 Okay. Can you hear me well with the microphone? Is that okay? So what I thought to do is I tell you two stories. One story we have done on cell cell adhesion and development, and the other one on cell migration. I think I have a total of one and a half hours, right? One and a half hours I have time. Okay. I can point only at one of these screens. Which one would be more convenient for you? Where are more people on this side? Okay. Okay. So I'm a developmental biologist. You know, it's an interest in sort of biophysics and cell biology. And what I would like to do is with her slides, give you a little introduction into the model organism we are working on. Just to give the physicists a bit of background on the model organism we study. Now, what we are interested in is a process in development which is called gastrulation. And it's essentially early embryonic development. And the organism you're looking at is zebrafish. And what you have here on the left side is a zebrafish embryo which is approximately three hours old and consists of a thousand cells sitting on top of a very big yolk cell. And the type of morphogenetic process we are trying to understand in the lab since, you know, 15 years is how you transform this cluster of cells into something which looks like a proper embryo which has a head structure, a tail structure, an anti-apostea polarity, a left-right polarity which you can't see really, a morphological visible polarity and a dos-ment polarity which is obvious in this picture here. So essentially we are trying to understand the transformation of these cluster cells into something which looks like an egg yolk. I put this movie in for the ones and probably everyone who has seen a fish embryo has seen this movie already, but some of you might not have seen a fish embryo. So I put that in here. It just shows you what gastrulation is about in a dynamic movie here and it starts essentially at the onset of zebrafish life. The first cleavage has happened up here. It's a partial cleavage which subdivides the blustermere up here and this is the yolk supply of the fish. And what is happening in the first ten hours of development shown in this movie here, you have initially a number of cleavages. It's just subdividing the volume of the initial cyguit up here into smaller parts and this is leading to the formation of the blusterm. The blusterm consists out of a thousand cells and morphogenesis sets in now. The blusterm starts to spread over the yolk cell as you can see here. You get mesoderm and endoderm cells to the inside. It's one specific type and these actoderm on the outside, you have convergence and then you have the extension of the body. I just run it one more time. Initially it's a simple cleavage process where you subdivided the volume into smaller parts. Then you have the blusterm and morphogenesis proper morphogenesis sets in now. The blusterm spreads over it. It's called epiphany. You get ingression of mesoderm and endoderm cells. You have convergence of cells for the global axis and extension of the body axis. And this is really essentially these different poses which you have seen here in the movie is what we are trying to understand in the lab. I put in these schematics here just to summarize a few of the key cellular events during a castellation of the fish embryo. And it starts here on the left side. This is the onset of castellation approximately three, three and a half hours old and then everything runs very quickly. This only takes seven hours and then they are basically done. I mean, that's very quick if you compare it to humans or other organisms. And just in the schematic diagram you can see down here the first type of movement as I mentioned before is spreading, it's tissue spreading essentially and what happens here in tissue spreading is that you reduce the thickness of the tissue along one axis and you extend the tissue along the remaining two axes. And this process of epibly as it is called is presumably driven, I will talk about that tomorrow in a bit more detail. It's driven by two different processes. One is spreading of an epithelial cell layer on the outside which is called the enveloping cell layer. It's sort of a skin surrounding the blaster down and that undergoes active spreading. So it's a squamous epithelial cell layer which undergoes active spreading. At the same time you have to redistribute these deep cells below and the way they are redistributing is undergoing radi-intercalation. Essentially the cells deep within the blaster down move up into more superficial layers. You thin the tissue along its radial axis and you extend it along the remaining two axes. And the coordination between epithelial cell layer spreading and radial intercalation of deep cells is what epibly is about. I will tell you tomorrow in my second lecture how this process is being regulated. Another very important type of cellular rearrangements in gastrulation is essentially setting apart the different germ-layer progenitor cells. And you have three different types of germ-layer progenitor cells ectoderm, mesoderm and endoderm. And usually in an embryo ectoderm sits on the outside when you have mesoderm and then endoderm is on the inside. And you have to segregate these different cell types into distinct lanes. And the way that the fish embryo is doing it is you have an induction of mesoderm and endoderm progenitor cell fates in these marginal cells here that comes from a signal which is residing within equatorial regions of the oocyte as localized RNA being translated into protein acting on these marginal cells and then using mesoderm and endoderm cell fates. And once these cells are becoming mesoderm and endoderm they undergo something which is commonly called mesenchymal transition. With saying that with some caveat because these cells are never completely epithelial. They have pseudo epithelial but they don't have an epigabable clarity. But what these cells do is they become more mesenchymal, more migratory and the response to becoming mesenchymal migratory they are moving to the inside and forming the mesoderm germ-layer on the inside and the cells which are not ingressing are the ectoderms. We are basically forming out of a single sheet of cells you should form a double sheet of cells with mesoderm and endoderm on the inside and ectoderm on the outside. And while these cells are going to the inside you start to accumulate cells on the dorsal side by movement of cells from lateral regions and ventral regions over to the dorsal side where they are accumulating undergoing medial lateral intercalations as they are arriving at the dorsal axis here and thereby extending the body axis. Essentially these cells are streaming towards the dorsal side. They are undergoing intercalation which is very simplistic and that's a very hard one. Okay, so in the first part what I would like to speak about is how the different germ-layer progenitor cells are segregating within the embryo. How do you set apart mesoderm and endoderm from ectoderm essentially? Why is mesoderm and endoderm going to the inside and ectoderm stays on the outside? And I put this high magnification to photon laser scanning microscopy movie in here and showing you the process of ingression of mesoderm and endoderm cells right at the germing margin in this little window over here and what you might be able to see is cells moving into the inside migrating up in this direction here and these are the cells which are in the boundary between ectoderm and mesoderm would be right here along here. And interestingly, once these mesoderm and endoderm cells go to the inside you would not find a single cell which ever goes back into the ectoderm. So there is a complete segregation. You can attract all these cells and you don't find a single cell which would ever go back into the tissue they are coming from. And it's a quite interesting pose because if you look at the morphology of these cells they are very superficially they don't look much different from the cells up here. There is no basal laminar any other sort of structure which separates these two tissues but it is a complete separation and there is no intermixing. And what we are trying to understand is how this segregation and separation is functioning how mesoderm and endoderm cells are completely segregating from ectoderm cells and what maintains their separation because there is no structure which physically separates them. Now, what we did is to understand this process in a bit more detail and that's something we are typically doing in the lab is we go away from the complexity of the embryo we are taking cells out of the embryo and we are trying to see what they can do in a sort of self-organizing manner in culture and then we try to translate what we are finding into culture back into the embryo. Now what we did is in this case we took out ectoderm cells and labelled them in red and then mesoderm cells and labelled them in green single cells differentially labelled and then we are mixing these progenitor cells and putting them in different vows and we are asking would they be able to self-organize in distinct layers and the process what is happening is if you are putting them into culture is illustrated here you can see very nicely that the ectoderm cells go to the inside and the mesoderm and endoderm cells seem to surround the ectoderm cells but there is essentially in this process there is a complete separation between the endoderm cells perhaps in culture and this is self-organized there is no embryo or anything which gives some external cues anything this is a self-organized process I will come to that exactly I mean that's exactly what we are trying to understand is what makes these cells different and how do their differences ectoderm forms skin and nervous system and mesoderm and endoderm form muscles and digestive tract anything which is on the inside digestive tract is endoderm sorry? they are not essentially enough to actually moving able to one kind to differentiate so you cannot if you look at morphology it's very hard to distinguish between ectoderm and mesoderm and you have to look at marker genes here so you cannot morphologically they are not differentiated cells yet I am saying there is a possibility that probably they are all the same there is a kind of ectodermal world I mean the experiments so you think they are a spontaneous segregation of cells I am saying they are all the same there is a moment going on and after about one point they are all the same so in this segregation we know that the green cells are already mesoderm and they are putting them into culture so they are expressing mesoderm typical genes characteristic for mesoderm cells why the red cells are expressing genes characteristic for ectoderm cells so they are already different cell fates at this stage when we are starting so fate specification is not a result of some sort of segregation as you have suggested in the embryo it seems to be if you look at transgenic embryos which are driving GP under the promoter typical for mesoderm and endoderm is that the specification actually happens earlier than their ingression so specification is actually likely causing the ingression rather than the ingression causing the specification okay the basic question then is what makes them different and why do they segregate from each other and what we thought to understand that is the turn back to old studies by Steinberg and colleagues which yes okay which you are probably aware of that Steinberg proposed the tissues behave like in liquids in this case which have a deep surface tension and what we speculated based on these studies and there are a couple of other studies on Xenopause and amphibian gastrulation that possibly ectoderm has a different surface tension from mesoderm and endoderm and since these tissues have different surface tensions they separate from each other based on the difference in surface tension so basically we followed up the hypothesis that there might be a differential surface tension which causes tissue segregation and that the tissue which has a higher surface tension in this case it would be ectoderm goes to the inside and it's being surrounded by tissues with a lower surface tension there is some sort of heterotypic adhesion between mesoderm and ectoderm there was the idea so what we wanted to know is the ability of ectoderm aggregates of cells to form a very cohesive cluster with a high surface tension is different from mesoderm and endoderm aggregates so we wanted to compare how cell adhesion in homotypic aggregates is being established in the different germ layers to see if there is any difference in surface tension and if this difference in surface tension might then drive segregation of these germ layers so what we did is we applied the essay by not looking at very many cells but looking at the smallest possible tissue you can look at which would be two cells contacting each other and we are basically comparing them to ectoderm cells in comparison to mesoderm cells and to endoderm cells and try to find out if the ability to form a cohesive cluster of cells enlarge their cell cell contact area if they have different potential in doing so so what we did and this is what we did together with Frank who is in the audience can I increase the volume I'm not sure if I can I can put that up perhaps do you think is that better I can try to speak a bit louder but I have a cold but I can try tell me can you understand me in the back any problems okay so what we thought is we are looking at the stufflets of cells and we are comparing ectoderm daplets to homotypic ectoderm daplets to homotypic mesoderm and endoderm daplets we are looking at the stufflets the formation of the cell cell contact as a function of the interfacial tangents which would exist in such a daplet and we assume there are two different types of tangents interfacial tangents one is called by the actamizing cortex and the other one is an adhesion and basically when we are looking at a daplet which has a stable morphology then we would assume that these tangents are in equilibrium here at these contact points or the contact line around the cell cell contact and we assume that the morphology would then tell us about the distribution of the interfacial tangents here and what we are comparing here is an cortical tangent at the cell medium interface on the outside and at the cell cell interface and an adhesion tangent omega which goes in the opposite direction because the adhesion tangent tries to increase the cell cell contact size while the cortical tangent at the cell cell interface tries to reduce the cell cell contact size so we are opposite to each other and then we have these two cortical tangents at the cell medium interface so what we want to do is to see how the morphology of ectoderm and endoderm daplets differs from each other so we want to measure these cortical tangents and eventually arrive at the adhesion tangent and see if there is any difference in the morphology is the difference of morphology due to differences in cortical tangent end or adhesion tangent because we assume here a specific adhesion between these and this is cation immediate cell cell adhesion we assume in coupling between these cells so if you wouldn't have any adhesion molecules they would not form any cell cell contact our gamma cm would be here the cortical tangent at the cell medium interface ok for the surface tangent of these cells ok so the first thing we did is we looked at the morphology of ectoderm daplets for this mesoderm and endoderm daplets as a function of contact time and what we measured is the contact angle which I indicated before up here is the angle pepper this one here is an independent of the volume of these cells and what we find is if you're looking at the contact angle that ectoderm has a higher contact angle over time than mesoderm and endoderm daplets would have indicating that the contact size ectoderm or ectoderm cells are more able to form large contacts than mesoderm and endoderm cells would be right ok so that indicated already that ectoderm that's consistent with the idea that ectoderm aggregates have a higher surface tangent than mesoderm and endoderm aggregates ok and then we want to measure these different entities to see what contributes to this difference or what causes this difference between ectoderm, mesoderm, and endoderm and the first experiment we did here is we used an AFM microscope we put a plastic bead onto the cantilever and then we did a force indentation curve by pressing the bead onto the surface of single progenitor cells to measure essentially the cortical tension at the cell medium interface and we did that for single progenitor so ectoderm progenitor cells were the single mesoderm progenitor cells and endoderm progenitor cells and what we found is that ectoderm has the highest gamma Cm or cortical tension at the cell medium interface followed by mesoderm and endoderm and as I control the interfered with myosin 2 phosphorylation in this activity and therefore actamized in cortical tension and we arrived at very low cortical tension in all these 3 cases which is indistinguishable indicating that ectoderm has a higher cortical actamized independent surface tension on the outside gamma Cm, then mesoderm and endoderm right? then we wanted to see, we wanted to go from gamma Cm to gamma CC the cortical tension at the cell cell interface and the way we did that is an experiment which there only are a PhD student in the lab came up with and what he did is he created this triplet of cells here this would be a homotypic triplet containing either 3 ectoderm or 3 mesoderm cells, he holds them with 2 pipettes on these outer cells and then he pulls on one of these sides here and then they're separating either here or here and once they're separating I can show you the experiment, what you see is that this cell deforms with the former cell cell contact side and this deformation is an indication about the difference in cortical tension at the cell cell or the cell medium interface assuming that pressure is being homogeneous within the cell, it deforms to an extent and cortical tension at the cell medium interface and the cell cell interface here so by looking at the deformation in the different triplets ectoderm versus mesoderm versus endoderm we can learn something about the ratio of cortical tension at the cell medium versus the cell cell interface and once we have, since we have measured the cell medium interface and the FM experiment we learn something about gamma CC and the different progenitor which delay which delay for like, it takes about 15 seconds Aha Yes, it's not instantaneous that's true, yeah Honestly, we have not really thought about it I mean, there could be, I mean, the whole thing Yes, I mean, it should be instantaneously deformed but I mean, sometimes what we are seeing is that there are errors between these two cells and I'm not sure if they would explain why that is actually like an interesting idea Ah, the medium on the outside is just a culture medium so it's DMF12 in this case, it's water Assuming that the M, you would have interstitial fluid which has the same composition as water then, you know, that would be an equivalent you know, suppression, yeah I show you more evidence what is actually happening at the contact of the adhesion molecules I will come to that for the adhesion molecules and the redistribution of adhesion molecules during the separation process Yeah, can about the viscosity of the cells or the droplets the cells are in the medium the medium is water let me see, yeah I'm not sure why that is looping so you are asking about the viscosity of the medium it's water it's just water essentially it's water plus some supplements in there, some proteins it will take some time because it has this you know, push the water away, right I mean that could partially explain the delay I guess yeah, okay so what we essentially arrived then here is a measurement of so we directly measured the cortical tension at the cell medium interface we derived the cortical tension at the cell cell interface by the ratio of these two cortical tensions here we have the morphology and the contact angle here and then we can derive the adhesion tension we can do that for ectoderm cells and what we find out and what is common to all these things is if you compare the adhesion tension to the cortical tension at cell medium interface the adhesion tension is very very small indicating that what has been assumed as one of the key functions of adhesion expanding the cell through contact by providing a negative tension here is actually only marginally contributing to the shape of these droplets here what really matters is the ratio of cortical tension at the cell medium to the cell cell interface and this ratio is going to be smaller if you compare Cc over Cm gamma Cc over gamma Cm this is smaller in ectoderm cells compared to mesoderm and endoderm in simple words what the ectoderm cells do is they have a higher cortical tension at the outside to start with and they disassemble the ectomizing cortex to a very low level and in mesoderm and endoderm cells you start with a lower cortical tension at the cell medium interface to a similar degree but the ratio is going to be different then because you start with a higher cortical tension compared to mesoderm and endoderm so what really sets apart mesoderm and endoderm cells from ectoderm cells is the degree of cortical tension once these cells are starting to fuse and then what adhesion is doing is disassembling the ectomizing cortex via a process which I will come to and leading to reduction of gamma Cc at this interface here the adhesion tension is marginal yes I mean if you look at you can look at actin and mysid2 and you see that mysid2 is stronger localizing to the cortex in ectoderm compared to mesoderm and endoderm mesoderm and endoderm cells are being induced by nodal TGFeta signals and what we speculate here is that nodal TGFeta signaling reduce cortical tension in these progenitor cells in a very global way by probably interfering with the endoderm and endoderm so it's thought to be an adhesion tension which is leading to contact expansion so it should point out as indicated here that would be on the gate here because it's extending the contact it would be a negative tension in this case and the idea is behind this negative tension is that adhesion molecules are staggering up and they are extending the contact by forming sort of aggregates of adhesion molecules which extend the contact which is one of the ideas but if you look at the localization of adhesion molecules you will see immediately that this is actually not the case so you think they look different from the I mean this is just the ratio here of gamma cc to gamma cm here and this is omega over but do you mean that's different from what we are seeing here? it's the same values we are using here oh yes I think they are significantly different here the ratio here ok so what we conclude from here is the difference in the contact angle and the surface tangents determined by the different ratios of the contact angle so it doesn't matter in the original assumed function adhesion molecules extending the contact seems to be not the dominant function here but it seems to be adhesion molecules with using cortical tangents which determines how big the contact becomes ok and adhesion tends to only marginal contributes to the delta contact relation ok so then we were asking what are adhesion molecules doing this closes here in one function I indicated already that they must be used to a certain degree by a signaling function of the actualizing context of the cell cell contact ok but what we wanted to know is then how the strength of the cell cell contact is determined and what the function of adhesion molecules might be in determining the mechanical strength of the cell cell contact so what we did is to determine the cell cell adhesion force between two cells as we took types again we are taking one cell here, we are taking another cell then we are putting them together we are leaving them together for a certain contact time and then we are trying to separate them again by aspirating this cell with increasing step-by-step increasing the pressure in the pipette here ok and then once we are reaching a critical pressure we can actually separate them and this gives us then the separation force which is a function of the diameter of the pipette and the pressure which is applied the negative pressure in the pipette which we apply so we can measure the adhesion force in homotypic templates for ectoderm compared to mesoderm and endoderm to find out if the larger contact we are seeing actually in these relates to a stronger contact than a mechanical or with a more difficult to separate contact in the pipette yeah the ways you see this is essentially BLEPS and I will come in my second part today if I will arrive there and explain how these BLEP-like protrusions are happening in these cells and these are sort of traveling they are called Circus Movement which is a protrusion which travels around the circumference of these cells so in dissociation of the plus membrane from the cortex and then it's being pulled back on one edge then it travels on the other edge but I will show you a better picture ok so here we are looking at the separation force and we are looking at the separation force a function of contact time again we are going up to 10 minutes here and we are comparing ectoderm douplets to endoderm and mesoderm and endoderm douplets ectoderm douplets the contact becomes stronger as a function of time while the contact becomes as a larger interestingly in mesoderm the contact strength so the separation force becomes weaker and in endoderm it stays pretty constant but it's weaker than mesoderm initially and ectoderm over the full period of time so we thought initially perhaps the contact strength is just a function of the contact size and if you are assuming you have a homogeneity the same density of adhesion molecules could simply be that if the contact is larger it's harder to separate because you have a larger contact so what we did is we normalized the separation force by the contact radius to see if that is explaining why we are seeing a difference and still if you are looking at the separation that's divided by the contact radius that ectoderm is larger than mesoderm indicating that the size of the contact alone cannot explain why these contacts are so much stronger in ectoderm compared to mesoderm and endoderm so we wanted to find out what else and one the most intuitive explanation and this case we normalized the radius actually but we assumed it's going to be round the whole thing but we have so the radius is scary with the area because we assume it's a circular contact in this case but we have not subdivided by the area but we could do it by the area I come to that I come to that so what we thought is the difference it might be the density of Cartesian molecules at the contact which might be different assuming that ectoderm would have more perhaps express as higher levels of Cartesian than mesoderm and endoderm so what we did is we looked at the amount of Cartesian at the plasma membrane using a antibody against all classical Cartiurans in what we found which was counter-attuitive at this stage is that it appeared if you are looking here right in the embryo the mesoderm would be on the inside of Cartiurans at the plasma membrane mesoderm and endoderm seem to have more Cartiurans at the plasma membrane than ectoderm so opposite to what we thought the cell which has a weaker contact actually appears to have slightly more Cartiurans at the plasma membrane so the amount of Cartiurans difference in the amount of Cartiurans cannot explain by ectoderm has a mechanically stronger contact than mesoderm and endoderm so we want to know what determines the high contact strength in ectoderm and what we did is we looked at the a very simplified version of the Ephesian complex between two cells in this stripped-down version it would consist of Cartiurans and Ephesian molecules which bind to better Cartinian better Cartinian binds to alpha-Cartinian and alpha-Cartinian links into the sector skeleton so you are coupling the two cortices of these cells via this link consisting of alpha-Cartinian, beta-Cartinian and Cartiurans right and then we thought how strong the strength of this chain between ectoderm and mesoderm and endoderm and the way we want to see how strong that is is pulling on it and seeing where it ruptures and if it ruptures earlier in let's say mesoderm then compared to ectoderm okay so what we did is we did an experiment first we looked at the localization of these different components and what we find here with an ectoderm antibody is that most of the ectoderms are localizing actually to the cell-cell contact edge I'll show you one more time and this is exactly the point where the two cortices actually need to be attached to each other assuming that the atomizing cortex is largely disassembled at the cell-cell contact right so the adhesion molecules in this complex localizes where you need to mechanically actually link these two contractor cortices from the two contacting cells and then we did we looked also in Divo and you know right in the end you can very nicely see that all these rings in this case it's better that you have, you know this is a very nice localization of beta-catenin right at this ring like at the edge of the cell-cell contact so you don't have a homogeneous distribution of adhesion molecules, adhesion complex over the cell-cell contact but everything sort of localizes to the edge of the cell-cell contact and then we did the experiment which I told you before if it will come okay let me go back okay so now we're looking here at two cells I hope you can see that can we put the lights down is that possible perhaps not this is good, yeah, perfect so you might be able to see these two cells here and they're expressing a version of N-caterion with the CP-tact and N-caterion localizes to the cell-cell contact at the margin of the cell-cell contact and you want to know when we're pulling on it where would N-caterion end up does it stay, you know, is it separating at the you know, Katerion-Katerion binding and then you would have it essentially in the two separating cells or perhaps it rupturing further downstream and then you would have it like in the middle between these two separating cells okay so that's the experiment we're pulling them apart using pipet and you see very nicely that N-caterion floats now they're being coated N-caterion goes right and then you have it at the base of a plasma membrane tether which connects these two cells okay so N-caterion is not just staying behind where it was before at the cell-cell contact edge but it sort of flows and it ruptures presumably it ruptures from the atomizing cortex and flows into the plasma membrane tether or in the base of the plasma membrane you can see it a bit nicer if you look at better Katerion-GP again you're separating these two cells and then you have a tether in between these two cells and you have a blob of better Katerion right in the middle of this tether here indicating that you pull out Katerion plus better Katerion and then they flow into this tether and you leave behind presumably the actin side of the skeleton if you're looking at alpha Katerion it looks different you're separating them and alpha Katerion essentially stays not before now you pull it apart but you don't see alpha Katerion really at a tether between these two cells and similar if you're looking at actin using a uterine teri actin doesn't go into these tethers these are plasma membrane tethers which are not filled with actin right so from these movies I should be in both cells so actin is in both cells have an actin cortex why do you say it's reaching only one cell you don't see that you don't see actin in the outside you should be in both cells I mean this is just an imaging artifact not sure what that is okay it quickly goes here okay so this is just a quantification of what I tried to tell you from these movies is if you look this will be the contact before the separation and then it is completely separated with the contact radius if you look at Katerion in the beta Katerion you can see that they go right and they are accumulating at the diminishing cells of contact till they are in the tether while if you look at the concentration of alpha Katerion and actin then this is not happening right they are just staying where they were before they are not changing their localization during the pulling process so it appears as if the weakest link in the Katerion appears to be between alpha Katerion and beta Katerion so if you pull on this thing it breaks between alpha and beta Katerion and what we then conclude also is that this linkage must be or presumably is stronger in ectoderm cells compared to mucoderm and mucoderm cells and that explains why ectoderm have a mechanically stronger contact compared to mucoderm and mucoderm this is just showing you that this is not just in culture artifacts but looking within the embryo there is nothing more than that much and you can see nicely that n-Katerion goes into the tether between two cells so that's something that happens within the embryo these are mesoderm cells between the embryo expressing n-Katerion GP a fusion of ectoderm the problem is that ectoderm doesn't move so much relative to each other so what happens so mesoderm and endoderm migrate approximately 2 micrometer per minute and ectoderm perhaps 1.5 1 micrometer but they are moving in opposite directions so one moves up and the other one moves down so you're thinking about heterotypic adhesion now between ectoderm and mesoderm or you're thinking about homotypic adhesion so we have not looked at heterotypic adhesion between ectoderm and mesoderm or ectoderm and endoderm so what is the relation about mesoderm and mesoderm that you're thinking about together and do you really recommend it to the students and do you just imagine they're talking about it so would be the speed or separation in vivo be similar to the speed or separation we have in our experiments here when we're pulling them apart because they have this shear flow and some they have they're like they're taking too far but you're asking how ectoderm cells move relative to other ectoderm cells, right, to mesoderm cells. And you're assuming there's some heterotypic adhesion between them, and that would lead to some transient contact formation. You have not looked at heterotypic adhesions at all. You're just looking here at homotypic adhesions. So you're looking only at ecto-ecto-meso-meso-and-endo. We have done some initial studies and it seems to be that if you're looking at a heterotypic adhesion between ecto-meso-to-force for corticotendron and the ratio of corticotendron and force between what ectoderm cells are doing and what mesoderm cells are doing, it seems to be more dominated by the weaker cell than by the stronger cell. So the weaker cell limits probably the adhesions. Why? Why is it the weakest, I mean, presumably the binding strength between alpha and beta cutinene is weaker than between alpha-cutinene and cutterene or between alpha-cutinene and some molecules which link into the cytoskeleton. That's a molecular interaction, and I think it has been reported, binding affinities have been reported, and I think they are conflicting reports in literature. Some of them would indicate that the binding strength of alpha-to-beta-cutinene is weaker than the binding strength of alpha-cutinene to the cytoskeleton or beta-cutinene to cutterones. Okay, so the one experiment to see actually if this binding strength is meaningful, I mean, we are speculating now that ectoderm has a stronger anchoring strength to the corticocytoskeleton than mesoderm and endoderm, presumably we are having a stronger alpha-beta-cutinene interaction, and what we want to know is if this binding strength matters for ectoderm forming larger contacts than mesoderm and endoderm, is this binding strength important to bind? And the way we want to interfere with the binding strength is in a very drastic manner. What we do is we take a cation molecule and then we are cutting off their cytoplasmic tails. So they have still a trans-membrane domain, and they have an exocellular domain, but they don't have a cytoplasmic domain. They can essentially not couple to the corticocytoskeleton. What we then want to do is we want to take out the endogenous caterins, fulling caterins in ectoderm cells, as well as in mesoderm and endoderm, and replace them with a truncated version, right? And then see if they all have the same truncated version, they all would not be able to link to the corticocytoskeleton, would ectoderm still form a larger contact than mesoderm and endoderm? That would sort of indicate that the binding strength into the corticocytoskeleton is meaningful and required for ectoderm forming larger contacts. Okay, that's the experiment. And that's what we did is, and this is just illustrating what the, and, you know, just look at, this year, you were looking at the separation force and the upper one and you're looking at the contact angle or contact size in the lower one down here. And what we compare now in the control experiment is you take out the endogenous version of caterin and replace it with a fullings version of caterin, right? If you replace now e-caterin with a fullings caterin II, and I can explain why we are using caterin II and not caterin I in this case, then what happens is that ectoderm cells form a larger vessel contact angle compared to endoderm cells. If you replace the ectoderms now with a truncated version of caterin II, then ectoderms form a similar small contact than endoderms. So the ability of ectoderm forming larger contacts depends critical on the anchoring ability of caterins to the corticocytoskeleton. Similar if you look at the, I said here that this is the separation force and this was the contact angle and that's exactly the same dark here. Interestingly, you still have, in the case of, you know, that truncated version, you still have some contact forming indicating that there perhaps are other caterins which we have not knocked down in these cells and which might mediate some residual cell cell adhesion in these cells. Okay, then we want to know if, you know, in case you have the truncated version and the cell cell contact is approximately the same between ectoderms, the ectoderm doesn't, isn't different anymore to mesoderm and endoderm, would that affect their segregation behavior and culture, right? And that's what the, the Tia is looking now in a control experiment. You mix ectoderm and endoderm cells and you get a very nice segregation usually. And if you use that truncated, if you take this truncated version, this, the sec, segregation of these pretenders cells doesn't work anymore. Okay, so what we conclude here is that ectoderm mediated adhesion mechanically happens with contractor cortices of contacting pretenders cells. That the coupling strength of the cauterion scales with the cortical contractility of the different pretenders cell types, indeterminate by the cypher scale anchoring strength of the cauterion. Meaning, when you have a very strong and contractor cortex, you have to make sure that the, the coupling strength of the cauterons actually scale with the cortical tendon in these cells. You have to, you need a very strong coupling strength to hold these, and, and so, so you, you, you need a, a stronger coupling strength than ectoderm compared to mesoderm and endoderm. And the difference between the pretenders cells and contractor formation cells often depend on the cypher scale anchoring of the cauterion. Okay, so, so just to summarize it, what we, what we think is the, the reason why ectoderm segregates from mesoderm and endoderm is partially determined by differential surface tensions of ectoderm compared to mesoderm and endoderm. The differential surface tension comes from, you know, the fact that ectoderm forms in homotypic ectoderm as far as the form larger cell-cell contact, the more stable cell-cell contact like mesoderm and endoderm. And the mechanical strengths and the size of the contact critically depends on the anchoring strengths of ectoderm to the cortical cypher skeleton. So they need to be rigidly coupled to the cortical cypher skeleton in order to allow ectoderm to form large cell-cell contact. Yeah, okay, and there is, I mean, perhaps we can discuss it probably, you know, for a longer time in the evening, but there are loads of different ideas how the Cateron complex actually anchors to the actin cypher skeleton. Some people assume that alpha-cutinene exists in two forms, in a dimeric form, in a monomeric form, and only the dimeric form can actually bind to the cortical cypher skeleton. In the meantime, this model has been changed and now what is generally believed that alpha-cutinene even bound to better cutinene can directly bind to the cortical cypher skeleton if the actin filaments are pre-stressed. Now, what else has been assumed is that alpha-cutinene when bound to better cutinene in the monomeric form cannot directly bound to the actin actin-actomized in cypher skeleton, but it can do so via linker proteins, like vinculin or ethylene or other proteins which bind to alpha-cutinene and then link to the cortical cypher skeleton. But, you know, we can discuss what the evidence is, but, you know, either it directly binds or it needs other coupling molecules to bind to the cortical cypher. So, you mean during the ingression process? Yeah. I mean, I think what perhaps what you're referring to is actually, is there actually sorting happening in the MVR? Do you have, actually, a mixed condition actin-actomized in an endoderm? And if I, perhaps I should go back and show you. This might be, okay. So, so, so, okay. If you look at this situation here, this is really the ingression process, right? And, you know, it doesn't, it's not a salt and pepper. You don't have actoderm randomly intermixed with mesoderm and endoderm. And then they are segregating out. What you have is actually, you have a population of cells up here which has been actoderm and they receive now a signal which comes from the arc cell and makes these cells more and more mesoderm and endoderm. So there's a sort of continuous transformation of these cells into a different cell fate. And once they are mesoderm and endoderm up here and they're expressing markers typically for mesoderm and endoderm, then they are moving to the inside. My saying that is what I think the differential surface tension is doing is keeping these two layers apart from each other rather than actually driving the ingression process. I think the ingression process is more driven by these cells becoming migratory mesenchymal and then actually migrating to the inside, right? So the second, the involution or ingression process seems to be more driven by a form of active cell migration. Monthly on the inside, you have to make sure that they are not just going back to the tissue they're coming from. And this separation of the different germ layers might be driven by differences in surface tension between these tissues. Okay, so that's actually the precise. I mean, you know, I think that's the most honest version of what is happening there. Yeah. If mesoderm can become active again or yeah, it's in again, I mean, I could talk about it but I'm not sure if I can do it on Wednesday is how the different cell pates are being induced. Mesoderm and endoderm, how they have been maintained once these cells are on the inside. And one critical factor which allows mesoderm cells to remain mesoderm once they're going to the inside is actually cell cell contacts. They have to adhere to each other and there has to be some form of auto-crine or pyrocrine-cycling between cells which allows them to maintain their mesoderm and endoderm cell pates. If you keep them in isolation, they become eventually, again, they lose their mesoderm fate and they could potentially become active again. But once these cells are going to the inside, they are in contact with each other and they are cycling going on between these cells which sort of maintains and promotes their cell pates. They have acquired in the Germany. In the self-organization essay? Yeah. Yeah. So here I have to go into technical details. What we do here is we induce a stable population of mesoderm cells which express actually the mesoderm-inducing factor by themselves. It's a nodal-teacher-better signal. These cells will always remain mesoderm, no matter what, right? If they're in contact or not, it doesn't matter because they're expressing so much of the stuff which induces them in the first place that they will always remain mesoderm and endoderm. The other cells which you have taken as active are cells from a mutant which cannot respond to mesoderm and to the mesoderm-endoderm-inducing factor nodal and they will always remain actoderm because they are just insensitive to any factor which would induce mesoderm and endoderm in this. So it's sort of an artificial assay which allows us to keep them completely separate and have no switch in cell fate during the seclusion process. Okay, so how much time do I have left? How many hours? So I can try to tell you a bit about cell migration as well. Yeah. 40 minutes. 40 minutes? Okay, that will be enough. Okay, so I want to tell you a bit about the mechanic about the cell migration and development and specifically, I wanted to introduce the types of cell migration you see in the embryo. And again, we are looking now at the mesoderm and endoderm which has gone to the inside after ingression and we're looking how mesoderm and endoderm but then the cells are migrating on the york cell between the actoderm on the outside and the york cell on the inside side. And you can see very nicely these cells as they are migrating up. It's a sheet, a coherent sheet of cells that's electrically undergoing migration with cells as the leading edge being very protrusive, right? And what we wanted to understand in this study is how some migration is being triggered in the embryo and which different types of migration you have in the embryo. This very much looks like a mesenchymal type of migration where cells are forming active rich protrusions, lamelopodia, phylopodia and they're crawling over there. Either using the actoderm or the york cells are substraten, they're crawling and moving forward. How fast they're migrating up to two micrometer per minute. Yeah. Okay, so in a high magnification we're looking now at these cells which are coming to the inside and are migratory and now this cluster of cells just moving from the left to the right side in this movie here. And I have, Alba, the PhD student in the lab has done this, she labeled the cluster memory in red and act in in green just to show which kind of protrusions are being formed in these other migrating. You might be able to see here these kind of lamelopodia green protrusions up here but you also see occasionally these membrane bleps. You can see it now. I think there's one forming down here. There's a blep forming. So you have all sorts of different types of protrusions. You have more actin rich, lamelopodia, pseudopodia, phylopodia protrusions and then you have these, you're initially acting devoid membrane bleps which repolymerize actin and then these bleps are being pulled back, right? So I would say lamelopodia, phylopodia and bleps are the most frequent protrusions types. You can quantify that during the migration. Again, you're looking here at a blep which would be a membrane without actin initially. Then a phylopodium containing actin and a lamelopodium which is filled with actin a very broader flat sheet. And if you look at the percentage of these different protrusions types, surprisingly the bleps are the most frequent ones followed by lamelopodia and then phylopodia in these types of migration. Once these mesoderm cells are going to the inside. We're actually looking here at axial, anterior axial mesoderm cells just for the sake of completeness. Okay, so what we want to know is we want to find a way how we can change the distribution of protrusions and see if changing the distribution would have an effect on the migratory ability of these cells assuming that they might have specific functions in migration life. And what we did is we increased further the percentage of bleps at the expense of lamelopodia and phylopodia and the way we are doing it is genetically interfering with a molecule which is called S-rin and S-rin couples the actin, atomizing cortex to the plasma membrane and we can down-regulate S-rin by using antisense morpholino oligonucleotides or using a dominant negative version of S-rin. In this case we are reducing cortical to plasma membrane attachment and then we're reducing that by getting an increased number of bleps at the expense of lamelopodia and phylopodia in these cells. And what we found when we are doing these experiments is I'm not sure what a computer is doing with all the time is that the direction of persistence of these cells is going down, then we are treating them with dominant negative S-rin and S-rin morpholinos and okay, and the net speed is going down. So these cells are migrating in a less directional manner and they're migrating slower once we have more bleps at the expense of lamelopodia and phylopodia indicating that for directed and fast migration these cells need lamelopodia and phylopodia and once we are switching into bleph-like protrusions they are not as directional and not as fast in their migratory activity. No, and I can tell you why but this was only a starting experiment into actually what we wanted to know is we wanted to see how these different protusion types are being formed and what Verena in the lab then did is she thought what they usually do is take cells out of the embryo mesoderm and endoderm cells put them into culture on different substrates and ask which protrusions can they form and how would these cells then migrate in the presence of different protrusions. Now, this is what you have seen before and even in these pipette experiments these circular bleps, can you see that? This is what a cell, a progenitor cell, a mesoderm or endoderm progenitor cell when you put it on a pyronectin coated substrate this is what they do for a couple of hours, very boring. They're just forming this flap and it's called circus behavior and they're just not very adhesive and then they have this flap like protrusion form which is circulating around there and can go counterclockwise or clockwise. Okay, so that was not particularly encouraging but then what Verena did then is she increased and she improved the culture conditions by changing the pyronectin substrate in which these cells are sitting and if she made it long enough then what was happening is that these cells which were usually just forming this boring flap they're becoming now more adhesive and you can see that they're forming this chain of migratory cells which looks more like what is happening within the embryo where you have migrating cells moving as a sort of a cohesive sheet of cells, right? So she can get something which looks a bit closer to what cells will do. Then she did one experiment this was basically done by accident she added serum to the medium usually we do all these studies in serum free culture medium and she added serum to these cells and I'll just show you what then happened this and she can add a serum component LPA and what is happening then is this would be these mesenchymal migratory cells. Now she added serum and what she found is a complete transformation of these cells which we have never seen before and we can see now, whoops. And in this case it is acting all the acting is gonna be at the back of these cells and they have a very large flap-like protrusion form they're very stable flap-like protrusion form that really happens instantaneous when you increase cortical contractility by adding serum or LPA to the medium. So that was quite interesting and she got quite interested in that I was very skeptical if that has any meaningful relation to what is happening within the embryo. What she then did is she described these cells in a bit more detail what she found is that what you've seen before in this movie is that acting goes to the back end predominately accumulates in the back end of these cells and then they have this stable flap-like protrusion in the front end which seems to be largely devoid of the acting and migratory. This is a typical appearance in a BSE image here of these cells where all the stuff is on the back and the front is a huge balloon in the front which is very, very stable. So the first thing she asked then is how would this polarity emerge in these cells? And what she did is and the first thing is she determined the dynamics of this polarization process and what she did is she takes one of these cultures which consist of mesenchymal cells which have lamina podia and photopodia and then she adds a time point zero LPA to the medium with very small concentrations and then she's asking how quickly these cells are polarizing and within 10 minutes a very large proportion of these cells have transformed into these stable flap-like cells. So essentially if you wait for long enough all of these cells transform from this mesenchymal state into this weird, stable flap morphology as you can see up here. This is simply done by increasing cortical-actamizing contactility. The reason why we leave so is that LPA is a known ligand which binds to a G-protein-capped receptor and this G-protein-capped receptor with LPA receptor signifies G-proteins into row A and then it acts on to active polymerization and or-actamizing contraction. So we thought this branch of the LPA pathway is presumably what we activate in these cells and the reason why we believe so is shown in this experiment what she did here is she did the same what she has done before she has a control culture where all the cells are essentially non-polarized mesenchymal cells then she's adding LPA and she can transform a very large proportion of these cells into these stable flap cells and if she's doing the same experiment with plebistatin or with a rokinus inhibitor or depolymerizing actin she can inhibit this transformation, right? Indicating this transformation depends on actamizing contractility in these cells. Yeah, I come to that. We can, for example, we can just activate row A and take a constitutive active form of row A and you see the same essentially in a shotgun. So it all smells like increasing cortical contractility by whatever means, right? Serum, LPA, various ways. You get, you arrive at the same effect that you can transform these cells efficiently from a mesenchymal into a stable flap-like cell. But she then wants to know how this polarization relies on myosin-2 and actin redistribution of the cell and what she finds here is that if you see that myosin-2 goes all to the back of these cells and you have this very stable myosin-2 free from them which she called a stable flap. So myosin-2 goes to the back end during this polarization process. And so she gets a very strong cortical enrichment of myosin-2 in the back end of these cells. So what she speculated here is that there's, you know, depending on the, you know, an increasing, increasing cortical contractility, you can transform non-polarized cells which are mesenchymal, in this case, they are just non-cells, into either cells which are dynamically blabbing at medium contractility levels into a cell which has one pertrusive stable front left at very high contractility levels, right? So you go from a mesenchymal cell into a spontaneous blabbing cell into a stable back cell depending on the level of cortical contractility you have in these cells. And what she wanted then to know is if she can induce this polarization by locally triggering cortical contractility. And the way she's doing it, she's taking a pipette, she blows LPA up to one end of these cells and usually what is happening is that at the end she blows this LPA in this case but she can use serum as well as myosin-2 is accumulating and this protrudes this left front and points away from the pipette. So in principle, you can polarize these cells by locally strongly activating cortical contractility in these cells. Then you get, you know, you can determine the polarity of these cells. She also wanted to know if there are different ways of triggering cortic contractility in one way which has been demonstrated by Matthew Piel in a compilating study in Paris. He showed that if you take cells from different cell lines and you strongly compress them, put them under spatial confinement, you trigger octomizing contractility in these cells and you actually can transform them into something like these cells. He showed that independently. So we thought, you know, perhaps we can do it independently of LPA or serum or, you know, activating raw A but just putting them under spatial confinement and that's exactly what we're finding when we're putting them under strong spatial confinement we can transform them into the stable left cell with the back end. I mean, they are quite squeezed in this case but you can reliably do exactly the same what Matthew has shown for the different cell types. Now we wanted to know if this transformation depends on a certain cell fate in the embryo. So we took ectoderm cells, mesoderm cells and endoderm cells and he compared their ability to transform under, you know, in the presence of LPA by increasing cortic contractility and the take home message is it doesn't matter what these cells are. If they're mesoderm, ectoderm or endoderm irrespective of their primary cell fate, you can always trigger a large degree of transformation in the cell for simply up-regulating cortic contractility. So it's not a cell fate, it doesn't depend on the fate of these cells. Now, the question really she was interested in is migration and she wanted to know if these cells, yep? There's a lot of ask for my advice. Invent the full information then, yeah. Actually they become migratory right away because, I mean, is there another single in which you could do apical constriction of birth and then migration in this context or is this probably the matter that I'm not adding now? So you're wondering why in Drosophila under high cortic contractility level these cells are not becoming, I mean, the first difference is that all these cells we are looking at here are non-epithelial cells, right? These are non-proper epithelial cells. We're looking at some form of mesenchymal cells to start this, right? And interestingly, the only cell type in the embryo which you cannot transform into a stable black cell are these outside EVL enveloping layer cells which are the only truly epithelial cells. So from an epithelium to go into these stable black cells it's quite hard. I mean, that doesn't work very, very likely. Because of the epithelial polarization I think and, you know, perhaps the adherence functions which makes it harder. Okay, so if you want to know how these stable black cells would migrate and, you know, what is well known in migrating cells that actin is being polymerized to the front end and then you have a backflow of actin which is resisted by the friction to the outside environment of these cells which leads to the protruded force of actin pushing for the side. I mean, that's the general model of this actin polymerization backflow, retrogart flow triggering migration. So if you wanted to see if there, you know, how actin and myosin dynamically distribute you can see in these cells here that you have a very fast flow of actin which is between 20 and 30 micrometer per minute to the back of these cells, right? I mean, you have a very, you know, there's continuous polymerization, myosin as well, you see these myosin mini flaments flowing backwards in these stable black cells, right? And that already, you know, sort of indicates that these cells have a potential of migrating. You just need to put them in the right environment where there's some friction where they could actually migrate. So what she did is she quantified these flows in the first place, but then she put them in confinement and she not just put them sitting on a one month update but she put them in a certain confinement where she put an agro plate up here and the glass down here. And when they have some friction to the agros and to the glass down there, then you can actually get them into a migratory phase and I'll show you that here. Now these cells are becoming migratory and they're migrating actually very, very fast. These cells in culture migrate up to 20, 30 micrometer per minute. This is more than 10 times as fast as you would find a single cell in the embryo tumor. That's something that looks weird, right? But they are super fast and that depends on and scales with the degree of atomizing flow, backward flow you have in these cells. You have a very fast atomizing backward flow which is resistive as a friction to the outer medium and then propels these cells, right? Like a caterpillar to a certain degree, okay? So now if you have a stationary cell which is glued here then and it's not in confinement when you just have this flow and this flow is not resisted by any friction and this is a cell which is migrating and where you have a fast flow resulting into a forward motion of these cells when they're not sticking to anything and when they're confined on those cells. Okay, so the type of migration they're doing seems to be a persistent random walk and essentially what is happening when these cells are migrating in one direction, they hardly change their direction anymore and it's very hard to see of them. You cannot give them any chemokine, they would not change their direction. Once they're running, they're running in the same direction. And their mean speed is something between 15 and 20 micrometer per minute, very, very fast. Okay, so after that, after this point, I mean one thing she wanted to see is do these cells need any specific adhesion? Do they need any sort of matrix adhesion, or do they need to be immediate matrix adhesion for migration of these cells? You must have some, I mean it looks like you might have a best able system with what you've taken throughout. It's not, it's not, it's not, it's not, it's not, it's not, it's not, it's not, it's not. I mean what is happening is you have some spontaneous polarization of these cells, assuming that there's no local conductivity induction in these cells, and once they have chosen a direction, they just run along this path. And the way they run within, I can show you that later in the endogenous environment, is they're looking for space. They run where there's space, and if there's no space, they just stop, right? I mean they go to more space, essentially. There's a mechanical difference. I mean once they are polarized, why are they known at the end of the course? I mean then you have to do this very fast, I mean how would you see them? You would see them by some chemokine signaling or something, right? But I don't think this, you never see any accumulation of any membrane proteins at the front end of these cells, because everything flows very fast backwards, right? If you look at integrins or anything, it's all sort of halfway back where the cell is, where the stuff is starting to accumulate, but at the front end, you hardly have anything really stably attached to the cluster membrane, because the cortex is constantly flowing very fast backwards. So I don't think molecularly you have anything which could build a sensory complex, a receptor complex which would allow the cell to stay already there. I mean that's the simplest explanation here. Exactly, so I think there's a continuous polymerization of actin and myosin intercalation and this network once it is polymerized, it's really starting to flow back, right? So the continuous polymerization and back flow at the same time. If you put actin and myosin together, what do you mean? Yeah, exactly, ah, you see that myosin is in between actin filaments, right? And seem to anchor to the actin filaments, but it's not directly overlapping, but it's somehow flowing together at the same speed at the actin cell. So if it's more, you mean more in cortical areas or on the bulk. I mean I assume it has to be some, I mean there has to be some back flow as well, right? I mean if it flows in one direction, you need some back flow in the middle. I think what she has done here, this is right at the, it's terf microscopy, so it's probably more on the cortex rather than the middle of the cell, she's imaging that. I guess there must be some proper flow, right? I mean there must be some sort of compensatory flow for the back flow you have on the outside side. Okay, so one question she asked is do you need any specific integrity meaning the adhesion and what she found in, this was quite interesting. So if you put cells at different substrates, you can put them either on polylysin or you can put them on pyronectin or you can put them on these PSA. In all these cases, it seems to be that specific adhesion to exocellular matrix is actually slowing down their migration and the best is if you're putting them on pack, you know, passivated substrates where specific adhesion is being actually minimized. There they actually love to, and they run fastest and as soon as you introduce specific adhesion, they slows down their migration speed. So the only thing they need is spatial confinement, high cortical contactility and low unspecific adhesion to a substrate and then they are really fast. Everything else is slowing it down. Okay, so what we learned from that is that it depends on spatial confinement, it's independent on specific adhesion and substrates photosynthetic polylysin and these A are to only slow it down and it's independent of endogenous cation expression and endogenous integrating better one expression in these cells. Now the obvious question is, is that all a culture artifact with any meaning for the actual situation within the embryo, do these cells exist? So what it is, we went through many, many movies we recorded over the last 15 years. Yeah, it's in the back end, but not directly sitting, not really on the back end, but perhaps in the last third of the cell. Okay, so we want to know, do these cells actually exist? So what it is, we screen, so all the movies we have recorded over the last 15 years on mesoderm and endoderm migration and what he ended up is one movie and this was just a complete accident. And look at this movie, you're looking again at mesoderm and endoderm cells migrating and there's one cell coming on the left side, which we obviously ignored at this date, which slightly looks like a, you know, I stay with that cell, very fast and migratory and be, you know, couldn't understand what the cell was actually doing, but you know, that sort of indicated that these cells might actually exist within the embryo. We asked the Japanese poster, Kitoshi, to actually start recording many, many movies and see if he can actually find these cells in different conditions within the embryo. And what he then consistently found is actually that these cells exist. You can see another cell, I think it's migrating here now, very fast, you know, peeping mesoderm and endoderm. And he did many of these movies and you know, he consistently finds that there are spontaneous emergence of these cells within the embryo at random locations. It's hard to predict where they would emerge, but you do find them in the actual situation within the embryo. Now he wanted to have a way he can actually induce them in the embryo to see if, you know, cortical contractility is actually involved. And what he did is a very simple experiment where he looks at, you know, the blastoderm of, you know, pre-gustular state embryo and he uses a little lesion wound up here. And when you're wounding the embryo, you trigger cortical contractility for wound closure. And what he found is when he's doing these experiments is that he actually can trigger these cells. You see that? There's one cell running through here and there's another one over there, right? So by simply up-regulating cortical contractility through this wounding, he can actually induce these cells within the embryo. He can also, and that's another experiment where he used a constitutive active version of row A, just up-regulating row A activity and those optimizing contractility in these cells, he can actually transform cells within the embryo. I think that's shown in this movie here. There you have one of these cells which is over-expressing constitutive active row A. The cell speed of these cells is much faster than any other cell within the embryo. Again, it's a bit slower than what we see in culture but it goes up to 10 micrometer per minute. And if you look at myosin true localization, of course, it's harder to see within the embryo but you see backflow of myosin in this case here which looks similar to what you see in culture where you have this consistent, very fast backflow of active and myosin in these microteins cells. Okay, so we know that these cells exist in the embryo and the obvious question is in which sort of environment could they actually be physiologically relevant? What is their function? The short answer is we don't know what their function is in the embryo but they have different possibilities how they could function. One possibility would be if you do a wounding where you actually injure the embryo, what you usually get is a wound closure. You get a contraction of cells and a very high density of cells. In one way you can reduce this high local and this local high density of cells would be dissipating cells out of the wounding area. So one function could be that you use this very fast migratory mode by getting rid of cells out of the wounding area and redistributing them in the embryo. Another sort of interesting observation came by talking to cancer cell viologists and they told us when they looked at our movies that they looked strikingly similar to metastatic cells leaving primary tumors. And they were very excited about it and they're still very excited about it is what it is when we did one, one to see if we can mimic a primary tumor in the embryo but it is if you have x-plant and you're pressing on the x-plant treating it with LPN we can occasionally see cells leaving at the margin of these x-plants and actually showing the stable blood migration by simply having a large aggregate of cells treating them with high contactility and then at the edge you can transform cells into these stable blood cells. So one possibility might be and that's something other people are looking into now is that in the primary tumor you trigger local octomizing contactility which leads to some of these cells transforming it to these stable blep cells leaving the primary tumor migrating as single cells and being metastatic. Okay, so what I want to conclude on that is that for gender cells in culture can be transformed into another part of migratory amniotic cell type that uses the stable blood-like contusions. This transformation is independent of the primary cell fate. Doesn't matter if they're muted in the end or exoderm and the migration mode before if they are bleeding or mesenchymal and the amniotic cell of migratory in any other cell in the embryo and this depends strictly on spatial confinement and it's independent of specific attrition. And that's it.