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Cell signalling in development: Notch signalling and establishment of the embryonic body plan £7.49
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Cell signalling in development: Notch signalling and establishment of the embryonic body plan

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Cell signalling in development: Notch signalling and establishment of the embryonic body plan

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  • February 18, 2021
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  • 2020/2021
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torchiatantoine
In the last set of slides, what we talked about was the role of Notch in regulating the sulfate choice of
progenitor cells, contributing daughters to either the developing skeleton or the developing spinal
cord. And what we're gonna do today is look at, a potential role of Notch in one of those two tissues
once they have formed. So here we're looking at a slightly older chicken embryo than in the last
lesson slides, human looking at a 3.5 day chicken embryo. And if we take a cross-section through that
embryo at the level here of these yellow dotted lines. Then what we see over on the right is this sort
of donut shape that represents the spinal cord, the developing neural tube that will develop in the
spinal cord. And what we know is that lining the lumen, just here in the center of that overall region.
Lining the lumen in this sort of beige area is the neural progenitors. And then out towards the outer
periphery of that structure are the differentiated neurons. And we know and have known for a long
time now that Notch plays an important role in preventing the neural progenitors from
differentiating too quickly. And so maintaining some of those progenitors and preventing precocious
differentiation to form neurons.

But what I want to think about today is where the Notch plays an earlier role in setting up the pattern
of those progenitors such that they can generate different types of neurons. So initially, what
happens is that neural plates or that neural tubes at the neural plate that folds up to form this neural
tube is made up of equivalent progenitors that don't express a specific marker that's represented
here by the green immunohistochemistry result. And so here we have an antibody that recognizes
that a PACS protein, which is expressed by cells that are in the middle of that neural epithelium here.
And initially that gene is expressed by all of those progenitors. But gradually there are patterns of
different types of progenitor setup that express different markers. The red marker bear or the blue
marker, or indeed this purple marker. And the expression of those markers is established through the
activity of a signal that's released by this structure here, the blue circle, which is the notochord that
we referred to in the last set of slides.

So what happens is the notochord releases a signal that's called Sonic Hedgehog (SHH). And sonic
hedgehog is then able to diffuse through this tissue and setting up therefore what we call a
morphogen gradient. So cells far away from the source. Up here, we'll see less concentration of Sonic
Hedgehog then cells close to the source down here. So in that way you set up this concentration
gradient across that developing neural tube. And the cells turn on the expression of different
markers. The green one, the red one, the blue one, and the purple one in response to different
concentrations of sonic hedgehog. In addition then to the strength of signal, strength of Sonic
Hedgehog signal that cells receive. It's also the duration of sonic hedgehog signaling that is important
in setting up that pattern of neural progenitors. So if we look here at the schematics in this square up
here, what that's representing is expression of ventral markers in that neural tube over time. So time
is along the bottom axis here. And so early on, the notochord releases low levels of Sonic Hedgehog.
So at time ten here. And the cells in the ventral region at the neural tube turn on expression of this
orange marker called OLIG . And as time progresses, levels of Sonic Hedgehog are increased. So
higher levels released by the notochord. Then the cells in the ventral region of the spinal cord turn on
this red marker called NKX2.2 and the cells adjacent to the ventral region see slightly lower levels of
sonic hedgehog because it diffuses away from the source. And so they turned on the orange signal
that was previously expressed down in the ventral region, and this time progressively their father.
Then what happens is the notochord releases even higher levels of sonic hedgehog. And so at that
point, the cells in the ventral neural cheap actually turn on Sonic Hedgehog itself and not as a marker
of a population of cells that are called floor plate cells.

So what that means is that as son-in-law, Sonic Hedgehog concentration increases and as the
duration of exposure to sonic hedgehog changes, then cells at the ventral neural tube take on

, actually different fates. So initially, as levels of Sonic Hedgehog rise, then cells in that ventral region
turn on the expression of markers of motor neurons, which is the OLIG marker up here. They then
turn off the OLIG marker and turn on NKX2.2 which is a marker of the P3 domain here. And then as
levels reach even higher, they turn on the expression of sonic hedgehog, which is a marker of the
floor plate. So the character of the neural progenitors in this ventral region changes as the time at
concentration of sonic hedgehog that those cells are exposed to changes. And actually the floor plate
itself is a very curious cell population because although it requires the very highest levels of
Hedgehog. And what we know is that if you expose levels of if you express the neural tube to even
higher levels of Sonic Hedgehog, then in actual fact, those cells will turn off the floor plate marker
and will actually revert to a P3 phenotype. So you actually need to stop exposure to high levels of
Hedgehog to maintain that floor plate faint.

So here on the right we are seeing the expression profile of this gene called Ptc1 (cPtc11), which is
both a target of a Hedgehog pathway and a receptor for the pathway. And what you will see is that
early stages. So, so these images on the right hand side, here and here and here. In fact cross-
sections at these three levels of the embryo here. And so at the level C, which is the caudal must
imager region of the embryo, we see only low levels of Ptc1 are turned on as levels of Hedgehog
around slowly released from the notochord at this slightly higher level of In your axis. Then here we
see slightly higher levels of Ptc1 in that ventral region. And then at the level of a here where you're
seeing the very highest and longest levels of exposure to sonic hedgehog. What you see that in this
region, that you want to become a floor plate, you actually have to turn off responsiveness to Sonic
Hedgehog. And that is exemplified here by the loss of the expression of the, of the Hedgehog target
Ptc1 in the most ventral region that will become floor plate. But you remain hedgehog responsive in
this domain, which becomes a P3 domain and expresses P3 markers such as NKX2.2. All of that to
explain that the ventral pattern in the neural tube is completely reliant on exposure to this
morphogen called sonic hedgehog. Now, I was wanting to know whether Notch plays a role in this
set up of this progenitor profile. And so what was the clue that that might be the case?

Well, if we look at the expression profile of a Notch target now, and that is exactly an example of
that, is for example, the cHairy2 gene. Then we can see that at very early stages of neurogenesis,
when we don't yet have a neural tube, but in fact we have a neural plate as exemplified here. Before.
That neural plate is folded up to form a neural tube, you can see that expression of the Notch target
CHairy2 is expressed in this region that will become the ventral region of the neural tube. In exactly
the same way that this hedgehog target cFoxa2 is expressed in that domain. And sure enough, when
that neural plate eventually folds up to form the neural tube, then CHairy2 to expression, the Notch
target remains on in that ventral region of the neural tube. And that is actually very similar to the
expression profile I just showed you for the hedgehog targets cPtc11. Expression profile of Hedgehog
targets seem to match that of Notch targets. And what I also told you is that at later stages, so
further up the axis of the chicken embryo Ptc1 is now downregulated in that third most ventral
region of the neural tube. Because the cells need to lose responsivity to hedgehog. And actually we
see that CHairy2 is also lost from that domain at later stages. So the expression profile at the Notch
target is beautifully matching that of the hedgehog target its own in the ventral region at early
stages, lost in the ventral region at later stages. So that suggests that perhaps the Notch target may
be responding in some way to sonic hedgehog morphogen signal.

So we decided to test that. And what we did was to take a piece of neuro epithelium at this level that
would never normally be exposed to sonic hedgehog because this is the domain that is exposed to
Sonic Hedgehog. And we cultured this little piece of normal epithelium, either in the absence or in
the presence of Sonic Hedgehog. And then we look to see the expression of Hairy, the Notch target.

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