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

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
  • 5
  • 2020/2021
  • Lecture notes
  • Kd
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torchiatantoine
I left you last time with a cliffhanger, which was that we were going to be looking at the molecular
biology underlying and regulating the period of clock gene oscillations. And I left you with this idea
that has been put forward by mathematical models in the field, which is that the periodicity is
potentially regulated by the short half lives, by the instability of the negative regulators. And that's
something that we wanted to investigate. So we've seen this slide before and what we chose to do
was rather than actually looking at the negative regulator in the notch pathway, we wanted to see if
we could manipulate the stability of the positive regulator, which is also very unstable and which
affects all of notch clock genes, and see whether that changed the periodicity of those oscillations. So
the particular component that we decided to look at was the notch intracellular domain. So this piece
of the pathway down here, once the receptor and the ligand interact, the intracellular cleaved piece,
which we've spoken about before, called NICD. So the idea was we wanted to see if we could
manipulate the stability of NICD and look at clock gene expression. So what do we know already
about the regulation of NICD stability?

Well, this is representing the C terminal end of NICD, a region called the PEST Main, which is
important for regulation of NICD stability. We know that because mutations found in this region of
the notch protein leads to increased levels of NICD. We also know that residues in this region are
phosphorylated and that phosphorylation event leads to recruitment of the STFE3 ligase complex,
but then targets NICD for degradation. But interestingly, despite the plethora of different roles notch
in development and in disease, very little is known about the actual sort of details of what kinases
are involved in that phosphorylation and what residues are being targeted. There are a number of
publications that have identified on this slide which have pointed to some potential kinases, but the
evidence behind a lot of that was fairly weak. So we want to come in and take a systematic approach
to looking at this. So the first thing we did, given the fact that the strongest evidence in the field was
that CDK8 may be involved in this regulation, was to use an essay that I'll describe on the next slide.

So what we do is to take the caudal region of the embryo, so this region down here and what we're
able to do then is split that in two so that we have these two identical explants here. And what that
gives us is a beautiful internal control. So we can then, for example, culture one side as a control, so
control media. And to the other side we add an inhibitor of specific pathways that we're interested in
looking at. And then what we're able to do is to look at how the dynamic clock gene expression is
affected. And so the inhibitor that we chose to look at was a CDK, a broad spectrum, a set of broad
spectrum CDK inhibitors. And of course, if the expression profile of the clock genes looks to be
retarded compared to the control side, that could be because it's going slower for gene expression or
that it's stopped. So to distinguish between those outcomes, what we can do is to then having found
one that affects the pace of genes, is to treat both sides with the inhibitor and then fix one of them
after three hours, for example, and cut to the other one for half a clock cycle, which is four to five
minutes in the chick and then see whether you see a difference in the expression profile of a clock
gene in the two sides. And if you do, that suggests the dynamic expression is still continuing, but it's
going slower.

So we did that assay and we did it in both a chick and in the mouse, and as I say, what we did was to
remove the tail end of the embryo, cut it in half, use one half as a control and to the other side as an
inhibitor. And one of the inhibitors that we use, this is a positive control for recognizing the antibody
against NICD was to use an inhibitor against gamma secretase. So that's what this GSI means, gamma
secretase inhibitor. And you can see that in me, this is a Western blot looking with either chicken
NICD antibody that we raised in the lab or tubulin as a loading control. What you can see is that in
the side that is treated with a gamma secretase inhibitor, you lose the NICD band, which is present
here in the control side. Loading control shows you that protein is present in both lanes. So that

, shows us that you're looking at the right band. And then these are lies from four different pulled
samples. ABCDE on the left hand side is the presomitic mesoderm with no CDK inhibitor. And on the
right hand side in each of those four sets of Blots is the PSM treated with CDK inhibitor. And what
you will see looking here at the NIC band is that in the inhibitor treated side, in each of those cases
you can see that there is a lot more NICD in the presence of CDK inhibitor. So that suggests that as
published by others, that CDKs appear to be involved in regulating levels of NICD. And so the idea
would be that if you are using these inhibiters, you stop phosphorylation by CDK and therefore
preventing degradation of NICD. So we then did a possum chase, I say, to look at how quickly and
NICD levels were degraded. And what you can see here is that in the presence of Cyclohexamide,
which prevents production of new proteins, you can see that during a time course within looking at
NICD by Westernblot NICD is degraded very quickly. But when you do that same type, of course, in
the presence of CDK inhibitors, that NICD remains more stable. So these higher levels of NICD that
we see in the presence of CDK inhibitors is due to the increased stability of NICD. So what happens
then to clock genes under those circumstances? So here we are using Yasini described to you before
we're looking at this in chick. So this is just a normal control, half embryo cultured for three hours.
And this is the other half of that embryo cultured in the presence of a CDK inhibitor, which we know
is leading to higher levels of NICD. So where you see high levels of NICD, what we're seeing is that
this side of the explant is stuck at the end of an oscillation cycle shown here on the right, whereas
the untreated side has started a new has gone right the way round and started a new wave of
expression. And so is ahead in terms of clock oscillation cycles. It could be, of course, that this one
has just stopped oscillating. But we know that that's not the case because we did the other essay
described to you, which is to now treat both sides with the inhibitor, but then fix one, this one on the
left and the one on the right acculturate for further time and culture. And what we can see is the
expression profile on those two sides is different. We've gone from the phase two here on the right
up to a phase three. So the expression profile is moving, but it's moving more slowly. So in the
presence of CDK inhibitors, we see increased levels of NICD and the clock oscillations are going
slower.

The other thing that we saw in the essay is that the size of the somites was bigger, and that's exactly
what the mathematical models would predict, because less of the president is himself passing the
determination front during one oscillation.

So what we then wanted to know was what the actual detail of that NICD regulation by CDK was. And
so this is the work of a student, Francesca, in the lab. And so what we know already from what had
been published was that NICD, when it enters the nucleus, it forms a complex with other proteins
such as CSL, Mammal, Skip, and shortly after that, that NICD is phopharylated, that phosphorylation
event recruit's Fbxw7, which is part of the STFE3 ligase complex. And what that leads to is to
degradation of NICD by the proteasome.

So what Francesca did was to use mass spectrometry to identify all of the phosphorylated residues in
NICD. So here is the sequence of NICD and green all of the sites that Francesca found were
phosphorylated and two of them had previously been identified by other labs. So what Francesca
then did was to generate forms of NICD, fused GFP in which she mutated each of these sites. And
then what she did was to do an immuno precipitation, pulling down GFP, so pulling down her
construct from the transfer of NTN3 cells and then to do a Western blot with a Fbxw7. And what she
was able to see is that mutations of Serin 2205 had no effect when it was changed into an alanine
have no effect on the interaction with Fbxw7. The same with 2527 when you change seeming to
alanine that it makes no effect. And the same here with 2538. when we mutated serine 2513 to an
alanine, what she saw was then a complete loss of this interaction with a Fbxw7. So if you mutate

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