedent
for the notochord
and the signaling aspects of the Spemann organizer
in invertebrate development,
so it's a real question,
a real mystery,
how did we get our chord?
So, let's go back to the Spemann experiment
and vertebrate-specific development.
This is an experiment done in the 1920s
by Spemann and Mangold,
and it was an experiment that...
was the most famous experiment in the history of
developmental biology, maybe,
and still in the history of developmental biology.
And the basic experiment
was that they knew that one part of the egg,
or the early embryo,
would generate the belly
and one part of the early embryo would generate the back.
And they took a little bit of tissue
in the gastrula stage of the early embryo,
just when there are no structures
-- or obviously apparent --
and the moved this tissue,
which was from the back, just a little bit of it,
onto a recipient embryo
and put it on its belly.
And then the embryo developed,
and what they sort of half-expected, undoubtedly,
was that that tissue might or might not
revert to belly features,
or it might form a little piece of the back
on the belly.
But the result was very striking:
it produced conjoined twins,
transforming the belly tissue
into an entire embryo,
including a central nervous systems,
and the somites,
and the notochord,
and all those things
-- you can see in the bottom in the histological section --
a mirror image of the other side.
And most of those tissues, almost all those tissues,
arose from tissues
that were respecified by some sort of signal
that came from this transplanted tissue.
So, that seemed to be...
that transplant tissue must have been really very, very special.
We actually now know
that though it is special,
it really is special in
eliciting an existing response
from the surrounding tissues,
which are normally repressed
from forming the body axis.
So the body axis could form anywhere,
and this is a local region of derepression.
So, okay, but anyways...
and this tissue, this signaling tissue,
gives rise to the notochord.
So the notochord
contains signaling elements,
but it also is an anatomical structure
that derives from that tissue,
and since the notochord is
the iconic structure of a vertebrate,
we want to know where it came from.
How did it arise?
So, what about the notochord?
The crux of Bateson's argument
-- remember, Bateson was this guy
who studied... beautiful studies of hemichordates in the 1880s
and then went on to become a famous geneticist --
he argued that
the hemichordate was essentially a chordate,
and he based it on the fact that the structure,
which is now called the stomochord,
was in fact a notochord.
Well, we've looked,
and we've looked for all these characteristic genes
that are found in the notochord:
chordin, which I've talked a lot about,
noggin, admp, brachyury, hedgehog.
And we have a small problem, as they say:
they're not in the notochord,
and they're not absent from the animal,
they're just somewhere else.
So, the notochord...
the stomochord is clearly not a notochord,
at least by all those criteria.
Well alright, now,
the notochord is the product of the Spemann organizer,
and the Spemann organizer, as we said,
is something that signals many, many different types of tissues,
so we maybe show still ask the question:
does it have a Spemann organizer?
Because the Spemann organizer, as I said,
is a complex signaling center.
And if we look at these
genes that are present
in the region of the notochord...
of the stomochord before it actually forms,
such as foxE2, or dmbx, or otx,
these genes, which are involved in signaling,
are present in the right place and the right time.
So the interpretation is
that even though the stomochord is not a notochord,
it has a signaling center,
it has an organizer.
And this is...
now, what is really the organizer?
Well, we know a lot more about the organizer
than we did in Spemann's time,
and we know that's it really
a tripartite structure
formed of three kinds of signaling centers,
shown on here in the frog picture,
that are pasted together
and held together
and work together in the vertebrate embryo.
There's a trunk-tail organizer,
this is the part that gives rise to the notochord.
Then there's a head organizer
that gives rise to the brain
and to the tissues surrounding the brain.
And then finally,
there's an anterior endomesodermal organizer
that gives rise to very anterior tissues
and things also in the mesoderm.
And in the vertebrate,
these get stretched out
and pulled in all sorts of directions,
but held in some adjacency.
Well, what kind of an organizer does a hemichordate have?
Well, it doesn't have the trunk-tail organizer,
even though it has these genes that...
those trunk-tail genes
are never associated with the stomochord structure.
Instead, it has some of the genes,
or maybe most of the genes,
that are found in the head organizer
and in the endomesodermal organizer.
What basically we're saying is
that the organizer, as we know it,
this tripartite structure,
was assembled from separate organizers,
which are now somewhat separate,
and sometimes completely separate,
in the hemichordate.
So for example,
if we look at brachyury, which is the classic trunk organizer,
it's present near the posterior end
and remains posterior
in the whole course of development.
If we look at goosecoid,
which is a typical anterior prechordalmesoderm organizer
that organizes the anterior part of the embryo
and induces the brain and that sort of thing,
it's present in the stomochord region
and stays in the regions that we would expect it.
And hex,
which is a gene in the very endomesodermal organizer,
it also stays in those sorts of structures as well.
So, what can we conclude
about the basic organizer
or the Spemann organizer?
The vertebrate organizer is in reality
a composite of three signaling centers,
which come together in the hemichordate.
Two of them are already associated with each other
in the hemichordate.
That is, the most anterior signaling centers,
but the posterior signaling center,
the one that gives rise to the trunk-tail,
is not associated with that same structure.
And either it came apart or,
in fact, most likely, it was assembled together
in the vertebrate lineage.
The vertebrate organizer is very complex
because it conflates dorsal-ventral
and anterior-posterior signaling.
The disaggregated hemichordate organizer
corresponds to separate signaling centers
in more primitive bilateral organisms.
So we now can look at this basic body
as quite simplified, the hemichordate,
but having all the features
that we hope to try to explain
in the chordate.
First of all,
it has this dorsal-ventral asymmetry,
that is, the BMP-chordin gradient,
and that is fundamental.
And what's happened underneath all that
has been reorganization of the mouth
and nervous system,
but the really fundamental signaling pathways
were already there and present,
not only in the hemichordate,
but in more primitive animals
and all the way up through vertebrates.
In the A-P dimension,
I've talked mostly about,
you know, the detailed underlying patterning genes.
But what I haven't talked about,
which we have very good evidence for,
is a cascade of Wnt genes
and inhibitors of Wnt genes,
which are placed in the animal
just as they are, really,
in the chordate.
That is, Wnts that are very posterior,
Wnts that are present
at the midbrain-hindbrain boundary,
Wnts that are present
somewhere in between,
and also inhibitors of the Wnt pathway,
the secreted Frizzled-related proteins shown in pink,
which also tend to sharpen
and limit the Wnt pathway,
and the dkk or dickkopf-like proteins,
which also limit the Wnt pathway.
So this basic Wnt signaling mechanism,
which I haven't talked about,
lays out the basic A-P axis, anterior-posterior axis,
and BMP lays out the dorsal-ventral axis.
And in the vertebrate lineage,
there's a specific relationship
with these two axes
and the presence of nodal
on the left side of vertebrates
and on the right side of hemichordates.
So now,
not only can we begin to explain,
you know, with some level of certainty,
but certainly not complete certainty,
how the basic features of the chordate body plan
arose and where they came from.
But we can also extrapolate backwards
to begin to see
what was common with this body plan
and the body plan
that is demonstrated in other invertebrate organisms
like flies, and annelids, and such things,
to get a glimpse of what that first bilateral animal
must have looked like
around 600 million years ago.
And here we have a picture of it,
it's only a drawing, it's not a real picture,
but it has the features that this animal
must have had,
so that these things could be maintained,
preserved with such high fidelity
in all these other lineages.
Well, what did it have?
Well, it may have had a centralized nervous system,
eventually, that's what's shown here,
or it may have a delocalized nervous system
that went essentially around the body.
It would have had a fairly complete set of hox genes,
which kind of pattern the anterior-posterior axis,
and this patterning would have been done,
would have been generated via a Wnt pathway
as it is in so many animals.
And it would have had a dorsal-ventral axis
where it had a dorsal heart,
just like in the invertebrates
like Drosophila,
which would have been sitting up
on a BMP-rich side of the animal,
and a ventral anti-BMP pattern,
which was where the mouth is located
in this animal.
There are some images in the fossil record
of burrows in the mud
of an animal about a millimeter in size,
and that may have been...
but no fossilized remains of the animal itself,
so this may be the animal
that was burrowing through the mud
before the major Cambrian explosion.
So, one of the questions
I wanted to get back to to summarize,
is what is unique to the chordates?
Everybody wants to know what's unique about themselves.
Some people are concerned with what's unique
about them as individuals,
some people are concerned about what's unique of them
as a nation or as a religious group,
and I'm much broader in my feeling.
I'm happy to know what's unique about us chordates,
I really relate to all them as stand up as
a chordate chauvinist,
and wallow in our accomplishments
over the last 530 million years.
So what can we really be proud of?
Well, we inverted our dorsal-ventral axis.
We moved our mouth to the opposite ventral side.
We centralized our nervous system to the dorsal side,
and I don't know why we did any of those things,
but those are our accomplishments.
Along with that,
we centralized the mesoderm to the dorsal side,
we evolved the notochord
from the gut midline,
and concentrated all these structures by convergent extensions.
And we clustered antagonists of BMP and Wnts
into the notochord and endomesoderm
to form an organizer.
That is, we took all these separate signaling centers
and we packed them together
in a group of cells that was gonna stretch out and move
through the animal during its development.
Well, I would like to
thank all of you for listening to this,
I think it's been a historical talk
in the sense that we've tried to recreate
the history of our phylum,
and even beyond that
to our whole superphylum of the deuterostomes.
And I'd like to talk a little, for a moment,
about the evolution of this project,
because it came about
through really three people
who got together for an intense month of work
and then subsequent work during the course of the year
at Woods Hole, Massachusetts,
and the Marine Biological Laboratory,
and I'm very indebted
to their facilities and their support over there
for making this all possible.
John Gerhart, who is a professor at UC Berkeley,
who has done a lot of work on vertebrate embryology,
and myself
-- he and I have worked together for a long time,
as I mentioned in the introduction --
and Chris Lowe,
who is a very independent postdoc
who is now a faculty member at the University of Chicago.
And this was done on the cheap, in a sense.
We didn't have any grants for this,
we did it in spare money we could get together,
but with a lot of help with our friends,
and it turns out there were people who recognized
this was an interesting thing to do,
and provided us with all sorts of help.
So I wanna just sort of
go through a few of these people.
At Harvard Medical School, Kristen Kwan, who was a student,
did a lot of work with us in the summers.
Bob Freeman did bioinformatics,
absolutely essential to try to interpret these genes.
At Berkeley, Mike Wu did a lot of the molecular biology,
he's a technician there.
At the University of Chicago, the following:
Jochi Aronowicz,
Imogen Hurley,
Steve Green,
Marcin Wzila,
and Ellyn Farelly
were graduate students and technicians there
who came for some summers
and contributed a great deal to this work.
And then, to do this molecular analysis,
the basic idea, as I said,
was just to get a list of genes
and look through them and say,
these are the ones we wanna look at.
And so I want to just basically point out that
Eric Lander and Nicole Stange-Thorman, at MIT,
did the EST project for us,
and we had some real help from
Chris Gruber, at Express Genomics,
in making libraries.
And the microinjection of these eggs,
even though they're quite big,
they're very difficult to microinject
and the most successful people at it,
and the ones who contributed so much were
Mark Tersaki and Rachael Norris and Michelle Roux.
So this has been a village project,
and I think everybody just sort of felt
it was worth putting their efforts into something
that was a little bit unusual.
So thanks very much.