 OK, good morning, everyone. So I would like to continue on what I basically started to do in the first two lectures. And what I would like to speak about now is not spreading of the blastodermal of the york cell, as we have seen, epithelial spreading, and deep cell intercalation. But what I would like to introduce here is the interaction between the different forming germ layers. So essentially, the function of tissue interaction in morphogenesis. And the type of tissues which are formed in these early stages of zebrafish development are illustrated again in this schematic diagram. You're essentially forming two types of tissues, one tissue which is on the inside, and will become the mesoderm and endoderm muscle and digestive tract. And then the tissue which is not ingressing at the germing margin and which is becoming acted on. And if you look already at this schematic diagram, you can see that these tissues are directly adjacent to each other. And so at the end, they're moving in opposite directions. Internal tissue goes up to the animal pole, and the external tissue goes down. And you have an interface between these two tissues. And that's assuming that these tissues are directly in contact with each other, but moving in opposite directions would already imply that there might be some mechanical interactions between these tissues, which might be important for their morphogenetic behavior in the process of transformation. OK, so what I would like to present you now and this is all of course unpublished is the potential impact of mesoderm which is coming in in the schematic diagram. You can see it here if you're looking right at the axis of the developing embryo. And the green tissue over here and the green tissue in the actual embryo is mesoderm and endoderm. And its anterior axiomesoderm and endoderm, which has ingressed at the germing margin down here and is now migrating in a straight path up to the animal pole, as you can see here in the schematic diagram. This is all happening below the layer of ectoderm. And the ectoderm layer you have seen yesterday is spreading around the oxal and moves in the opposite direction. So you're just looking through the ectoderm onto the mesoderm here, but you have two different flows of tissues. You have the red flow, which is the ectoderm tissue, which is above this green tissue. And then you have this finger of green tissue, which is moving up in the opposite direction of the ectoderm. And what I would like to sort of present you data on is how these different movements sort of interact with each other and which kind of feedback exists between these tissues during oscillation, mechanical interaction predominantly. Now, just to illustrate, this is what, and I think I showed you this movie before, this is what this anterior axiomesoderm looks like in a real embryo. I can just run it once more. It comes from germing margin and then they're migrating up. And they're migrating relatively fast and they're migrating a cohesive sheet of cells. It's essentially a one to two-layered sheet of cells, which is migratory at the leading edge. And they're coming from the germing margin and they're migrating the straight paths up to the animal core. And above is the ectoderm you can't see in this movie, which moves in the opposite direction. So the motivation of looking at the potential or the sort of the first indication that there might be actually an interesting interaction, morphogenetic interaction between these different tissues comes from an observation where we again taking advantage of a mutant embryo. The mutant embryo, the wild-hat situation is shown up here and this is a mutant embryo and this mutant embryo, and I think I mentioned it in my first talk, is a mutant which is called maternal zygotic one-irpinate mutant embryos. These embryos don't form a mesoderm and endoderm. They don't have an internal cell layer. They only have epiderm on the outside. And if you just look at the end of constellation then the body axis has formed in a wild-hat embryo and you compare the length and the shape of the body axis in a wild-hat embryo to the mutant embryo, you can already see that the mutant embryo has a very shortened body axis and thickened body axis compared to a wild-hat embryo at the same stage. And that if you would consider that in this embryo there's no mesoderm and endoderm already sort of points to the possibility that the presence of mesoderm is required for proper extension of the body axis in a wild-hat embryo because you don't have that in these mutant embryos. It's of course a pure assumption in this case but it's definitely worth looking at. Specifically what we were looking at is the ectoderm which is at the axis and which overlies the mesoderms of the tissue which is not stained here and this tissue down here, they're giving rise to the anterior neuroplate, to the anterior edge of the new ectoderm which is essentially forming the brain later on. If you look at the position of the future brain which is marked here by the expression of a gene which is specifically expressed in these progenitor cells, you can see in a wild-hat embryo it's up there at the animal pole and if you look in a mutant embryo which has no mesoderm and endoderm then this anterior end of the neuroplate is moved very strongly back to the posterior end of the embryo. Indicating that the brain is actually forming where the tail should be of the fish embryo in a wild-hat situation. So positioning of the, or patterning or positioning or shaping of the neuroplate is very strongly affected if you don't have mesoderm and endoderm. Okay, so what we hypothesized is that this anterior axiomesoderm which is called precold plate from now on we'll just call it precold plate. This finger, this green finger which moves to the inside. That this is required for new actoderm cell movements which again are required for proper extension and shape acquisition of the neuroplate within a wild-hat embryo, okay? So that was basically the starting hypothesis and the way the addressing it was first just looking at the movement of actoderm cells in an embryo which doesn't have any mesoderm and endoderm. And what is happening and I just run that once more we are looking now only at these red arrows here and we are just trying to understand how actoderm cells are moving if there's no mesoderm and endoderm in a mutant embryo, right? In these MZ1-acrylate mutant embryos. What you can see is that these actoderm cells are all moving in a straight pass downwards towards the natural pole. As you would suggest from the tibial movements because the whole tissue is sort of moving downwards and the actoderm moves with the tissue downwards, right? There's no interference with mesoderm or endoderm. As soon as you are now looking in an embryo and a wild-hat embryo which has this finger these pre-carbled plate cells moving in the opposite direction, the movement of the neactoderm looks quite different. You can see it over here. The mesoderm is coming from down here. These are the black dots and they're moving up here and what you're inducing within the neactoderm is essentially redirecting the movement of neactoderm cells which is downwards directed. Now upwards and you're inducing these large vortex movements within the neactoderm. So it appears as if the upward movement of the pre-carbled plate redirects the downward or stops or slows down very strongly the downward movement of neactoderm cells leading to actually a redirection of neactoderm movements and the induction of large vortex movements within the neoplate. Okay, so that's a very interesting observation because it indicates that there might be a mechanical interaction between pre-carbled plate and neactoderm which leads to the specific movement pattern of neactoderm cells in a wild-head ampere. Okay, so I just show you the type of quantification we did to capture this phenomenon. What we are looking here is again, perhaps we are starting with a wild-head situation down here where we have only neactoderm cells which are moving essentially in a straight path downwards. You're now analyzing the movement velocity of cells within this window which I have indicated here and we are plotting that velocity as a function of time during gestulation essentially, right? What you see is that these cells are moving downwards and downwards is a positive velocity and they're moving at a speed of one to 1.5 micrometer per minute downwards. But it's a pretty homogeneous downward movement in the absence of mesendoderm. Now if you're looking at a wild-head ampere which has now mesendoderm cells then pre-carbled plate cells here, then you can see that these pre-carbled plate cells move in the opposite direction. They're moving upwards. They have a negative velocity and they're moving between two and four micrometer per minute upwards. And what they are doing is they're taking along the neactoderm cells which are just above an anterior of the neural plate and move it in the same direction upwards, right? So there you can see that this movement which usually is downward directed is now upward directed and goes together with the underlying pre-carbled plate. So we're again looking just at these cells in this window here indicating that the pre-carbled plate has a potential of redirecting the overlying actoderm cells in the direction of its own movement upwards. Okay, so from this analysis what we assume is that in wild-head pre-carbled plate movements which are directed towards the animal pole redirect neactoderm cell movements and inducing a large vortex movement within the neactoderm which again is required for proper extension of the neural plate and positioning of the anterior neural plate and brain tissue at later stages of development. Okay, so that's the sort of conclusion on these initial descriptive experiments. What we wanted to know is if indeed there's a causative relationship, a functional relationship between pre-carbled plate movement and neactoderm movement. You could also assume that perhaps the pre-carbled plate is secreting any biochemical signal which does something on the ability of cells to move within the neactoderm. It's not the movement of the pre-carbled plate but there might be a signaling function of the pre-carbled plate, so. So we wanted to see if specifically movement of pre-carbled plate cells and anterior directed or animal directed movement of pre-carbled plate is causative for this redirection of the neactoderm cells above, right? So what we did is we turned to another mutant which is indicated down here. In this mutant, we knew from previous studies it's a mutant for a secreted ligand which is called wind 11 and wind 11 functions in a non-canonical wind signaling pathway. And given, I mean, the only thing you need to know is that in this mutant, anterior migration of pre-carbled plate cells is reduced. They are migrating at a slower pace and less directed up to the animal pool. That's the main defect you have in the mutant phase. We were wondering if now in the mutant embryo where the pre-carbled plate migration up there is being impaired if new actoderm cell movements would subsequently be affected. Now that's what is happening. We look at actoderm flows again in a mutant embryo and we're looking at these little window up here which is above the pre-carbled plate in a mutant embryo in which animal directed migration of pre-carbled plate cells is being inhibited. And what we find is that the pre-carbled plate moves at a slower pace up to the animal pool. It's only two micrometer per minute approximately. And it's less able to redirect the overlying actoderm cell movements than what you have seen before in a vulture embryo. When these cells are still moving downwards and they are not being really efficiently redirected and you don't induce very large vortex flows within the actoderm. So that sort of indicates that the relative movement of pre-carbled plate in the actoderm determines the effect of pre-carbled plate on the actoderm of a genesis. So the pre-carbled plate needs to move fast enough in order to induce these large vortex movements within the actoderm above. So it indicates that pre-carbled plate movement is critical and presumably not some signals which come from the pre-carbled plate when there's something on the actoderm. Now what we did is we, at this stage, we teamed up again with Guillaume Sardouille and a postdoc in Guillaume's lab, Sylvia Pridolan, to address the hypothesis which sort of results from these initial observations, these sort of phenomenological observations using Newton's and Weithab-Embius and what we assumed is that the type of mechanical interaction we have between the pre-carbled plate and the overlying actoderm is the generation of frictional forces. And we assume that frictional forces arise at the interface between the pre-carbled plate and the actoderm and that these frictional forces are responsible for the effect of pre-carbled plate on the actoderm cell movement site. And to test if this is sort of a plausible assumption because it's still an assumption, you don't know if these are frictional forces or if there's any other type of interaction we have between these tissues. But it is, together with Guillaume, we formulate this erratic model which is based on the principle of viscous fluid motion and we consider the new actoderm as a 2D viscous compressible fluid moving in the opposite direction to pre-carbled plate cells, which again, they're modeled as a rectangular element. In this case, it's a simplification, it's actually around cluster cells exerting a frictional force on the new actoderm. So this is sort of a simplified version of what we think could happen there and we want to see if the predictions from this, from simulations merging from this theoretical model would be able to match our experimental observations. So if that is essentially a plausible assumption that the interaction is mechanically an interaction, the force generation is a friction origin. Now, I show you two cases where we did that. Again, look at wild-type ambios in which we know that the pre-carbled plate moves up in a relatively fast velocity, two to four micrometer per minute, and we compare that to mutant ambios in which the pre-carbled plate movement is reduced up to two micrometer per minute, up onto two micrometer per minute. Then what we are doing also, and then just explain, I don't have the slide for that, is we are looking, so what we are doing is we are taking a wild-type ambio, and then we are subtracting only the pivley movements in the wild-type ambios. So we want to take away all the effects of the pivley movement standards by subtracting the pivley movements which we record in an ambio which doesn't have any pre-carbled plate from the wild-type ambio. Then we are taking away all the pivley movements and we are just looking at the effect of pre-carbled plate on-cell movements within the new actoderms, that basically the effect pre-carbled plate does on movement within the new actoderm, irrespective of the amount of the pivley movements which happens in a wild-type ambio. I hope that is clear. So we are subtracting the pivley movements, we are just looking at the effect of pre-carbled plate on redirecting movements within the new actoderm. Okay, so what we are finding here is, and this is a fund-dimension representation where we are just looking at movement along this axis from, you know, basically along the circumference here, along the circumference here, outlined here, and what we are seeing is that there's an effect of the pre-carbled plate on redirecting the movement of the new actoderm cells, which the red lines are the theoretical predictions and the blue dots are the experimental observations. We can do exactly the same here in a mutant ambio in which the experimental effect is being reduced, as I showed you before, and the model predictions actually fit very nicely again the experimental observations here. So what we conclude from this close match between the theoretical predictions to our experimental observation is that the plausibility that these are frictional forces which do the job, which mediate the effect of pre-carbled plate on the actoderm is a plausible prediction, and that in principle, the assumption that frictional forces are doing the job is sufficient to explain the observations we have here. We cannot exclude that there are other effects, but we are just arguing here that in principle, just assuming that there's mechanical interaction leading to the generation of frictional forces is sufficient to explain the effect of pre-carbled plate on the actoderm cell movement. The other thing which we can actually derive from these simulations, and that's sort of important if you want to understand which type of interaction, how these frictional forces are being generated, we can sort of derive a friction coefficient. Now, the only thing we can, the only thing but the range of friction coefficient we can arrive is pretty large here because we don't know exactly about the tissue viscosity of the new actoderm during all stages of its interaction. But we can sort of assume that there's a range of friction coefficients which is likely to be important for the interaction between pre-carbled plate and the actoderm. And by this range of friction coefficient, we can learn something about the interaction between the pre-carbled plate and the overlaid in the actoderm. And the most likely explanation based on this friction coefficient would be some chemical reaction. And in this case, it would be cell cell adhesion which might be mediated by a cell cell adhesion molecules such as classical cation. Okay? So we wanted to see if indeed this generation of frictional forces between pre-carbled plate and the actoderm is mediated by cell cell adhesion, direct interaction between pre-carbled plate cells and overlaid in the actoderm cells via cation mediated adhesion. So we first looked at this interface in some cross-section where we are staining the tissue with an antibody against e-caterin. E-caterin would be down here, the red. We're looking at e-caterin localization. What we are finding is that e-caterin is localized in actoderm cells above here at the plasmembrane of actoderm cells. We also find e-caterin to be expressed in pre-carbled plate cells at the plasmembrane. And importantly, we find e-caterin to be localized at the interface between pre-carbled plate and the actoderm cells, indicating that there's likely a caterin-mediated cell cell interaction at the interface between pre-carbled plate and the actoderm cells. And that this interaction, this e-caterin-mediated interaction might be responsible for the generation of frictional forces which we assume to mediate the effect of pre-carbled plate on the actoderm. Now, we can test this assumption. And what we did is we did an experiment which I need to explain in a slightly bit more detail. So what we undertook in this experiment is we took a mutant, the mutant which I introduced before this Maternosecotic Granite Pinnot mutant embryo, which doesn't have any mesoderm and endodermal genital cells. And we wanted to rescue new actoderm movements in this mutant embryo by putting back transplanted mesoderm and endodermal genital cells. So we are trying to rescue, to re-induce these vortex flows within the new actoderm by putting back mesoderm and endoderm into a mutant which doesn't form it in dodging as we. So that's the experiment you can do that. And that would be a Maternosecotic Granite Pinnot mutant embryo. And then what Michi did in this experiment, he transplanted pre-carbled plate for the cells which he took from a donor embryo, puts it into this embryo, and then he's asking if these cells, once they have been transplanted, are able to redirect the movement of new actoderm cells about. And what he finds in these experiments, and this is just sort of summarized over here in these velocity profiles that these pre-carbled plate cells, the green ones, are moving at least for a period of time up to the animal pole. And once, and during the time when they are moving up to the animal pole, they are able to redirect transiently actoderm cells up to the animal pole as well. So you can partially and transiently rescue new actoderm cell movements, re-induce these large vortex movements of new actoderm by just putting back pre-carbled plate cells into mutant embryo. Now, why do I introduce this experiment? Because what you can now do is you can take pre-carbled plate cells which are wild-type, which express ecaterin, and which could possibly form caterin-mediated cell cell adhesion to overlying new actoderm cells. But you can also take now pre-carbled plate cells in which you impair the expression of ecaterin. And then you could ask, would ecaterin expression in pre-carbled plate cells be required for the activity of pre-carbled plate cells to redirect new actoderm cells? OK. That's sort of typical, if you want to, developmental biology experiment you can do. And that's shown down here. So what we are doing is now we are not transplanting wild-type pre-carbled plate cells, but we are transplanting pre-carbled plate cells in which we have specifically down-regulated the expression of ecaterin. So they're expressing very low levels of ecaterin at the plus membrane. You're putting these cells back into an embryo, into one of these mutant embryos. And then we are asking if these cells would be able to redirect the movement of the overlying actoderm cells. Now, first of all, if you're putting these ecaterin and ecaterin morphin cells, which are not expressing pre-carbled plate cells, which are not expressing ecaterin, into an m-zeta-wp embryo, these cells are still migrating up to the animal pole for a period of time. So they are not defective in migration and they still can go up, that's right. But if you look at the effect on the overlying actoderm cells, this seems to be very strongly reduced. And compared to over there, the actoderm cells above move nearly the same velocity at the underlying pre-carbled plate cells. In case we are putting now pre-carbled plate cells in, which are not expressing ecaterin or express very reduced levels of ecaterin, then this effect is very strongly reduced. Suggesting that you need ecaterin at least on the side of the pre-carbled plate for the pre-carbled plate in using via frictional forces a redirection of new actoderm cell movements above the pre-carbled plate. OK, so this is all done within the embryo and you still might wonder if that is actually explaining everything. And what we wanted to do is we wanted to sort of design a reduced experiment where we can actually test the hypothesis in more directly. And what we did in this experiment, again, that's an experiment. Michael Smutting, the lab has done is he wanted to sort of generate an artificial situation where he induces new actoderm, redirects new actoderm cell movements by not using pre-carbled plate tissues, not live cells. But what he's using now are plastic beads, which have approximately the size of cells within the new pre-carbled plate. He coats these plastic beads with ecaterin actodomains. And then he's gluing the plastic beads onto a cantilever. He takes a tissue of new actoderm which he cuts out of the embryo. He glues this tissue of new actoderm onto a stage which he can move in both directions here. And then he's trying to move the new actoderm in one direction and these beads in the opposite direction, as he would have it in the embryo when the pre-carbled plate moves up and the actoderm moves down. And he wants to see if he can generate frictional forces at the interface between these beads and the tissue block of new actoderm down here. And if this interaction would indeed lead to redirection of new actoderm cells within this block which mimic the situation in a wild type embryo. So it's a very simplified version of what happens in a wild type embryo just to see if you don't use pre-carbled plate, but instead you're just using passive beads if you can do the same job. Is it a pure mechanical interaction? I think that's the question here. Now that's the outcome of these experiments. I'll just show you first the control experiment where he uses beads which are passivated, which are non-attensive. If he moves these beads, these would be the green, would be the movement of the beads in one direction. And if he moves the beads in one direction, he hardly does anything on actoderm movements which are below the beads in this case. But once he has coated these beads now with the actodomain of the caterin and he moves the beads in one direction, which is again shown here by the green curve, then he can very efficiently take along actoderm cells adjacent to the beads as the pre-carbled plate pass in a wild type embryo. And that's actually an important experiment because it sort of excludes still the possibility that what the pre-carbled plate is doing on the new actoderm might not only be to induce frictional forces and then redirect movement, but there still might be some sort of signaling function of the pre-carbled plate which might do the job and we cannot exclude it in the embryo, but we can exclude it with such an experiment where instead of the pre-carbled plate, we take plastic beads which certainly cannot signal. I mean, the only thing they do is, in this case, they're coated with liquid here, yes. Ah, here, it's the same direction. So they both move in the minus section here, in minus velocity. They, I mean, perhaps I should show that they are being redirected in the same direction. And in a wild type embryo, let me quickly see that. Yes, I mean, that's what happened. You can see they're moving in the same direction here, so. So we're just looking at the new actoderm cells which are anterior and above the pre-carbled plate. These cells are being redirected by the pre-carbled plate. They are now moving upwards in the same direction. The other cells which are outside of the pre-carbled plate, they're still moving downwards. And that induces actually these large robotics. So you locally redirect the movement of the actoderm cells and that leads to robotics and the rest of the actoderms are moving down. Right? So this is a complete ex-mevo experiment. What we are doing is all done in culture. So we are taking a tissue block which we are cutting out of the embryo and the actoderm tissue block. You're gluing, we're taking it into a culture dish which is filled with the culture medium in the MF12. You're placing it on a phytonectin substrate to make it adherent. And then we're coming with a cantilever in this case and on which we glue the plastic beads and we are sliding the plastic beads over the surface of this tissue block and sliding the tissue block at the same time in the opposite direction. So you have like in the embryo where the pre-carbled plate which would be the beads move in one direction and the actoderm moves in the other direction. And now we're looking at the local interaction and the cell movements right at the interface between the beads and the new actoderm. I'm clicking the girls like that. Okay, so what we conclude on this part then is that pre-carbled plate cells have the potential of redirecting new actoderm cell movements. That's very clear from all the experiments I've shown you. That friction forces are likely to arise at the interface between pre-carbled plate and the new actoderm and that these friction forces are in principle sufficient to explain the observed effect of pre-carbled plate cell movements on the actoderm cell movements. And there's a relative speed of pre-carbled plate where those new actoderm is critical and that ecatier immediate friction at the tissue interface is also critical for the generation of friction forces and boost the effect of pre-carbled plate on the actoderm cell movements. Perhaps I should tell you about one experiment that I don't have here and slides in here. And that is actually what we wanted to know is if by generating a friction force at this interface if we would induce some shear forces within the tissues which are adjacent to this interface, right? And one experiment we did is we looked at movements not on the side of the new actoderm but we are now looking at movements on the side of the pre-carbled plate. The pre-carbled plate is not a single layer but it's up to two to three cell layer thick and cell diameter thick. And what it is we looked to what degree pre-carbled plate cell movements change as a distance from this interface. Assuming that the pre-carbled plate cells would be mostly affected by new actoderm cells right at the interface and they would be less affected when you go deep into the pre-carbled plate away from this interface. And that's exactly what we are seeing that pre-carbled plate cell movements, if you look at the velocity of pre-carbled plate cell movements which is upwards, they are moving slower when they are right at the interface and they're moving faster up to the admiral pole when they are further away from the interface. Again, supporting the assumption that there's friction forces leading to some shear within the tissue and differential movement velocities as a function of distance to this interface. And interestingly, you can also look then in cases where you reduce cation expression within the pre-carbled plate or within the embryo. You can see that this effect, that the distance that pre-carbled plate cells change the velocity as a function of distance to the interface is being abolished in cases where you don't have cation anymore. So that's another supporting evidence that indeed there's friction forces leading to some shear within the tissue within the pre-carbled plate tissue in this case. Okay, so now I would like to tell you a little bit and we're going away from pure mechanics here into cell plate specification. I think that's another important topic in development biology is in the end, I think we have heard it yesterday from Thomas as well, is how different cell plates are being induced within the embryo. And in particular what I would like to speak about is the potential interaction between morphogenetic movements on cell and tissue level and the induction of different cell plates within the embryo. And just to not complicate things too much, again, we're looking at this one tissue, I showed you before, which is this pre-carbled plate. This anterior axiomisandidone tissue, it's just very convenient to look at it. It's a discreet tissue with approximately 100 cells. We are asking how the fate of these cells within the pre-carbled plate is being induced and how rearrangement of cells within the pre-carbled plate might affect the fate acquisition. Okay, I'll show you just as a sort of a primer for this problem. I'll show you a movie of the pre-carbled plate. It migrates, you can see it here. It migrates from the German margin, which is down here, up to the animal pole, which is up here. And this is a transgenic embryo, which is expressing RFP under the control of a promoter, which is called goose grit. You don't have to remember now all the details, but essentially, red shows you the fate pre-carbled plate cells take on, which is their pre-carbled plate progenitor cell fate. And the green cells are actually cells which are adjacent to the pre-carbled plate, which become endoderm, which are not part of the pre-carbled plate eventually. So what you're looking at is basically a decision of these cells either to become pre-carbled plate or to become not pre-carbled plate and then they're becoming endoderm. So either to be red or to become green, okay? And so what you see here, that the pre-carbled plate is migrating up, and you can already see that the few cells, few of these red cells might be leaving the pre-carbled plate and they are turning them into green cells, you know, they're here, you have a few green cells. The majority of cells are, remains within the pre-carbled plate and remains red. But this already indicates that cells have two choices here. They either stay within the pre-carbled plate and acquire pre-carbled plate cell fate or they leave the pre-carbled plate and then they're becoming endoderm cells and become green. So you have a binary cell fate decision either to be pre-carbled plate or to become endoderm. And that's sort of an interesting observation. We wanted to see how the interaction of cells within the pre-carbled plate would affect this decision of cells would determine if cells remain within the pre-carbled plate or leave the pre-carbled plate, okay? So this is just showing you there the different cell plates which are being induced. And so we got, in particular, we got interested in this problem when we looked at the relationship between cells of contact formation of cells within the pre-carbled plate and the acquisition of pre-carbled plate cell plates. And what we did here is we plotted the mean contact duration between pre-carbled plate cells which we can measure within the pre-carbled plate. So basically we're looking at the accumulation of GFP under the promoter, which I mentioned before, Goose Good, which is a promoter which is typical for pre-carbled plate cell fate specification. So this would indicate to what degree these cells are becoming pre-carbled plate, acquire a cell fate of pre-carbled plate, and this is their contact duration. You can see that cells which are forming very stable and long lasting contacts acquire higher levels of EGFP and those are more likely to become pre-carbled plate progenitor cells which have very short contacts, very loosely contacting each other. They acquire lower levels of EGFP and the likelihood of leaving the pre-carbled plate and not becoming pre-carbled plate progenitor cells is being reduced. So there seems to be a correlation between contact duration within the pre-carbled plate and cell fate specification. And that's sort of an interesting problem you can experimentally address and then further elucidate. So cell cell contact duration scales with a level of two-square GP expression in the pre-carbled plate progenitor cells. Now, what we wanted to do is to find out if there is indeed a functional interaction between contact formation and the acquisition of a certain cell fate. But we did this and what we usually do in the previous experiment I showed you as well is we sort of reduce the complexity of the embryo by taking cells out of the embryo and taking them into culture and see how these cells in culture would behave and then see what the Nandani culture, how far it can apply to the situation within the embryo. Now, specifically what we do in this experiment is this experiment we've done by Vanessa Barona and PhD student in the lab is she took out these pre-carbled plate cells which are indicated here in green, dissociates the pre-carbled plate cells and takes them into culture. Now she's looking at two different types of arrangement of cells. In one case, she looks at singer cells which are isolated from all neighboring cells. They are just sitting there, singer cells. And she looks at cells which have made a contact with another cell. So she looks at doublets for the singer cells. And then she's asking how would these cells accumulate GP under the control of the goose to promote as a readout of which fate these cells are acquiring over time in culture? The question would be, would the cells of contact affect EGP accumulation? And would that be sort of a further indication that contact formation is important for GP accumulation and fate acquisition of these cells? Now there are the results that just show you quickly the control down here where you compare singer cells to doublets and you're just looking now at EGP accumulation under the promoter of better acting which should have nothing to do with other specifications, uniformly expressed in the embryo. What we find is that EGP accumulates in singer cells as much as in doublet cells so it's independent of cell cell contact formation. But if you look now at EGP accumulation under the control of the use good promoter, you can see that this looks quite different. Singer cells acquire over time a lower level of EGP compared to cells which are in contact with each other. That sort of supports the original claim or assumption we had that contact formation facilitates fate acquisition of pre-continued cells. Okay, so that's what we promote, what we conclude as a contact formation promote goose good GP expression and pre-continued plate cells and do presumably fate acquisition of pre-continued cells. Now the question we ask of course is how is that being achieved? How can contact formation affect fate specification? I mean these are very different processes and what has contact formation to do with pre-continued plate cell fate specification? If you look at pre-continued plate cell fate specification that's a problem that has been addressed in many studies before and they looked at the various signaling pathways which are responsible for pre-continued plate cells acquiring their specific fate and particularly there's one signaling pathway which I think I mentioned before which is called the nodal TGF-patter signaling pathway. I just show you down here how the signaling pathway looks like. You have a ligand which is called nodal or nodal related and this ligand binds to a receptor and in fact these are two receptors, the type one and the type two receptor. These receptors open binding of the ligand dimerize. They lead to phosphorylation of a SMAT2-4 dimer which once phosphorylated goes to the nucleus and acts as a transcriptional co-activator which triggers gene expression specific for pre-continued plate cells in which triggers pre-continued plate cell fate specification. So that's the sort of commonly accepted version where nodal signaling triggers pre-continued plate cell fate specification by specifically switching on target gene expression which is, which then lets the pre-continued plate cells becoming pre-continued plate. Okay, so how can we address if nodal signaling has anything to do with contact formation and pre-continued plate cell fate specification? If nodal signaling is actually affected by a cell cell contact formation and might then mediate the effect of contact formation plate specification. Now that's an experiment I need to explain a bit more. It's not particularly complicated. Up here we're looking at the experiment I showed you before, we, I mean the answer took out these pre-continued plate cells, green cells from a transgenic embryo, put them into culture and what she's doing then is she's looking how single cells versus doublet cells would acquire, would accumulate GP as a readout of fate acquisition in these cells and what she finds and I showed you that before that in doublets the accumulation of EGP is higher than in single cells. That's the experiment up here. Now she's doing another experiment specifically address the contribution of nodal signaling in this effect. She takes now cells from a maternal so Gothic one-eyed pinnet mutant embryo. Now these mutant embryos, and I indicated that in the previous part of my talk, they cannot, they basically are insensitive to nodal signals. They cannot respond to endogenous nodal signals and since they're insensitive to nodal signals they don't form mesoderm and endoderm the gender cells, right? So if she takes these cells and they cannot respond to nodal signaling they will not become pre-cordal plate for sure. Now she's using a little trick. She's taking these cells and she's exposing them to another ligand which is called activin. And this ligand can induce pre-cordal plate cell fates in these cells irrespective that these cells are usually insensitive to endogenous nodal signals. So she can artificially turn these, turn a robotic one-eyed pinnet mutant cells into pre-cordal plate by adding this activin, this ligand which is not a zebrafish ligand but you just get a recombinant protein and you can induce pre-cordal plate cell fates. Now she's taking cells out from a mutant embryo which is usually not pre-cordal plate, takes them into culture, then she's exposing them to activin up to a certain stage and then she's taking activin out of the medium. These cells are now pre-cordal plate but from now on they don't see any nodal signaling anymore because inductionously they cannot recognize their own produced nodal signal, okay? Now she can compare singer cells and doublets which are pre-cordal plate but which are insensitive to nodal signaling, any sort of nodal signaling which might go on between the contacting cells. On this case you can see over here that singer cells and doublets cells acquire the same level of EGFP indicating that the effect of contact formation on the acquisition of cell fates, the accumulation of GFP and the acquisition of cell fates depends on the continuous presence of activin within the culture medium because if you take the same experiment up here and now you just leave activin in the medium you can see that you get this effect back that doublets acquire higher levels of GFP than singer cells indicating that you need signaling to be on in these cells for the effect of contact formation on cell fates specification, okay? So you need to have a functional nodal signaling pathway in order for contact formation promoting fate acquisition in these cells. That's the take a message from this experiment. I hope that is clear. Questions? A bit of a complicated experiment, yeah. Sorry? What is the basic? Oh, pack sorting. This is just, you know, what you do is you basically sort the cell out of an embryo by the level of the fluorescence. You just want to get the green cells and not the other cells. Fluorescence assisted cell sorting, it's called. Okay, so yeah. So you're comparing is actually singer cells to doublets in the first place. But what we also do now is we are comparing mutant cells to wild up cells. And what are you referring to now? Yeah, I'm coming at this point. What we are doing at the moment is we are not looking at a binary self decision. We are just looking at the amount of GFP under the control of a specific promoter which is required for one cell fate, which is pre-carded plate. We are asking to what degree the promoter activity and those likelihood of these cells to become pre-carded plate is controlled by contact information. But we are not yet asking if these cells would eventually fall into endoderm or pre-carded plate. That comes a bit later. So we don't know yet anything about a binary cell fate decision. I mean, I took this movie only in as a primer, but we are looking at this only, the only readout is now GFP accumulation under a promoter which is usually on in pre-carded plate for them in the cells. As a readout to what degree these cells are becoming pre-carded plate. That's sort of an estimate to what degree you keep, to what you do influence the likelihood of these cells turning into pre-carded plate. But I'm not saying that the alternative fate is now endoderm. That comes a bit later. How do we know that there's no third fate? I mean, that's the two fates we can distinguish based on our previous studies on this topic that there is essentially endoderm and pre-carded plate in this region. There might be differences between different subtypes of endoderm or perhaps there might be subdivisions within the pre-carded plate, I don't know. I sort of simplify the problem and say there's pre-carded plate in endoderm and that's what we can recognize at the moment. I'm not saying I'm not excluding that there's a finer subdivision which we don't see yet. Say it again. Is it, it's not accurate as assisted cell sorting because you essentially sort cells according to the level of fluorescence they have. So you don't activate these cells necessarily. Okay, so one more slide on the nodal signaling. I hope that was clear. It's a bit, you know, it's biology, but what you wanted to know is why is contact, how is contact formation actually interfering with nodal signaling or facilitating nodal signaling as a function of contact formation, right? How is contact formation sort of modulating nodal signaling activity in these cells? That's the, you know, the question you need to ask. What we found is if you look at different components of the nodal signaling pathway and I mentioned before there's a ligand, there's a receptor, you find that the ligand together with the receptor specifically localizes ethyl cell contact. That's something you don't see if you just have a single cell and it's pretty uniformly distributed. It seems to be an accumulation and concentration of the ligand and the receptor in actually endosomal compartments which are signaling compartments and ethyl cell contacts. So one possibility from these localization studies is that contact formation enhances nodal signaling by putting all the nodal signaling apparatus together ethyl cell contacts, putting all the components right in place and thereby facilitating the interaction and nodal signaling in these cells. Now again, this is a test of a prediction to see if nodal signaling is indeed increased in contacting cells or the cells which don't have a contact. That's something you can do. You can look as a readout of nodal signaling and perhaps I should very quickly go back and just remind you. A readout of nodal signaling is actually the translocation of this dimer, SMAT2, SMAT4 into the nucleus. If you have nodal signaling on, this dimer goes to the nucleus and regulates transcription. So you can look at the localization of this dimer into the nucleus as a readout of the signaling activity in singer cells versus tablet of cells. Okay, now if you do that now and we assume that nodal signaling is being enhanced by contact formation, that's what we actually find if you're looking at the nuclear localization of SMAT2 in tablets compared to singer cells, that seems to be enhanced indicating that nodal signaling is stronger, it's more active in cells which are forming a contact versus cells which are unable to form a contact. Okay, so what we conclude now is that cell cell contact formation affects pre-cautotate cell state specification by promoting nodal signaling in contacting cells versus cells which don't form a contact. Now in most of these interactions, there's feedback and we wanted to see if there's a possible feedback, update specification on contact formation and which type of feedback is a negative or positive feedback. Are we looking here at a feedback loop, a potential feedback loop? So what we wanted to see is if pre-cautotates have its specification affect cells and contact formation as well. Now the experiment we did here, and again, this is an experiment which Vanessa did, is a relatively simple experiment but I need to explain it carefully. So what she's doing is she's taken an embryo and then she's injecting the embryo with RNA which encodes for a nodal ligand and dodge-ness nodal ligand, which is called nodal related too. Now you have an embryo which is expressing the ligand within all cells of the embryo because she's injecting at one cell stage and the RNA distributes pretty uniformly in all the cells. And if I say uniformly, there's always variations in the expression level of this ligand. So you have occasionally cells which are expressing more ligand and cells which are expressing less ligand. What you can do is now, you can do this flux sorting and you can sort these cells according to the level of nodal related to EGP fusion proteins so the level of ligand they're expressing. You can have a fraction of cells which are expressing a lot of nodal ligand versus a fraction of cells which are expressing perhaps half of the level of nodal ligand versus a fraction of cells which express very low levels of nodal ligand. These are just the variations of nodal ligand expression in such an experiment where you uniformly inject RNA and then hope that all cells would express the same level, but they don't. So you have essentially three different fractions of cells which have different levels of nodal ligand and presumably nodal signaling activity in these cells. And you can ask how would the level of nodal ligand which are expressing these cells relate to the ability of these cells to form contacts of different size and stability? And what Vanessa found is if you compare now these three conditions, in this case we have this relates to, I think these numbers actually one. So this is the highest level of nodal signaling and this is the lowest level of nodal signaling and this would be the other round to the three, two, one. And what she finds is that the cells which are expressing the highest level of nodal signal are forming the largest contact and the cells which are expressing lower levels of nodal signaling forming smaller contacts indicating that the level of nodal signaling scales with the size of the contact these cells are forming. Now the question is, how is nodal signaling affecting cell cell contact formation? And there are two poses you can look at and I think we talked about it yesterday. There are two molecular poses by which you can control cell cell contact formation. One is by modulating the contactivity of the actual myosin cortex of cells which are forming a contact and the other one is by modulating the amount of cation expression in these cells, cell cell adhesion molecules. But she finds evidence of both of these mechanisms if she has cells expressing a lot of nodal ligands, the expression of pan cation, the expression of classical cation in these cells seems to be enhanced and similar if she looks at the phosphorylation level and activity of myosin two, the motor for optimizing contraction that seems to be enhanced as well as a function of the level of nodal signaling expressed in these cells. So it's likely that nodal signaling enhances there's a positive feedback loop of nodal signaling on cell cell contact formation and which is mediated by the effect of nodal signaling in regulating the amount of cation expressed in these cells and the level of cortical contactivity present in these cells. Okay, so what we are facing now is a positive feedback loop where cell cell adhesion, cell cell contact formation affects fate specification and fate specification affects cell cell contact formation. And both of these processes are mediated via one key signaling pathway which is nodal signaling. Here we're assuming that contact formation affects nodal, increases nodal signaling by polarizing all the nodal signaling components to cell cell contacts which leads to enhanced fate specification and once these cells are becoming more pre-carded plate, they have higher levels of nodal signaling and this high level of nodal signaling also enhances contact formation. So it's sort of a double positive feedback loop here. Now, this is something again one can experimentally address if this positive feedback loop would exist then you can break it in different ways. One way of interfering with this feedback loop is interfering with the ability of cells to form cell cell contacts. And once you are interfering with the ability of cells of forming cell cell contacts then this should have an effect on their fate specification, right? Because then this feedback loop would not be present and the likelihood that they're expressing they're becoming pre-carded plate would be reduced. Now, the experiment which Vanessa did here in this case is she does a transportation experiment. She takes a donor embryo which is a wild-head embryo which has a pre-carded plate which is labeled here in green, expresses gluuscular GP. And then she's putting cells into the pre-carded plate from two different donor embryos. In one case, she takes control cells which should perfectly integrate into the pre-carded plate and become pre-carded plate like the host cells. And the other case, she takes cells in which she has reduced the level of cartilage expression. So these cells should be less able to form stable cell cell contacts compared to the control cells. And then she's comparing the accumulation of GP within control cells, whether it's cells which are less able to form stable cell cell contacts. What she's finding, and this is still a work in progress, but there is a clear tendency in this direction that cauterine cells, they're forming less stable contacts and consequently they're accumulating less EGP compared to wild-head cells which have a higher tendency to form stable contacts and higher amounts of EGP accumulation. You can also use another experiment to see if this feedback loop between contact formation and surface specification actually exists within the pre-carded plate and that would be directly modulating nodal signaling within the pre-carded plate. So directly interfering with the level of nodal signaling in pre-carded plate cells. That's not an easy task and what you have to use for that is technique which is called optogenetics where you can trigger the signaling pathway in this case by light and you can shine light specifically onto pre-carded plate cells in a subset of pre-carded plate cells and thereby triggering nodal signaling. In this case, what we are doing is we are using two modified nodal receptors, active receptor one and active receptor two B, one B and two B, which are fused to a left domain and this left domain dimerizes when light is being shined on them. So we can trigger receptor dimerization and thereby nodal signaling activity by simply shining light onto cells which are expressing this opto-nodal receptor. Okay, that's just a technique which is pretty common now in cell biology and development biology. Now what we are doing now is a very similar experiment to what I explained before about the Caterium cells. We have a transplanting mixture of control cells and then cells which are expressing this opto-nodal receptor. What we want to do is we want to specifically trigger nodal signaling in these transplanted cells by light and then see if increased nodal signaling affects contact formation and pre-covetate cell fate specification in these cells as you would predict from this feedback loop. Now that's the experiment you put these cells in. The red cells are the opto-nodal and the purple cells are the control cells and you see that cells, once you trigger nodal signaling form significantly, form contacts with a significantly higher contact duration and they also acquire then higher levels of chief P so the likelihood of becoming precovetal plate is being enhanced by simply increasing nodal signaling in these cells. So that sort of confirms that nodal signaling is a key ingredient for this feedback loop between contact formation and cell fate specification. Okay, so to see if that is this assumption that there's a positive feedback loop is a plodal assumption, we teamed up these two theorists, Moritz Lang and Kallen Goet and what they is, they build a stochastic model on a couple of, based on a couple of parameters which should determine the interaction we are proposing in our model. So the different parameters which are experimentally accessible is actually the unbinding rate of cells within the precovetal plate, the binding rate of cells within the precovetal plate and then the introduced constant which describes the accumulation of nodal signaling components as cells are compact. We call it an activation of cells are compact. And then we have two parameters which are important for nodal signaling within the cell and these are the kinetics of SMAT 2-4 dimers to go into the nucleus and out of the nucleus again. That describes the nodal signaling activity and this describes the cell contact formation. And we assume in this model that what nodal signaling is doing, it's leading to the constriction of an effector molecule which we don't know yet, the identity of which we don't know and we don't care really, but we are saying that this effector molecule affects the unbinding rate of cells. So that's the feedback we are introducing that it blocks the unbinding and thereby promotes binding ourselves. So that's basically the positive feedback loop that we have sort of explained before. The contact formation is positively influencing nodal signaling and nodal signaling is positively influencing contact formation. Okay, so you can test this model and you can test it on the, first on the V2 data which are more accessible in terms of quantification. What we're looking in the upper panel is now the amount of SMAT 2 accumulating in doublets versus single cells. And down here we are looking at the accumulation of GP under the control of the Goose Good Promoter in the single cells versus doublets. And what you find here are again, experimental observations. These are measurements of SMAT 2 nuclear localization in doublets versus single cells. And this is the measurement, the dots here, the measurement of GP accumulation in doublets versus single cells. What we find is again, for these in V2 experiments, when you're setting the parameters which you're getting from, measuring unbinding and binding rates of precarotate cells in SMAT 2 dynamics in and outshutting out of the nucleus, taking values, comparing them to values which have been published in the literature, you're getting to a very close agreement of the theoretical predictions to the experimental observations. Now the real challenge is moving these in V2 data into the embryo and see by using exactly the same parameter values would be able to explain the relationship between contact duration and EGP accumulation also within the precarotate of an intact embryo. Now that's what we did in the next. What we find and that was quite pleasing to see is that if you plot now the theoretical predictions to the experimental observations, the contact duration is plotted against the EGP accumulation. You see a very nice fit between these two, between the prediction and the experimental observations indicating that first of all, the in V2 experiments are telling for the in V2 situation and that the model assumption of this positive feedback loop is a positive model assumption. Now the other thing which emerges from this model is is a bi-stability and that comes to the binary surface specification issue. If we are keeping the effector molecule concentration constant and then look at steady state of active contacts, we get one curve which is the blue one and if we are keeping the active contacts constant then look at steady state for the effector molecule production then we're getting another curve and there are three intersection points, two which are unstable here and then one in the middle here. So that sort of indicates that we are looking at a bi-stable fate decision here that cells could become either pre-coupled plate and they have very stable contacts and have very high notice signaling activity or they have very, very short contacts and they become something else. Okay, so I just put this movie in again to show you that there are two different cell fates which I indicated before already. These are these pre-coupled plate cells become rep and pre-coupled plate or you see a few of these cells leaving. They have very little contact or no contacts anymore. They are basically in isolation and they're becoming ended up. So that's sort of an interesting prediction from the model that there's a bi-stability and that you can subdivide cells into different cell fates that according to cells either staying within the pre-coupled plate have very stable contacts or falling out of the pre-coupled plate and have very little contact. Now we can look first of all at the distribution of these two cell fates and of markers of these two cell fates and if you would have binary by stable distribution then you would say that you have some sort of bimodal distribution of these markers and indeed if you're looking at the expression of SOC17 and goose good within the pre-coupled plate or surrounding the tissue of the pre-coupled plate you have either cells which are expressing a lot of SOC17 and endoderm and very little levels of goose good or the other way around here cells are expressing a lot of goose good and very little SOC17. So either cells are pre-coupled plate they're expressing goose good or they are endoderm and they're expressing SOC17. Now we wanted to see if we can actually transform cells by activating or inactivating our feedback loop if we are able to transform pre-coupled plate cells into endoderm or vice versa endoderm into pre-coupled plate and according to our model that should be possible and by the way we did an experiment here where we're looking at a wild type embryo which has a pre-coupled plate which goes up here and then we are taking advantage of our opto nodal receptor so we can trigger now nodal signaling and we are triggering nodal signaling only in half of the embryo only in this half here, right? So we are triggering a topic nodal signaling and by triggering a topic nodal signaling we are activating this positive feedback loop and we should actually then predict that we are getting more pre-coupled plate and less endoderm cells so we are basically switching endoderm into pre-coupled plate or the other way around said that we are inhibiting the likelihood of pre-coupled plate to transform into endoderm. Now that's what we did experiment in and that's what we're getting. What we find indeed is that the number of SOC17 cells on the site where we have induced nodal signaling goes down so endoderm is being reduced and at the same time pre-coupled plate is being enhanced. We can do an opposite experiment and this is a bit more difficult to explain the only take a message you should take home and this is a mutant in which we can reduce the duration of nodal signaling uniformly within the pre-coupled plate, okay? That's the only thing you need to know. So we are reducing partially reducing nodal signaling within the pre-coupled plate and we are asking would that lead to more endoderm and less pre-coupled plate, right? I mean we're doing the opposite of what we have done before by increasing nodal signaling, we're reducing nodal signaling now and what we are finding here is again that endoderm is being up-regulated in these cases and these mutant embryos and pre-coupled plate is being down-regulated. So according to the duration of nodal signaling or the dose of nodal signaling you have in these cells, cells either fall into the groove of cells which are very stable contacts and become pre-coupled plate or very low cell-cell contact, single cells becoming endoderm. So we also, and this is an independent study which we published earlier this year, is the sort of identified a signaling pathway or a gene regulatory pathway by which this interaction, by which nodal signaling leads to either cells becoming goose good positive or becoming SOC17 positive by assuming that there's actually that goose good itself which is transcription factor, typically for pre-coupled plate, suppresses the transcription of endoderm promoter which is important for cells becoming endoderm SOC17. If you assume that then with increasing nodal signaling duration, goose good becomes dominant and goose good can suppress SOC17 and these cells are becoming goose good positive while then you inhibit nodal signaling then goose good is not sufficiently expressed and then these cells are turning into SOC17 cells. That's just the gene regulatory pathway and underlying this binary self-accessification you have here which depends on nodal signaling and contact formation. Now can we address the influence of contact formation and nodal signaling right within the pre-coupled plate on self-accessification as well and Vanessa did two experiments to show that. First of all, she showed that contact duration is clearly different in cells which are either becoming pre-coupled plate or endoderm pre-coupled plate cells have contacts of much more stable contacts where endoderm cells have very short lasting contacts that's predicted from our theoretical considerations and then she did two experiments I just show you them here. One is she essentially repeated the experiments I showed you before where she is transplanting a mix of cells which are cut here in negative and they cannot form contacts where those control cells and then she's asking from these transplanted cells how many cells are becoming pre-coupled plate and how many become endoderm. In a control situation the majority of cells becomes pre-coupled plate and the minority of cells becomes endoderm. If you're interfering now with cell cell contact formation the proportional cells which become endoderm is being increased. That's again indicated by this binary plate decision that cells which are less able to form stable contacts have a higher likelihood of falling into endoderm plate. She can do a very similar experiment and she has to add here more numbers on it but again if she is transplanting a mix between control cells and cells in which she activates a topic nodal signaling then the control cells have a certain likelihood of becoming endoderm versus pre-coupled plate and if you activate nodal signaling these cells the proportion of pre-coupled plate is increased at the expense of endoderm. So when you use nodal signaling you induce contact formation and those you drive cells into pre-coupled plate cell plate instead of endoderm. Yeah, yes, yeah. I mean the surface tension would be reduced for sure, right? Why do you ask? Yeah, yeah, absolutely. So if you interfere with Catheon expression the surface tension of these endoderm aggregates would be reduced. Yes, so the cells morphology and I mean they are just forming less contacts and consequently the morphology of cells would look different. I mean they're more isolated cells which you know occasionally contact each other while in the presence of Catheon these cells are making contacts and sort of increase their sense of contact areas and the shape of these cells would change. Okay. She did one interesting experiment which was sort of triggered again by this model we had here. She wanted to see if, okay. So what would happen if she is taking out cells from a donor embryo and the only difference she's doing she's keeping one population of cells she is taking out, one pre-calipate cells. She's keeping them in isolation for a prolonged period of time. In this case I think it was 20 minutes. And then she's taking other cells which she has not isolated for such a long time and then she's asking is the degree of cell isolation having a negative impact on the nodal signaling activity of these cells and lose the fate specification of these cells later on, right? The assumption would be if you take cells out you keep them in isolation they will down-regulate their nodal signaling passes as a function of time. They would be less able to form stable cell-cell contacts and they would be less likely to become pre-calipate. That's the experiment she did here. She took out control cells, directly used them from front of them and then she took out cells from another embryo, kept them in the pipe for 20 minutes. Now they are not making any cell-cell contacts. Putting the mixture back into the embryo but she finds that indeed the contact in their cells which she has kept in isolation which has down-regulated the endogenous nodal signaling activity because they have been kept for 20 minutes in isolation that these cells have a higher likelihood to turn into endoderm than pre-calipate. Okay, I think that's all I can tell you here about pre-calipate self-explication. What's the, Stefano, what's the time? How much time? 15? 15 to 20 million, very good. So I wanted to tell you something which is really in the making just as a sort of, you know to stimulate some discussion on it I need to connect this computer now because it's not my talk. It's a talk of a PhD student which I have not given yet and I have to see if I can do that. I think it might be something, Stefano, you might be interested in. So I put that in for you actually so you have to listen, unfortunately. Not sure if anything comes here. Is that you background, Kala? Is that you background? Is that you background? Aha, okay. Kala, you have to help me. And I think you have to mirror that. No? Yes, we have. I think you have to mirror the displays and go down to the display. I can do it, I think. I think it's not doing it, I think what you have to do is exactly the same thing as here. Then mirror the display, yeah? And you have to, you know, you have to write the name, the name, the name, the name, and then you find it. Okay. Okay, so in the remaining 15 minutes, I just tell you about an ongoing project which I find very exciting and you can tell me what you think about it. It's far from being finished and certainly very far from being published at all. And what we are looking at here is now we are going back in development and we are looking essentially at the old site and the old site before it starts to cleave. And there's actually a very interesting phenomenon happening in seabed fish old sites before they cleave. This pose is called oplasmic streaming. And, you know, it represents, probably it's applicable to rearrangements and flows of cytoplasm within many cells. But in this case, what is happening is shown in this movie here and just look at it. This is the old site. And what you see here is the flow of oplasm which resides within the old site up to the animal pool where the blastomers are going to form. It's a very interesting process to just to show you these are york granules. And the york granules are a mixed strategy of proteins and lipids. And between these york granules at early stages is the oplasm distributed. And then what the embryo has to do is it has to redistribute these things. This is now shown in schematic diagram. It's not the best schematic diagram actually. But what is happening is you take all these enclosures here, these oplasmic enclosures. You move all the york granules to one side and you want to move all this clear zone to the animal half of the old site. And this part of the old site, the animal half of the blasto disk, is the part of the old site which gives rise to the embryo proper. This one is dividing and then the embryo forms and this is just the food supply. So essentially what you want to do is at this early stage here, you want to set apart the food supply from the part of the old site which gives rise to the embryo which is the oplasmic. And that's being done by streaming process where you basically squeeze out all these enclosures and make sure that they're ending up here. I just want to move one more time because I think the schematic diagram is actually not very accurate to show you what this process looks like. Now you can nicely see these are the streams you have here. There's one more round of streams now and all the stuff goes up here and what you left is with york granules down here and all the oplasmic up here. So it's a fascinating process to look at and you need to understand how that is being achieved. How do we do that? Okay, go here and then I show you now a case where what we have done is to visualize these streams in a slightly better way is the injected beads and the injected beads of different size and we have injected text run beads which are labeled in red, which have a diameter of open five micrometer and then we have injected larger beads. You are asking how these beads would be distributed by these flows via affection in this case. Now that's the experiment here. You have these pulsatile contractions initially and then the first cleavage is being induced up here and you induce these flows. You can very nicely see how these flows are being generated and how you flow up these labeled beads up to the region where the oplasm is aggregating and essentially you're ending up in an area which has all your granules down here and all oplasm including the beads up here. Yeah, that was not a question, but yeah. Okay, so essentially you have a mixture in there which in a long case is what we call your granules and your granules are a mixture of proteins with helogenin and lipids. These could be phospholipids, cholesterol, anything. It's a complex composition of lipids and proteins which aggregating into these granules and the oplasm is essentially the cytoplasm of the cells which get rise to the embryo. They contain everything you have usually in a cytoplasm including the nucleus and the plasmatic reticulum, Golgi and so on and so forth, right? So the oplasm is what you see usually in culture cells of cytoplasm and the your granules is the food supply which has been at the earlier stages of ogenesis pumped into the oocyte that you can keep it as a reserve later on on which the embryo is feeding. Okay, so I'm coming to that. So the cleavages induce the streams. I come to that. Membrane where? In between here? No, there's no membrane. No, it's no membrane, it's open. No, there's no membrane. So there's no membrane. Doing early cleavages, these cells are connected on here, continues with the york cytoplasm. So what we wanted to do and what Shaya wanted to do is he wanted to see how these flows are, how these flows are looking like and what he did is a PIV analysis and probably all of you who just know what PIV is and he looks again, this is a slow motion now, this streams that are induced and he has a few particles in here and then he's mapping the flows. This is quite slow. And then he can do a PIV analysis to see how flows are being generated during this process of cytoplasmic streaming we have here, or oplasmic streaming. It's a bit difficult to recognize a pattern on these PIVs but you can take that apart and look in more detail at different regions of the oocyte. And what you find is that before streaming this oocyte is just pulsating. It goes to a series of pulsatile-actomizing contractions. And then once the first cleavage is being initiated, you induce streamings and the streamings seem to be particularly pronounced in central regions where the oplasm is streaming upwards. There seem to be a backflow on the margin of the oocyte where your granaries are moving in the opposite direction. Okay, so this is sort of shown here in by looking at chemographs, where we're looking at flows along the animal-vegetal axis as a function of time during this cooling process. And if you just look here that flows are being wrapped this positive flow which is directed upwards and blue is flow which is directed downwards. Okay? And you get upward flows in a progressing fashion initiated in animal regions and there the flows are being initiated which are upward directed and then this upward flows are moving downwards towards the vaginal pole. So you induce oplasm-extruming initially very close to the blaster disc which is already present and then this propagates down to the vaginal pole. So you initiate flows in an animal to vaginal direction. And if you look now at flows along the left-right axis or around the circumference in this case what you can see is that the stage when you induce flows in the middle which go upwards you can see that you get backflows on the margin which are directed downwards which are blue. So just in a nutshell what I'm trying to tell you here is that what you're having is flows which are going in central regions up which lead to the segregation of oplasm and your granaries and then you have some backward flows on the margin which are parting in the opposite direction. Okay, so we obviously wanted to know how these flows are being generated and one prime candidate for that is the optimized in cytoskeleton again and presumably optimized in contraction of the cytoskeleton and we did a couple of experiments where we interfered with the optimized in cytoskeleton and with its contractile ability by interfering with acting polymerization and with the activity of rokinus which activates roa and subsequently myosin activity. Now we are treating embryos with these inhibitors and we are asking how flows would be affected in these embryos and that's not too surprisingly what we find is that in cases where we interfere with the actin cytoskeleton always myosin activity the flows are very strongly or strongly affected indicating that you need actin and actin cytoskeleton and actin myosin contraction for proper induction of these oplasmic flows in embryos. Now this is just a summary of loads of different inhibitor experiments he has done, he has interfered with effecting by using effecting depolymerization he has interfered with rokinus to interfere with myosin activity he has looked at interfering with up to mediated branch active polymerization, filamentous active polymerization and again with myosin activity directly and in all these cases, just as a summary in all these cases when he inhibits when he interferes with the integrity of the actin cytoskeleton or its contractile properties he can very strongly interfere with the streaming of oplasmic oplas indicating that you need to have a contractile actin myosin network to induce this stream. Okay, so now the question is how would actin myosin induce these streamings and that's really what we all want to understand and the first thing is he looked at the dynamic distribution of actin during the oplasmic streaming process what you find is that there's actin which is at the cortex which you can very nicely see around here and then there's actin which is within the bulk of the cytoplasm and you can now see the streams are happening and you get the first cleavage up here then there's silent again and this team is being induced again once the next cleavage is being initiated so you get basically dynamic redistribution of the actin myosin cytoskeleton during the streams and that's something he analyzed in a bit more detail than in the subsequent slides to understand which kind of interaction are happening first of all what he looked is at the cortical actin myosin cytoskeleton and what he did is he looked at the intensity of actin at the cortex of these cells during different stages of oplasmic streaming and he also looked at the accumulation of myosin two at the cortex in the same ambios and he looks at stages before streaming and then during the streaming at the end of streaming and this is a chymograph that shows you the distribution of actin and the distribution of myosin at the animal pole and vegetable pole during the streaming process and what you find is that there's a pulsatile accumulation of actin at the animal pole then it goes away when streaming is setting in then it goes back to the cortex when streaming stops then it goes away from the cortex when streaming is on and so on and very similar but less clear myosin accumulation it seems to be that if you look at cortical actin accumulation that cortical actin decreases at the animal pole and streaming is setting in but he also then looked is not at cortical actin but at the actin within the bulk of the cytoplasm of the oocyte what he finds is actually you can just look down here a very interesting regulation where the cytoplasmic actin increases and the cortical actin decreases so if you look at the animal pole of the embryo cortical actin goes down streaming is setting in and at the same time when streaming is setting in you accumulate actin within the cytoplasm polymerized actin within the cytoplasm so actin appears to be in two different localizations it's either the cortex when streaming is off or it's in the cytoplasm when streaming is on okay that's just a summary of it and so he was wondering how do you how do you induce this fluctuation of actin from the cortex to the cytoplasm and what he found is that this is tightly correlating in a spatial temporal manner with the induction of the first metotic division, the first cleavage and what he finds is in metaphase and anaphase for metaphase to anaphase that the first metotic division and subsequent metotic divisions trigger the accumulation of actin within the bulk of the cytoplasm and the relocalization of actin from the cortex into the cytoplasm so that seems to be a self-cycle regulated effect on the redistribution of actin so what he concludes here is that actin tase he calls that actin tase is polymerized actin structures here that they appear and propagate during metaphase now what he really finds and you know that's just yeah true yeah yeah it starts at the nucleus and then it propagates out from the nucleus exactly that's what I'm trying to show you here and I think there's even a movie in here that I'm not taking wrong yes you can see it here and you know that with the nucleus and then you know you get this actin cytoplasmic polymerization which emerges from the nucleus during the division and spreads into the cytoplasm so you initiate a wave of actin polymerization at the animal pole where this cleavage is happening and this propagates from the animal down to the vaginal pole that's interesting because they have indicated that before when you look at the flow of overplasm the flow of overplasm is first induced at the animal pole and then this flow induction propagates from the animal to the vaginal pole so together with this polymerization of actin within the cytoplasm of bulk actin within the cytoplasm you induce overplasmic flows okay and that's very reminiscent to other studies by Timidgesen and others which have shown that the centrosome can actually act as a chemical signaling center and new data to support that as well I think so what we speculate here at this point is that the centrosomes trigger actin polymerization during the first cleavage and this actin polymerization then is required or you know functionally relates to the adduction of overplasmic streaming within the oocyte okay but then the question is where is where is the force created which is needed for this overplasmic streaming and he did an interesting experiment what he did is he took the embryo and then he generated two embryos out of that took the oocyte and he generated two pieces of oocyte took the animal pole cut it off and then take the vaginal pole and he asked can he induce overplasmic streaming in both of these things one contains the nucleus the other one doesn't contain the nucleus okay and surprisingly and that's something we cannot really explain if you look at this thing here you call it mini embryos and this would be the animal and you see very nice streams of overplasmic appear but even in this vaginal part which doesn't contain nucleus there's no division you can induce these streams so there's some sort of pre-pattern of the oocyte which goes all the way from the animal to the vaginal pole which is retained even if you take away the actual division and still you know this piece here this natural piece has a polarity I come to that I think that's a very good experiment so what we really wanted to know is what is triggering these flows there were two sort of main hypothesis which derived from what we have seen here I told you that cortical acting goes down at the animal pole and certain plasma acting increases and so one easy explanation would be that what you do is you reduce cortical tension appear by the polymerizing cortical acting this leads to a deformation of this region of the oocyte and together with the deformation you induce a stream and overplasm streams faster than your granules and then you get a segregation so there was basically a model and that had been proposed in previous studies yeah it's creating the segregation of your granules from overplasm we can perhaps you know I go through the experiments and then we can discuss this part a bit more I mean there are a couple of more experiments which might be then explaining what you're asking now this would be a sort of a very simple experiment that you change the surface tension at the animal pole by reducing cortical actamizing this leads to a local blood formation up here which would be the bluster disk and this induces streams within the oocyte which leads to the segregation okay that was an experiment we could actually address and the idea which Cheyenne had is actually creating a cortex-spoken embryo that basically what he's doing is he's putting an embryo into a very small confinement and he presses onto the embryo up it explodes now it has no cortex anymore what you find is that you still induce flows in cases where there's no intact actamizing cortex anymore you get very efficient flows in these extracts of o-plasm if you want so which contains your granules and o-plasm so that sort of tells you you don't need an intact actamizing cortex for inducing efficient segregation of your granules from o-plasm yeah no you have it already at this stage you have already the pronucleus up there so everything you have already a polarity along the animal-vegetable axis when all that happens the oocyte is pronucled the oocyte is seen when it comes to the q bar that's able to, and that's able to go on the end of the cytoplasm and that's the point there I am when you make it the cytoplasm that's when you get the cytoplasm yeah that's explaining why the vegetal embryo still has a polarity because it contains probably the information yeah I think that's a good point we should use your so the cortex-broken embryo sort of indicates that cortical actamizing is dispensable for the flows so what we want to really know is if this bulk actin polymerization is actually essential for the flow and so what we speculate at this point is that mysine 2 accumulates at the surface of your granules and that the constriction of the actamizing network prepares granules vegetally so if you think about a model where we have a sponge if you're compressing the your granules and squeeze out the o-plasm in a directional manner by having a wave of actin polymerization which progress from the animal flow down to the vegetable now if you look at these different movies that's you know some supporting information on that model here we're looking at the emergence of this actin polymerization wave which comes from here these are your granules and you can see that these your granules are very strongly moved downwards this would be vegetable and this is animal by the way arriving from the animal pool and moving downwards so you can nicely see that that you can move your granules by simply triggering actin polymerization and contraction in a wave-like fashion from the animal down to the vegetable and likewise you can look at mysine and again the wave would arrive from here and you move these your granules downwards and then there's some relaxation once the wave has passed but there's a net translocation of your granules downwards by this wave which propagates from the animal down to the vegetable okay so essentially what the you know I leave you here with this model because this is far from being finished what the proposes there is a polymerization wave which is initiated the animal pool by you know via the division machinery that propagates from the animal pool down to the vegetable this is a cytoplasmic actin bulk network contraction which by moving from the animal down to the vegetable contracts and moves together these your granules and thereby squeezes the O plus in the opposite direction of us the reason why you get streams in the media region and not in more lateral regions might be that you have on top of it in an intact and you you have this deformation up here which might bias the flow into more center regions where this marginal region and that's pretty much all I wanted to tell you thank you