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0:00
- Olivier Puckry really does not need any introduction. He has been a pillar of the developmental biology field.
0:09
And he has done seminal work on cell differentiation, morphogenesis segmentation. And I am really looking forward to hear his presentation today. He is now, has a dual appointment at the Brigham and Women's Hospital and in the Department of Genetics at Harvard Medical School. And we are very happy to have him teach us about segmentation and body axis today. Thank you, Olivier.
0:45
- Thanks. Thank you, Vanessa. So I wasn't too sure what to do initially. Mara asked me to do a tutorial on development and I didn't want to do something too general. So what I thought I do is to illustrate a few basic concepts and using mostly work from my own lab over the years and try to describe some ideas which I think would be of interest for this audience. No equations, sorry.
1:25
So one of the things that I've been interested in for a very long time is what I call the cardinal features of the body plan. So if you think about most animals, they exhibit an elongated body axis. They are segmented. That means they are formed by a repetition of anatomical modules that be naturally symmetrical. And also they show some regionalization along the anterior-posterior axis. With a head and trunk and tail and so on. And so, I mean, if you think about these particular features
1:59
they are not non-trivial. I mean, we don't really understand why they've been selected and conserved throughout evolution. And they're established during early embryogenesis. As you can see here, this is like a two-day chicken embryo where you can already see the elongation of the body axis. You can see the segmentation of the future debris. The precursor is called somites. You can also see the bilateral symmetry which is very clear at this stage under the regionalization.
2:36
So now if you think about vertebrates, often people think about evolution as generating a huge diversity of form, which is true. But there's also a very striking conservation which underlies this diversity. And so here you can see these different vertebrate animals all show the features that I described. So they are bilaterally symmetrical, they are segmented and so on. And in fact, this extends even beyond vertebrates as you know well. In vertebrates like worms, for instance,
3:12
or flies are also elongated, segmented and bilaterally symmetrical.
3:18
So this can be traced back very, very early
3:25
during the Cambrian explosion. So we already have, for instance, in the Burgess shears which are more than 500 million years old, you already see fossils which have this kind of organization as shown for this creature called a hallucinogenia which exhibits all the particular organizational features that I described. So, I mean, again, this throws the idea that maybe why these features have been conserved is some sort of contingency that happens once it turned out to work well
3:59
and it's been conserved throughout evolution or whether there is some sort of selection pressure or some advantage, for instance, why being bilaterally symmetrical is advantageous because also you have to realize that coordinating this process between the developmental processes between the left and the right side is amazingly complicated and we don't understand it actually. So I think it's still an open and interesting debate.
4:29
So what I want to focus on today is more,
4:36
tell you a bit about the segmentation and the formation of the body plan and what I will concentrate on is what people call the vertebral formula, which is a characteristic of each species and which essentially describes the number of segments or vertebral segments that are found by anatomical regions. And so to understand how this vertebral formula is implemented during ambiogenesis and how it varies across evolution, it's important to understand how segmentation works.
5:15
So I will start with this, but then it's also important to understand how the body gets re-generalized. So that's how can evolution modulate the number of segments by anatomical domain and as you'll see, this is something which is to a large part devoted to a class of genes called the Hox genes.
5:37
So, yeah, so segmentation, that's essentially this idea that the body axis of most animals, vertebrates, but also invertebrates, is subdivided into periodic anatomical modules.
5:54
Invertebrates, this is very obvious at the level of the vertebral column.
5:59
Invertebrates, like annelids, it's seen at the level of the metameres that includes this parapodia, which are kind of legs.
6:13
And I'm going to focus mostly on how this pattern is established in invertebrates. In invertebrates, as you, just for the history, this segmentation has been very, very well studied in flies through the work of people, the pioneering work of people like Nusslein-Volhard and Wieschaus, who got the Nobel Prize for this in '95, were the first to transpose genetic screens to embryo and to study morphogenesis. And they identify a full genetic cascade called the segmentation cascade,
6:48
which ironically is not conserved in most animals. It's, flies are very evolved type of creatures. They have a very special type of development. I'll make a few reference to this in a bit, but in any case, this example, which has a very strong
7:16
epistemological value is, I mean, it's interesting because it's, again, it's not conserved. And you'll see in invertebrates, there is another process that has been implemented, which is also found in other invertebrates, non-flies. And this is what I want to describe. So the embryonic segments are called somites, as I mentioned. They derive from a tissue, which is called a paraxial mesoderm, which is this tissue in red with flags, the axial structures, the future spinal cord
7:50
and the underlying notochord. The embryonic somites are epithelial spheres in invertebrates that form from a mesenchymal tissue called the presomatic mesoderm. And the somites give rise to a large fraction of the musculoskeletal system, the axial skeleton, the ribs, which are clearly segmented structures, and also the skeletal muscles, all the skeletal muscles of the body. And also a few other derivatives. Some of them are segmented, some other not, like for instance, the brown fat,
8:24
which is involved in thermogenesis is not segmented. Endothelia, which participate to blood vessels are segmented and so on.
8:34
So the presomatic mesoderm itself is continuously formed during a process, which is called a gastrulation, where the cells, which are initially superficial in the embryo, ingress to form the mesoderm. So this process eventually starts in a structure called the primitive streak, which is also called the blastopore in lower vertebrates. And then it morphs into what's called the tail bud, which is, as I'll show you, a sort of engine that serves to produce cells to elongate the body axis.
9:12
So the primary segmentation of the body derives from the somites, but it also impacts the segmentation of other structures like the peripheral nervous system. And so here you can see a drawing representing all the peripheral nerves. You see that they are clearly segmented. There's one pair for each vertebral level. And this is implemented through a process called the resegmentation of the somite, which essentially describes the fact that vertebrae are formed by the fusion
9:48
of the anterior part of a somite to the posterior part of the consecutive somite. So there is a sort of shift between the definitive vertebra and the initial somite.
10:00
And this means that the two parts of the somites have different properties. And this is particularly marked at the level of the extracellular matrix, which is different between these two compartments, anterior and posterior. And what this results is that only the anterior part of the somite here is permissive for the migration of the axons of the nerves and the neural crest cells. And that's what is going to control this periodic organization of the nervous system.
10:34
So it's a secondary consequence of this organization. The other thing that I'll discuss a little bit because Paul mentioned it yesterday, is that in fact, and I never thought about this like this, it's actually very relevant to what you discuss between this double cusp and the homoclinic flips because the homoclinic flip is this idea here
11:02
that you can choose between two fates, anterior and posterior. But I'll get back to this.
11:10
So the question, an interesting question is, how do these embryonic somites form during embryogenesis? And they show a number of striking characteristics. Some of them relate to these initial cardinal features that I discussed. One which is very specific and original property of the somites is the rhithmicity of their production. So they are produced with a defined rhythm which is species specific. And that's one of the few embryonic structures that actually shows this type of property.
11:49
They also show directional production if you want. So their production follows the elongation axis, the anteroposterior axis.
12:01
One question you may ask is, what controls the total segment number? So each species has a defined number of somites and that's what controls the total number of vertebrae and hence the vertebral formula. And so I'll try to show you how this total number of segment is controlled in embryogenesis. And then the bilateral symmetry, which is a very well illustrated at the level of the somites as you can see here. And so I'm not going to discuss bilateral symmetry.
12:35
It's a very interesting property of most animals which belong to the plate of the bilateria. And we know very little about how this is established and how this is maintained and coordinated during development. - [Attendee] Olivier, but in the context of the somatogenesis, is bilateral symmetry evident because there's an oscillator on both sides and these two are coupled? So it's not a single oscillator. - We don't know whether they're coupled. - [Attendee] I see. - That's the whole-
13:06
- [Attendee] The observation is that they're synchronized. - That's the whole, they're synchronized but we have no idea whether they are coupled. And that's pretty mysterious.
13:16
So this is just a more dynamic illustration of the process of somatogenesis where you can see the rhythmic addition of pairs of somites which proceeds in a bilateral fashion at the tip of the prismatic mesoderm. You see that this is tightly coordinated to the axis elongation process. And I'll show you that this coordination is actually what controls the total number of segment in an embryo. And here is a chicken embryo where you can see this happening in real life
13:48
where you see here the rhythmic addition of somites at the tip. And you see that this is coordinated with this very striking elongation movements that are involved in the formation of the body axis.
14:01
What's worth noting also here, I'll come back to this but this structure here is called the primitive streak and becomes the tailbutt. And that's what generates most of the embryonic tissue.
14:26
So how can we understand somite formation? There's been a number of theoretical models that have been proposed over the years. The most famous one is called the clock and wavefront model. And this model was actually proposed in following up some work from Weddington that we heard about yesterday. And because at the time people after Turing's work and people were very excited by reaction diffusion. And the model was proposed in reaction to that because Jonathan Cook who's one of the authors
15:07
did experiments where he reduced the size of frog embryos and showed that this was leading to a reduction of the embryo. It was, sorry, it was leading to an embryo with the normal number of somites but smaller and suggesting that this could not be explained by a simple reaction diffusion mechanism in which you wouldn't have expected a change of the biochemical property of this mechanism, the mechanism involved in somitogenesis. So anyhow, it was a very ad hoc model
15:46
proposed by Chris Zeman who was a mathematician
15:51
working on the catastrophe, the theory of catastrophes that Paul discussed yesterday. And so the idea was that the formation of a somite was one curse catastrophe, which is,
16:06
and I don't want to get into this because I don't understand this very well. It's just a set of solutions from
16:16
the domino equations or something like that. But anyhow, the thing is that this type of catastrophe is also very similar to a bistable transition, for instance, in physics. And the idea that he was pushing is that somitogenesis was one such abrupt transition between a mesenchymal and an epithelial state. And then in order to make the catastrophe periodic, they just postulated the existence of an oscillator or clock. And that's why it's called the clock and wavefront model.
16:51
The wavefront is the system that essentially displaces the catastrophe in space. And it was proposed to be the normal maturation wave of the embryo, the maturation gradient, which is seen in vertebrate embryos. And so it's an interesting paper to read as it's not really mathematical in reality, it's very descriptive. And at the time it
17:21
was quite well known and then it kind of got buried because there was no experimental support for it for a long time.
17:35
And 20 years later or so, we felt that there are genes actually that show this kind of periodic expression, which was predicted in a model. And so these gene are expressed, as you can see here, this is a time window to form one somite. And in blue, that's their expression domain in the presomitic mesoderm, which appears as this traveling wave that you can see here also in real embryos. So traveling waves of transcription in the precursors of the somite
18:07
that anticipate the formation of each segment. And so the periodic nature of this process led us to propose the existence of this oscillator called a segmentation clock.
18:21
And work over the years led to the notion that this oscillator is a gene regulatory network
18:30
that is downstream of several main signaling pathways involving FGF, Notch and Wnt that oscillate with defined choreography.
18:43
So in mouse, for instance, FGF and Notch oscillate in synchrony and in opposite phase to Wnt.
18:50
And what happens is that the pathways are constitutively activated. So the input is always there. And that triggers the expression of negative feedback inhibitors.
19:03
The vast majority of what we call the cyclic genes that show this periodic transcription are negative feedback inhibitors able to shut down this pathway. And so the concept that's been proposed to explain these oscillations is that
19:29
they're triggered by this activation of the negative feedback inhibitors that shut down their own activation and eventually get degraded because they are not produced anymore and this can generate this kind of loops. But there's also been maybe a more precise model proposed which actually now we know very recently from work from a study that's been done in the US from the lab, from a PhD student in the lab that the model is wrong, but I'll show you nevertheless because this is the mainstream.
19:59
This is proposed the mainstream model for the oscillator of the clock. And that involves this transcription factor for called HER1 or HER7, which is a BHLH transcription factor at the repressor which can shut down its own expression, but after some delay. And so, and this transcription factor is constitutively activated downstream of signals from the pathways that I discussed. And so, and you can easily show mathematically that this type of circuit is able to generate oscillations.
20:36
And that was originally proposed by Julian Lewis as a pacemaker controlling the oscillations. But I won't show it to you, but we have now results that if you knock this out, you still get oscillations, they are just desynchronized.
20:53
So, then this clock was first identified in vertebrates
21:02
in the chicken embryo, and then in mouse, in zebrafish and many other vertebrate species. And it turns out that there is also some similar kind of mechanism at play in insects and so in vertebrates. And this was shown using tricks like the one we initially used in the chicken embryo where you split the embryo in two, it's two bilaterally symmetrical halves and you culture the two halves independently. And so, for instance, here you see this is one half which was fixed immediately.
21:34
This half was cultured for 30 minutes and you can see the progression of the oscillation from this pattern to this pattern. And if you do many of these experiments, you can reconstitute the entire oscillations. And so they applied this, and that was the group of Mikhailis Avrov, applied this to Tribolium, which is a short germ-borne insects that means like, unlike flies which have a very synchronized development, so all the segments appear at once.
22:02
In most insects, the segments are progressively generated like in vertebrates. And so when they did the same experiments, they saw the same kind of dynamic pattern and could establish the periodicity of some oscillator with different sets of genes in Tribolium.
22:24
It's also conserved in humans. And in humans, it's tied to a set of disease called congenital scoliosis. And all the genes that were identified in this classical linkage studies of this disease which are characterized by vertebral defects that can be seen on X-rays are genes that are associated to the segmentation clock.
22:52
And so the clock defines the periodicity, but as I said, there's another system called the wave- - [Attendee] Automatic symbiology, the seminar. Drogon morphogenesis low-glide. This is NFR-UVs. - [Attendee] Go , please, mute yourself.
23:08
- Sorry. - I'll let you have a go. Okay David. - In the hospital, we have code blue, code blue, I don't know.
23:19
So clock specifies the periodicity that's necessary to, you know, to run this process. But as I said, you need a wavefront that spaces the response to the clock. And so what became clear is that the wavefront is established by a system of posterior gradients that involves, again, the Wnt and the FGF pathway that are graded, as you can see here, this is the ligand of GFA, for instance. This is the response, goes for ERK. This is the response to Wnt signaling, which is with nuclear beta-catenin.
23:55
And so these two gradient peak in the tailbird and decrease as you move along the prismatic mesoderm, defining what we call the determination front, which is essentially a competence window to respond to the clock. And you'll understand on this movie. So here's the combination of the two processes, the clock in blue, which delivers these traveling pulses of signaling that move along the tissue, and then the posterior gradient, which gets the response to the clock.
24:29
So in other words, what happens is that only the cells that passed this determination front, so this signaling threshold, are competent to respond to the clock and they do so when they get hit by this wave of signaling. And so the system ensures the sequential and rhythmic formation of stripes of gene information, which are shown here in black. And so for instance, the transcription factor called MES2 is one of these genes downstream of the whole process. And it's going to set up the next step,
25:05
which is the specification of the anterior-posterior compartments and the epithelial rosette formation that is required to make the somites.
25:17
And just to come back to what Alan was asking, this is incredibly coordinated. I'll show you movies in a little bit. And we don't really understand how this is coordinated between the two sides.
25:33
These are such movies. So after this work, people were able to starting to develop different system to visualize the oscillations in embryos. So this was from Kageyama's work with a Luciferase reporter. We developed some GFP reporter, and then you can see in a real embryo how this looks like. See, this is really nice. You have these nice oscillations that take place. This is a dorsal view, and this is a lateral view where you see the left pre-somatic mesoderm.
26:04
What is also important to understand here is that what it shows is like the, it's a visualization of active signaling in the embryo. You can see that it's very coordinated. It's very dynamic because the timeframe here is short. I should have said these are mouse embryo. The clock oscillates with a two-hour period. In chicken, it's 90 minutes. As we'll see in a bit in human, it's five hours.
26:33
Most of this early work was done with actual embryos, but then work from Alexander Orlela in Germany
26:43
showed that you can actually transpose the system in vitro. What he did is just to cut tailbutt from these mouse reporters and put them in a dish, and then he showed that they formed this very nice, so they kind of spread and form a nice cellular structure. But they retained the oscillatory behavior of the embryo, and you can track the fate of the oscillations here. And so that's work that he actually did with Paul Francois that we heard yesterday, which really showed that the system
27:18
was amenable to in vitro studies, and that that was a real game-changer. And for instance, in the lab, in work that we did with Ola, we adapted the system and tried to
27:33
optimize it in order to keep the cells oscillating ideally forever, but we were not successful, but at least far longer than they normally do. Because in the original system, the cells were recapitulating their normal developmental schedule, which means they were doing a few oscillations and then stopping. The ideal system, if you want to understand the oscillations, is basically a cell line that will oscillate forever. And so here we were getting close to this, as you can see,
28:04
maintaining also this geometry of traveling waves, but by changing the geometry from an elongated rod to a disk, then we obtained these concentric waves. There are a lot of... yeah? - [Attendee] If it's okay for that, feel free to punt this for later. So relating to the talk yesterday,
28:28
you can imagine two ways that you exit this state. The first one is that so long as the correct signaling environment is present, the cells are trapped in this state, and then what happens normally in the embryo is that the external environment changes, the signaling changes, and then they exit the state. And this is consistent with this idea that there is a pre-existing landscape which is then deformed in response to signaling. But another view is that if you
28:50
maintain exactly the same signaling, then these cells have a finite clock. They have an internal state, and they hit the end of that state, and then they exit, in which case it's really not a fixed point which has a destabilized, which of these is- - So I think that's the first one, because clearly from an evolutionary standpoint, we know that there's a lot of variability. I'll show you. So in the mouse, for instance, or the chicken, they will, the cells in the presemitic mesoderm
29:19
undergo a defined number of oscillations. Like, usually it's somewhere, for mouse, it's 6. For chicken, it's 12, because the presemitic has different length. And so it varies a lot. In snakes, it's also, I'll show you. It's much more. So I don't think there is, they count a fixed number. We know that if you, in contrast, you know, there's this FGF gradient. We know that if you stop, if you block FGF, you kill the oscillations immediately. So clearly there's a requirement
29:50
for this signaling type of landscape to be present. And in the embryo, it changes. So it's so great. - [Attendee] Is the flip side true? If you maintain the correct signaling environment, can you get them to soften you? - So yeah, that's a good point. It's half true. Half true. So here in these experiments, what Alexei at the time was able to do is to very much extend the duration. So it went to 48 hours, whereas normally in the embryo, it's a window of a few hours. But it eventually stops.
30:24
But then you don't know whether this is because your conditions, your media conditions are, you know, suboptimal to maintain, actually, the health of the cells. Yeah. - [Attendee] Is there a cell division in these explants? - There is, yeah. - Okay. And is there determination? - So, but the- - The duration front is- - Yeah, but the, no, so the rhythm of the cell division is much slower. - [Attendee] Uh-huh. Like one day or something? - Something like that.
30:52
Less than that, but 12 hours or something like that. - Yeah. - But then- but there's no determination here. Like, the determination front- - Yeah. - [Attendee] I mean, it was in a different part of the embryo. - Here, there's no determination front. - Yeah, but I was not going to go into this, but that's one of the findings that we made, which was very surprising at the time, is that in the clock, there is also a frequency gradient. As you know, as the wave moves along the tissue, it slows down.
31:20
But here, there's not, because FGF is flat. And people thought that the gradient was important to establish the frequency gradient to generate the traveling wave. But here, we can uncouple the traveling wave from the frequency gradient. 'Cause there's no frequency gradient. - [Attendee] But you could tune FGF and tune the frequency. - We could, we could. But the fact, what was surprising is the fact that you can get the wave without the gradient. - [Attendee] Yeah, yeah, no, I understand.
31:51
I'm just also clarifying. - And this, we still don't understand this. And so, one of the conclusions of these experiments, which I'm not going to describe, and that was thanks to Ma's input, was that one could view the clock as an excitable system, where notch would be the stimulus. And in this analogy with the pendulum, you have a threshold, which we showed is also defined by another signaling called the YAP, the mechanosensing signaling. And so, and that explained...
32:22
we could explain some of the experiments that we had a very hard time to figure out. Namely, the fact that we can get oscillations in the absence of the ligand for notch. So, we can isolate the cells and get them to oscillate by lowering the excitability threshold. So, if we remove completely YAP signaling, the cells start to spontaneously oscillate, even in isolation.
32:49
But I don't want to spend too much time on this. I would like to try to go to the end, if I can. Just want to say that... so, then we moved to human embryos and focused on somatogenesis, which is something which is not understood, because it happens between three and four weeks, so time when women don't even know they're pregnant, usually. So, there is no data on this. And so, somatogenesis happens, yeah, between 20 and 36 or so. And so, the only way to learn about this
33:24
is to use pluripotent stem cells. So, we started to develop protocol to differentiate pluripotent stem cells to the paraxial mesoderm. And this worked really well. So, we are able to generate cultures which are almost pure of posterior presomitic mesoderm, so the tissue where you see the oscillations, in two days and with a very simple treatment involving Wnt activation and BMP inhibition. And the differentiating cells recapitulate the normal sequence.
33:56
So, that means they first form tailbone precursors and then presomitic mesoderm. And then work from Margarita Diaz-Cuadros when she was in the lab showed that if you introduce a fluorescent reporter for the cyclic gene in these cells and differentiate them to presomitic mesoderm, they will recapitulate the oscillations.
34:17
So, this was, of course, a game changer for us because instead of micro-dissecting 5,000 cells in embryos, now you can generate 10 centimeters plate of synchronized oscillating cells. And so, in humans, she showed that the period of the oscillation is slower, it's five hours, which is also an interesting question which I'm not going to dwell upon. But in mouse, the oscillation is two hours. And for both species, it happens at 37 degrees, which means that there are some genetic mechanisms
34:51
that control the pace of development. And we know that a mouse develops in 21 days and it lives two years. Human develop in 280 and live 80 years. So, these are mechanisms that are actively controlled genetically because they happen also at the same temperature and we don't understand well how it works. We showed that this has to do with metabolism, but that's what we know at this stage.
35:19
So, these were the 2D systems, but Yuchuan was a postdoc in the lab, developed 3D systems where he adapted the protocol to non-adherent conditions, and so that leads to formation of the same tissue, but in a sphere. And then what you can do is you can seed these spheres of prismatic mesoderm onto an adherent substrate, such that it's going to form a small dome that will spread. And you will hear work from Benhui in the lab because there's a wetting mechanism and a dewetting, which takes place.
35:56
I mean, there's an interesting physics that takes place in these experiments. And the beauty of the systems here is that you can introduce reporters, fluorescent reporters that allow to track the cells with a very nice resolution. And here, for instance, you have two synchronized movies, one which shows in green the oscillations of the clock and then in purple, the reaching of the determination front. And then the one on this side will show the onset of a gene called PAX3,
36:29
which marks epithelial somite formation. So here you see the concentric waves of the clock. So there are two waves in this, or three, maybe in this one, you reach the front. And once you pass the determination front, then you will form the epithelial somites. And this is what you see here. And you see that the culture organizes into this striking epithelial rosettes. So it's a very interesting system because, I mean,
36:59
you know, there are some interesting questions you may ask about how this form, how do they organize and how do they get patterns and so on. What is also interesting, and I'll show you in more detail, the second system that helps understand this, is that this disconnects space and time because here there's no space element. It's purely a time progression. So it shows how you can uncouple developmental modules using this kind of in vitro system. And I think this is something quite fascinating.
37:35
So now we can use also this to study the anterior-posterior compartmentalization of the somites, which I mentioned earlier, which is important on the vertebrae, as I said, and the peripheral nervous system. And so what Paul was discussing yesterday about the slot, it directly relates to that. And the question which has been on for a long time is whether after segment determination, say at the end of this oscillation process, do you specify a whole segment which subsequently becomes divided
38:12
into anterior and posterior? And that is a homoclinic flip.
38:18
Or do you specify directly the anterior and posterior fades? And that's what's important for segmentation, which is this double cusp mechanism.
38:29
And so, and I actually didn't realize that this work, when he mentioned it yesterday, that it could relate to this, because it's been mostly discussed in the context of the second part of his talk with the work from James Briscoe's lab and Eric C. Chan and others.
38:47
So, but if you want to study this in the embryo, what you can do is engineer reporters for genes that are specifically expressed in the posterior and the anterior compartments. And you see using these somitoids that the cells do acquire these two identities. But interestingly, they segregate in different rosettes, which is not what we see in the embryo, because in the embryo, rosettes have either an anterior or posterior identity.
39:17
And Yu-Chuan studied this, and what he found is that when this gene MES2 is activated,
39:26
in reality, it's activated in a salt and pepper fashion. So it's not all the cells that activated equally,
39:35
meaning that they reached the determination front, but there is some sort of difference with high and low expressors. And this is work that actually we did, all the quantification part was done with Stefano D'Italia, who's going to speak later next week. And then what happens is that
39:56
you have this initial salt and pepper specification of the cells, and that leads to a sorting mechanism, which you can see here, where you see that the dots mark the high-expressor cells. You see that they will exit this orange area, and this sorting mechanism is what leads to the formation of these domains with anterior and posterior identity. - [Attendee] Anterior inward, posterior outward? - No, I'll show you in one second. Anterior is along the AP axis, so you have the somites,
40:35
it's a further subdivision of the... - [Attendee] Right, right, right. And now in this radial... - We haven't looked at this yet, so Yuchuan is looking at this. You mean like, is there... - [Attendee] Presumably the AP axis gets mapped to a sort of like luminal or inward-outward axis - Not, no, because they form independent rosettes. - [Attendee] Okay. - They're going to form their own rosettes, which is not what you see in the embryo. - I see.
41:05
- So that's another case where you have also uncoupling, you know, between the epithelial process formation and the specification of the anterior-posterior compartment, which I think are really nice. Yeah.
41:19
- [Attendee] Is there some sort of a planar polarity?
41:24
- There is some sort of directionality because the cells, the anterior cells, know that they have to move anteriorly and the posterior ones know that they have to move posteriorly. I don't know if I would call it planar polarity.
41:39
But there is this, yeah. Yeah. - [Attendee] So what is the source, what is the cause of the sorting? I mean, is there any differential addition? - So they, after the 72 hours, oh, sorry, during this phase when they start to activate MS2, they do express a lot of adhesion molecules. And this is trans-science. So it only lasts, so the sorting property is trans-science. If you take the cells later, after they're sorted, they cannot sort again. - [Attendee] So one of the cell types
42:15
just has an overexpression or like transient overexpression of an adhesion molecule. - Yeah, exactly. - Other types, nothing changes. - Yeah. Well no they express different codes of adhesion molecules. So you can sort the cells using these reporters and you'll see that the repertoire of adhesion proteins that they express is different. - [Attendee] Are these N-cadherins? - There are some cadherins differently, some efferins, also some protocadherins. It's a very complex repertoire, actually.
42:45
We could not single, what we could single out is the activation of RAC, which is, so one of the genes is TiAM1, which is a RAC activator, which is specifically expressed in one of the two compartments. And if you force expression of TiAM1, you will activate RAC and you suppress completely the sorting mechanism.
43:07
So we have some hints, but... - [Attendee] But if you, okay. If you stain for RAC in the, at the end of that movie. - Yeah, we haven't done the real cell biology, actually. That's where, you know, then we and Neutrona are moving now.
43:27
- One more question. - Yeah. - [Attendee] Does the differential RAC gene expression, does that make you think there's also differential motility between the two cells? - Probably, probably. The difficulty, it's a bit trivial, but you know, here you can easily track the black cells, but the white ones is much more difficult. So we don't have a good reporter for the posterior compartment. So that's, we could really efficiently track only on the fraction of cells, but it's probably, I mean, yeah,
43:57
I think what you're getting at is like, there's one population which is actively moving out of the other and that's very possible, but yeah. - [Attendee] So in the initial salt and pepper formation, initial condition and start, is there some sort of lateral inhibition that led to it? - Yeah, so that's a very good point. That's the idea we have. That's what you try and is testing now because this is all downstream of notch, you know, and MS2 is a notch target
44:24
and you get this salt and pepper expression. And so we really think this is lateral inhibition happening. And, but what's interesting is that the model that was proposed was suggesting that you have a full expression of MS2 in the segment as a readout of the segmentation clock. And that's what we, and then that you have a down regulation of MS2 in the posterior compartment as a secondary process that they express with complex genetics. But using this system
44:52
where you have a very good cellular resolution, we saw that's not what happened. Even though, you know, it's an artificial system and then I'll show you if we, now it developed also some system where, which are much closer to the natural geometry of the embryo. And there we can image the same type of sorting in this, this experiment. So this is what you try and call the segmentoids. And this is inspired from the gastroids that you may have heard about.
45:19
And the difference is that you just generate an aggregate of prismatic mesoderm and you put it in matri-gel. And so it breaks symmetry and forms this elongated rod of tissue. And you see here that in contrast to the somatoids, now you have nice anterior-posterior segments that form and that remain confined within one somite. So very much like the normal geometry of the embryo.
45:45
And you can generate this kind of movies. So here is a, that's a triple reporter where you have the oscillations here, the formation, the determination front and the segregation of the anterior-posterior compartments. You can see the design. This is the clock oscillation. You see that the structure elongates. And here it's, you can see the sorting. In fact, you can track those cells and see the sorting taking place, leading to the segregation of the anterior and posterior compartments.
46:13
So I think it's a really cool system because it's pure self-organization. There's no, you know, there's nothing around those surrounding tissues. And it was a bit ironical for me because I spent a big chunk of the beginning of my career studying the role of the surrounding structures on the differentiation. And now you can just take it and dump it into a matrix. It does exactly the same thing.
46:37
Yeah.
46:40
- [Attendee] So is the cell sorting in one particular segment complete before the other segment is formed? And that's what prevents it mixing between the segments? - Yeah, I'll show you this here. So recapitulation of what happens or how we understand the process. So at the end of the oscillation, so you activate notch into a segment-wide domain.
47:06
That's what the clock does to trigger this pulse of notch activation. This leads to mess-to expression, but mess-to expression in a salt-and-pepper fashion, probably downstream of a lateral inhibition type of mechanism. That triggers the sorting mechanism that moves quite quickly. So here we're talking about a 90-minute time window, so it's a fast process. Then the sorting takes place. At the end of the cycle, at the end of the 90 minutes, the cells lose the ability to sort.
47:40
And then the system is frozen, and then you move to the next pulse because the system has moved posteriorly because of the elongation process. And then it starts again. So in a sense, the clock is essentially a time-coordinator system. You can remove it. You will still have the process going on, but they will be disorganized. So the reason why they are very organized leading to these stripes and substripes is because of this oscillator.
48:13
And so just to emphasize- - [Attendee] Sorry, but in that, you can imagine that I'm sorting the opposites. What's ensuring the symmetry if it's broken or it doesn't? - Probably gradients. You know, there are these gradients of maturation, again, that give some sort of positional identity. So that's what we think. Like there is, for instance, there is retinoic acid coming from the somite
48:38
and FGF. - [Attendee] And the initial salt and pepper, can you already see that there is a bias towards more salt and more pepper? - No, we haven't, you know, tried to look at this resolution.
48:53
Yeah. - But if any, if,
48:56
I mean, I don't know whether it's possible, but to make the clock frequency go up, do you see this whole, you know, Ross-Ricardo law basically being disrupted because you're not giving enough time? - Sorry? - [Attendee] You make the clock tick faster, like as in the, do you see the two hops being formed between the somite now have a lot more, issues? - The problem is that we cannot really make the clock tick faster. We haven't, I mean, I'll show you one situation where this happens,
49:29
but experimentally it's not been really feasible. You can tweak a little bit, like, you know, 5%, 10%, but not something that will lead to a phenotype that you can really track.
49:42
So just here, the only point I want to make is that, so I showed you these two systems, somitoids and segmentoids. I think that what's nice is that they really illustrate how you can uncouple these different developmental modules. So here, for instance, you completely uncouple the process of somite formation, paraxomesoderm differentiation from axial elongation. Here, and in some ways, it's a bit like a fly embryo, if you want to think about it this way.
50:12
Here, you really recapitulate the entire process very accurately, which is pretty striking and raises a lot of questions on how such a system can self-organize. So I think these are really a core system to think and perform experiments on these questions. Aside from the physics standpoint, they're also very amenable to sophisticated experiments,
50:44
like to probe the mechanics, for instance, of the systems. So quickly, I don't know how much time do I have. I started a bit late. Do you think I can get 10 minutes? I'll try to, 'cause I want to show you how this system, which you can think about as a clock,
51:04
ties to another time-measuring system in the embryo, which is the Hox system. So if you, you know, I told you that there's a lot of diversity of the vertebral formula across vertebrates. And so, for instance, you can see this on this nice picture from Denis Dubrow, where it's a comparison between the chicken skeleton and the mouse skeleton. So you see immediately that chicken, they have a long neck, whereas mouse has only seven cervicals, like all mammals are seven cervicals,
51:36
pretty much all mammals. But the mouse is more thoracic, whereas the chicken has a small thoracic cage and so on. So how can you play on these anatomical parameters?
51:49
And we know that there is a gene family, which is critical for this, for the specification of the identity of these anatomical regions. And this is this Hox gene family, which contains, in mammals, 39 of these transcription factors. So they're BHLH, so they have a transcription factor containing a spatial helix, called the homeodomain. And what is very remarkable for this family is that they show a property, which is called collinearity, which means that their distribution along the chromosomes
52:33
reflects the position of their expression domain along the anterior-posterior axis of the embryo. So you have a sort of mapping of the anterior-posterior axis of the embryo at the level of the chromosome. And so this was first recognized in flies by Ed Lewis, who got the Nobel Prize with Einstein, Bullard, and Deschamps, which I mentioned already. And strikingly, it was found to be conserved in vertebrates and particularly in humans, except that they're the single cluster
53:08
or it's broken in drosophila, but it corresponds to one single cluster,
53:15
is duplicated into four clusters following two duplications of the genome in mammals.
53:24
And so in mammals, we also have this striking collinearity, meaning that the genes that have these low numbers,
53:34
like 1, 2, 3, are expressed very ideally, whereas the ones that have numbers like 9, 10, 11, are expressed very posteriorly. The numbers are called paralogues because they result from the duplication of this ancestral cluster. And there is also a very interesting other property in mammals, which is called the temporal collinearity, which means that the position of the genes along the chromosome also reflects the order of their expression during development, which was found by Denis Duboule.
54:09
And this is really important because it relates to the way the embryo is formed, meaning that early is anterior and late is posterior because it's a progressive sequence, so the posterior tissues are formed last, and so they express the more posterior host genes.
54:33
So one thing which is important to realize is that despite this very striking organization of this gene family, they've been identified almost 50 years ago, we know essentially, we know very, very little about their function, and we know very little about why this collinear arrangement has been conserved across evolution. I'm going to show you some ideas, but they are not completely established. But one of the very strong, one of the difficulty of working with this gene family
55:10
is their redundancy because you cannot knock out, you cannot, it's very difficult to eliminate their function. So far, people have eliminated one gene, two genes, maybe sometimes four genes. They've eliminated one cluster, two clusters, but then you cannot eliminate all of them because of the genetics. And I'm not going to show you this work, but in the lab with engineers, so Kongzhu has engineered lines where he's removed all the Hox clusters, and then you can use the systems that I've shown
55:42
to differentiate these cells because you don't deal with embryo lethality.
55:47
But anyhow, so what I want to show you is just how the,
55:55
this spatial collinearity of Hox genes is established. So this is a series of embryos where you can see that as you move
56:04
towards the five prime end of the cluster here, this is the Hox B cluster in chicken embryo, you can see that the anterior position of the expression domain progressively moves postially.
56:17
So this is the spatial collinearity. So how does it get established during embryogenesis? If you look at the expression of Hox genes, what you see is that they're first activated here on both sides of the primitive streak. So this is a section here through an embryo at this stage, which is a stage where you have gastrulation. So the tissue, the superficial tissue moves inside to form the mesoderm. And so it's this superficial cells that you see
56:44
that express the Hox genes in the salt and pepper fashion. And then the pattern consolidates, the cells start to reach the primitive streak, the positive cells, and then they start to ingress. And so when they ingress, the time when they ingress will define their anterior position. And I'll show you several examples of this.
57:07
What happens is that there is this temporal collinear activation, which I described, where B1 is activated first, B4 is expressed a bit later, but following the same sequence. So salt and pepper in the epiblast, and then consolidating, and then in the ingressing cells. B7 the same, and B9 and so on. You see, this is this temporal collinearity. So the same sequence, but displaced in time. And so what this implies is that the time at which the cells that express B1 is going to be before the time
57:46
at which the cells that express B4 ingress which is going to be before the time B7 and so on. Which means that the cells expressing B1 will be located more anteriorly than the cells that express B4, etc.
58:01
And this is shown here, like in a cartoon form. If you take the purple gene, which is here, and the green, which is here, you see purple one is expressed first according to the sequence I described. And so what happens is that when ingression begins, for the cells that express this gene, that marks the anterior border of expression, which here is going to be at some point one. But these cells that started to express this green gene later, they will follow the same logics,
58:34
but they will ingress later, and hence it will be positioned more posteriorly. And so that's how you can convert this time of the temporal collinearity into the spatial one. And so what we did a while ago was to study the function of these individual oxygens in this process, because they're expressed before the ingression. And so here what you can do is you can graft very tiny bits of the epiblast that overexpress different genes, so B4 labeled in red and B9 in green.
59:10
And you can track the fate of this graft. And what you see is that the cells that express the more anterior gene before, they ingress first.
59:20
And what happens is that they're going to be located more anteriorly. Because these two tissues are placed exactly at the same time, so they should be bilaterally symmetrical. Here the only thing you changed is that you expressed two different genes. And so what this suggests is that the HOX genes themselves are controlling the ingression of the cells, and by doing so they control the positioning of the cells along the anterior-posterior axis. And you can actually see this
59:51
when you section embryos at these stages, for instance, here. You see that the cells that express the B1, which is the anterior gene, they already ingressed, so they moved into the mesoderm. Whereas the other one that expressed B9, they retained in the epiblast. But they're not blocked, they're just delayed. And this is collinear, so it's a gradual... You can test many different HOX genes in pair in this way, and what you will find is that it's always the more posterior gene
1:00:25
that delays the ingression more. And so that suggests it's a system whereby you have the sort of dynamics that I showed you first. So, for instance, for this spin gene, that eventually, at some point, the cells enter. And then you have this staggered expression, so the next gene here starts to be expressed in the domain of the cells that already express the pink one. And then the pink cells, the cells that express only the pink cells, enter, so they're located more anteriorly.
1:00:59
And then, eventually, all the domain is going to express the green cells, and so it's going to be segregated from the cells that have ingressed, the pink cells that have ingressed. And so in this way, you can imagine how this kind of quantitative property control on the ingression properties of the cells is going to segregate the anterior and posterior domains of the HOX genes.
1:01:27
And so, very quickly, so snakes... I don't want to say much, except... Paul showed a nice picture yesterday. The only thing I will say is that one of the findings that we made in snakes looking at the segmentation clock is that they have a clock which is accelerated. The reason why they make so many somites is not because they grow more, which initially we thought, you know, you would imagine that if you want to make more segments, you just extend the elongation phase.
1:01:58
But that's not the way it works. The way it works is because they sped up the process. And so compared to mouse or chicken, what happens if you look at the cyclic genes, they have up to... You can see up to eight stripes of the traveling waves.
1:02:17
And so the conclusion is that if you look at the number of generations that you can calculate using cell cycle properties, which is required to make the axis of snakes, mouse, and chicken, you can see it's not so different, or at least doesn't explain this huge difference. But snakes, they make a lot more of smaller somites because of this acceleration of the clock.
1:02:42
And finally, I just want to tie this with the hox genes and just show you this because it's very important to understand well how the system works. So during gastrulation, early on, you have the cells that ingress from the mesoderm. They ingress from the epiblast, so this tissue that expresses early the hox genes and then they would ingress at the preemptive streak. Here, what you see is the primitive streak of an embryo. We labeled cells here with a fluorescent reporter.
1:03:17
And you will see these cells ingressing. See here the forming somites. You see the embryo is elongating. So it's very much like a plant, a meristem of a plant. If you want, you have a stem cell zone here which produces cells and it elongates at the same time. And so what I want to show you just to finish is what happens is that hox genes actually control this process. And they basically, they can tune it. And what I showed you, they can control this in a collinear fashion.
1:03:51
So the more the posterior of the gene, the more they're going to slow down the release of the cells and hence the, yeah. - [Attendee] Am I right in thinking that this is something that vertebrates have gained as functionality? I mean, do flies have, I mean, they have some sequential timing, I suppose, in the hox expression, but it's not, it's deployed sort of synchronously. - Flies maybe, but there are many,
1:04:21
many invertebrates that, I mean, there's a huge variability in the number of segments in invertebrates. If you think about centipedes, millipedes, and all this type of animals, so. - [Attendee] But I guess just me, the regulation of the ingression is a gained feature during. - Yeah, this is specific to vertebrates. I wouldn't argue that it works the same way in invertebrates at all. I mean, gastrulation is proceeding quite differently anyhow in invertebrates, yeah.
1:04:52
So okay.
1:04:56
And the other thing that's, which is important to have in mind is that a bit counterintuitively, this presomatic mesoderm is also the engine that controls elongation. And so we've made work, all the work and more recent ones with Ma, where we showed that this tissue, the cells are very actively motile, very much. And so the idea is that FGFX is like a temperature, effective temperature gradient. And this motility of the cells, which is very important for CRE,
1:05:37
is able to generate a force that's because of the confinement of the cells, that is going to be able to generate a force that is going to push to elongate the embryo posteriorly. And we measured this with cantilevers, for instance, and did some work to characterize the mechanics of this. But what's important is that to understand that this injection process of cells from the stem cell zone that I just showed is what also is going to control the elongation parameter of the embryo.
1:06:11
And now if you track the elongation process in time, which is what you can see here, this is a two-day movie of a chicken embryo, you can measure the speed of elongation and compare it to the speed of somatogenesis. What you see is that while the speed of somatogenesis is relatively constant, the speed of elongation first increases and then it decreases, and then at 25 somatogenesis essentially drops.
1:06:44
And this is going to lead to this,
1:06:50
which is if you measure the size of the presomatic mesoderm, so the posterior tissue where we see the oscillations, if you measure the changes of the size of this tissue in time, you see here that it first increases, and then when the embryo slows down, it starts to drop, because the somatogenesis is still constant. And so the simple interpretation of that is that what you have is a situation here where elongation slows down, where somatogenesis maintains at the same speed,
1:07:23
and that leads to a shrinking of the territory of the precursors. And so now you can quickly understand how this leads to the control of segment number. And so that's by controlling these two systems. So what we call here the injector, which is the tail bud or the primitive streak, which injects the cells that will feed
1:07:52
what's called the N-gene, which has these random movements that control the elongation. And these systems are controlled by Hox genes.
1:08:04
And so this is just an illustration of the expression of Hox genes in time.
1:08:10
These are the number of somites, which corresponds to the beginning of the slowing down of the elongation, and that corresponds to the beginning of the activation of the posterior Hox genes. As you can see here, there is a first window where there is expression of many genes, but it's not really relevant for the elongation. And what is important is this window here.
1:08:35
And it's been shown also that if you overexpress the terminal genes, what you do is you truncate the body axis. So sorry, I'm rushing on this, but I just want to show this because that's, I think, the important thing to remember, is that what happens in development is you have a crosstalk between these two systems, the clock and the Hox timer. And by adjusting the parameters in the two systems, and I've tried to show you a few examples of this, you can understand how you can end
1:09:08
up with a defined vertebral formula, which has a fixed number of segments for each anatomical domain. And that's really defined by the crosstalk between these two developmental modules. So I guess I'll stop here.
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