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I am Nicole Le Douarin.
I am a honorary professor at College de France in Paris.
I'm going to talk to you today about the
neural crest (because I am an embryologist)
and the role of this structure in the development and in the evolution of vertebrates.
The neural crest is a transitory structure
of the vertebrate embryo, and it is also a pluripotent structure.
It has been discovered by the German histologist, Wilhem His
in 1868 in the avian embryo.
By looking at the living embryo under the microscope,
Wilhem His noticed on the dorsal aspect of the neural tube,
a strand of cells lying there. And after a while, he saw
that the cells which constituted this structure
were moving away from the neural primordium
and were aggregating laterally to form the dorsal root ganglia.
This is why the first name that he attributed to this structure
was the Ganglionic crest.
This observation roused the interest of many embryologists at that time,
and they worked essentially on the neural crest
of lower vertebrates -- amphibians and fish.
And the work which was done during this period -- the first half of the twentieth century --
was summarized in a well-acknowledged monograph written by Sven Horstadius in 1950.
In the late 1960s, when I started to be interested in the neural crest,
very little was known about the role of this structure
in the embryogenesis of higher vertebrates.
And the reason for this is because in amniotic vertebrates,
the number of cells in the embryo becomes high,
and the cells which are migrating are rapidly indistinguishable
from the cells of the tissues through which they move.
This problem was overcome after I devised a cell marking technique
which can be applied in the avian embryo.
My talk today will be divided into three parts.
The first part will be the description and the use of the quail-chick marker system
and its use to study the ontogeny of the neural crest.
The second part will deal will the role of the neural crest in the
development of the head of vertebrates and also in the evolution of vertebrates.
The third part will concern the molecular control of the contribution of the neural crest
to cranio-facial and brain development.
The cell marking technique that I devised consisted of constructing
chimeras in ovo between two different species of birds:
the chick Gallus gallus, which was actually a common material for embryologists
at that time and another bird, Coturnix japonica,
the quail that you see on this side of the slide.
You see that the two birds, at birth, have different size,
and they also have different duration of incubation period.
Twenty-one days for the chick and seventeen days for the quail.
However, the size of the embryos during the first half of the incubation period,
where most of the important events in development take place,
is about the same in the two species.
The technique that I devised is based on an observation
that I made about the structure of the nucleus in these two species.
I noticed that, in all embryonic and adult cell types of the quail,
there is a particularity in the nucleus.
And this particularity consists in the fact that the heterochromatic DNA
is concentrated in the center of the nucleus
in a large mass which can be stained by a staining procedure which is specific for DNA
-- the Feulgen procedure. And this heterochromatic DNA is always associated
with the nucleolar RNP, making the nucleolus (which is unusual) Feulgen positive.
This does not exist in the chick, that you see in this part of the slide,
where, like in man, like in mouse, and like in most animal species,
the heterochromatin is evenly dispersed in the nucleoplasm during the interphase.
I had the idea of using this particularity of the quail nucleus
(which is a stable marker) to devise a cell marking technique to follow
the migration movements of cells during embryogenesis
and also to follow their fate.
The idea was to remove from the chick embryo, for example,
the part of the embryo, the fate of which is under scrutiny,
and to replace it with the equivalent region coming from a quail embryo
at exactly the same developmental stage.
These chimeras develop normally, and the quail cells which have been implanted
can be found in a time after grafting, thanks to the particularity of the nucleus of the quail
by using the Feulgen staining.
Now, it has been possible from 1975 to prepare monoclonal antibodies
which are specific for antigens which are present only on quail cells and not on chick cells.
One of those monoclonal antibodies, which is called QCPN,
for "Quail non-chick perinuclear antigen" has been prepared in the University of Michigan
by Carlson, and it is very much used to analyze these chimeric quail-chick embryos.
This technique can be combined with the use of molecular probes,
and transgenesis is also possible by using electroporation to introduce
nucleic acid into definite regions of the chick embryo.
One of the most interesting applications of the quail-chick marker system
was to study cells which have the property of migrating
inside the embryo during embryogenesis.
This is the case of the neural crest and also of hematopoetic cells.
And in my laboratory during the past years,
those subjects -- the neural crest and also the development of hematopoetic organs --
have been the main subjects that were under study.
The neural crest in the avian embryo
is derived, like in the other embryos, from the border
-- the lateral border -- of the neural primordium,
which are called the neural folds.
Closure of the neural tube starts at the level of the mesencephalon
that you can see here, and then proceeds rostrally and caudally
along the future spinal cord.
In the spinal cord, the neural folds are less conspicuous than at the head level.
However, the process of closure of the neural tube proceeds in the same way.
After fusion of the neural folds,
the neural crest cells (which are here) have lost their epithelial arrangement.
And they are rapidly going to migrate into the embryo along definite pathways,
at precise periods of time,
and they will settle in elected points in the embryo where they will differentiate
into a large variety of derivatives.
It has been shown in the recent years that, at this stage,
the neural crest cells can be distinguished from the cells of the neural primordium,
the cells of the neural tube, and also from the cells of the superficial ectoderm,
by the fact that they express a certain number of genes --
particularly transcription factors which are listed here.
For example, Slug, genes of the Sox family (Sox8, 9, and 10),
FoxD3 and also Pax3.
And they also carry a glycoprotein which has a glycosylated epitope
which is recognized by the monoclonal antibody HNK1.
The principle of the experiments that we have used
in order to study the neural crest cells and their fate and migration
is as follows. We take a chick embryo as a host,
and we remove at a definite region of the neural axis,
here, we remove the neural tube
which has just closed, but from which the neural crest cells have not yet started to migrate.
Then, we take a quail embryo which will be the donor embryo
and in which, we isolate the neural tube from exactly the same level
by subjecting this region to trypsinization -- in order that the neural tube
which is going to be grafted is not contaminated by other cells of different origins.
And the neural tube is then implanted into the chick host
at the site where the excision of its own neural tube has taken place.
And the next slide shows the real experiments
in which the chick embryo has been prepared for the graft by removing the tube.
You see here the notochord and on each side the somites.
And this is the neural tube of the quail, which is going to be implanted.
And you see on the top the neural crest cells.
And by 12 hours after implantation, you see that the neural tube
of the quail has developed normally.
It has been covered by the healing host ectoderm.
And in this slide, it is possible to distinguish the cells
coming from the neural crest of the quail
-- from the neural tube which has been implanted --
and which are migrating within the cells of the chick,
from which they are easy to distinguish because of the structure of their nucleus.
These chimeras develop normally. And in this case, the host embryo was
of the White Leghorn strain, which means it was completely devoid
of pigment. And you see that the only sign that this bird is a chimera is
the transverse stripe of quail-pigmented feathers.
These feathers, of course, belong to the chick, but they have been colonized
by the precursors of melanoblasts, which arise from the quail neural tube
which has been implanted into the chick.
These birds can hatch. You can see here a quail-chick neural chimera
which is three months old. It can walk, it can fly, it can compete for food.
Therefore, it has normal behavior, which means that the synapses
between the quail neurons and chick neurons, as well as the synapses
between the quail neurons and the chick muscles
are absolutely functional -- normal.
And this means that this chimeric model is quite reliable to study
the development of the nervous system and the development of the neural crest.
This technique -- these types of graft --
which were carried out systematically along the whole neural axis
by Marie-Aimee Telliet, Christian Le Lievre, and myself in the 70s
has led us to complete the list of
the neural crest derivatives.
The derivatives of the neural crest are numerous and diversified.
They include the neurons of the peripheral nervous system.
That means the sensory neurons, the sympathetic and parasympathetic
neurons, and also all the nervous system which is present
inside the enteric system -- that means the digestive tract.
The neural crest also provides this peripheral nervous system with glial cells --
that means with the satellite cells which accompany the neurons in the peripheral ganglia
and the axons in the peripheral nerves.
That means that the neural crest is the origin of the Schwann cells.
It is also the origin of the pigment cells of the body --
all the pigment cells except those of the pigmented retina.
It is the origin of endocrine cells -- the adrenal medulla cells
which produce epinephrine and norepinephrine
and also the so-called C cells of the thyroid gland --
that means the calcitonin-producing cells of the thyroid gland.
In addition to all these cell types, the neural crest is also,
most surprisingly, the origin of mesenchymal cells.
And they were called by the person who discovered them,
Julia Platt, at the end of the 19th century
mesectoderm to distinguish these mesenchymal cells
from the mesenchymal cells of the mesoderm
which are the mesenchymal cells which are all over the body
except at the head level, where the mesenchymal cells are derived
from the neural crest and then are called mesectoderm.
The cell types which arise from mesectoderm are cartilage and
bones. They form the connective tissue of the dermis,
connective tissue associated with striated muscles
which are at the head level, like the masticatory muscles, for example.
They form the vascular smooth muscle cells
and also the pericytes, and they form also adipocytes,
for example adipocytes in the dermis.
In addition to this list of derivatives that was derived from
... many of these cell types from our studies and studies which were done before
we were also able to distinguish from which level of the neural axis
all these different cell types arise.
And what is important is, as I said, that the mesectoderm
rises only from the neural tube which is at the head level.
That means the cephalic neural crest that you see here.
The TRON?; neural crest, as we have seen, does not give rise to mesenchymal cell types.
The cells which are going to become pigment cells
arise from the whole length of the neural axis.
And, as far as the ganglia of the peripheral nervous system are concerned,
their origin along the neural tube is regionalized.
And I'm going to give you an example of this.
As you can see here, we have indicated the gut on the left,
and the gut is interesting for us because it
is invaded by neural crest cells, which form two plexuses in the gut --
the myenteric plexus and the submucosa plexus.
And the origin of the cells of this plexus
are located anteriorly from the level of somite 1 to the level of somite 7.
The cells migrate massively from this region,
go to the pharynx, and when they're in the wall of the gut and the pharynx,
they migrate all along the gut, down to the cloaca.
There is a secondary supply of cells to form these gut plexuses
which arise from the lumbosacral region of the neural tube
and which concerns only the post-umbilical gut.
There is another region which is of interest.
This is the region which gives rise to the sympathetic ganglia,
and you see that the superior cervical ganglion
arises from cells which are at the level of somite 6.
The other region which is of interest concerns a region
from which the adrenal medulla arises.
And it is from this level which is from somite 18 to somite 21.
That means about the level of the wing in the birds.
And I'm going to show you experiments which are the result
of first the replacement of the neural tube at the level of somite 18 to 24.
And this is a section of the suprarenal gland of such a chimera.
You see, on the left, that there are strands of cells which are fluorescent.
And this fluorescence is the result of the so-called Falck and Hillap technique
which allows the catecholamines to be evidenced inside the cells.
So, those cells are cells which produce adrenaline and noradrenaline.
And the same section was subjected after to the Feulgen reaction.
And you see it on the right side, and if you
superimpose the two sections, you see that the cells which
contain catecholamines also contain the nucleolus of the quail.
And the cells which are between these strands of cells are catecholaminergic.
They are cells which have the chick nucleus.
And the cells which have the chick nucleus are derived from the chick embryo host,
and they are the cells which produce the corticosteroid of the suprarenal gland.
Now, let us see the replacement of the vagal region of the neural crest --
that means the anterior region from somite 1 to somite 7.
I call it the vagal region because this is the region from which
the roots of the vagus nerve which innervates the gut and forms
the pre-ganglionic fibers which innervates the plexus of the gut arise.
And this is the region from which the neural crest cells which are going to invade the gut
arise also. And I'm going to show you an experiment
which was done by Alan Burns in the laboratory.
And the same experiments were done by myself and also by Marie-Aimee Teillet.
You see the chick embryo on the left
in which the neural tube has been removed from somite 1 to somite 7.
And what you see between the somites
is the notochord. You see on the right the experiments
which consist in taking a quail embryo at exactly the same stage,
removing the same level of the neural tube,
and the neural tube which is red there is implanted in the chick after.
And you see the result of the experiment,
in which the quail cells were evidenced by the QCPN antibody.
You see how clean the experiment is and how well the neural tube
which has been grafted is incorporated into the host.
And you see here the migration of neural crest cells
which reach the ventral region, and you see finally part of the gut
-- the embryonic gut, of course --
in which the quail cells are stained with the QCPN antibody.
And you see that the submucosal plexus which is in tan
and the myenteric plexus are being formed on each side of the circular muscle layer.