How to Create a Body Axis
Transcript of Part 1: From Egg to Worm: How to Create a Body Axis
00:00:12.28 Hello and welcome to my first iBiology seminar. 00:00:17.00 My name is Geraldine Seydoux. 00:00:18.17 I'm a professor at Johns Hopkins University and 00:00:21.26 an Investigator with the Howard Hughes Medical Institute. 00:00:24.25 And today I'd like to discuss how embryos create body axes. 00:00:30.12 So, one of the most fascinating questions in biology is, how does a single cell, 00:00:37.14 the fertilized egg, create all of the different cell types that make up an adult organism? 00:00:45.06 And how all these different cell types become organized along the body axis? 00:00:50.12 So that cells that make up the head and cells that make up the tail end up at different 00:00:56.01 ends of the embryo. 00:00:57.16 So, today, I'm gonna go through some of the fundamental experiments that were used to 00:01:04.03 figure out how the body axis of a simple organism, the nematode Caenorhabditis elegans, is created. 00:01:13.05 So, here is C elegans. 00:01:15.27 This is an adult C elegans hermaphrodite. 00:01:19.24 And this short little video shows you one of the most amazing features of this organism 00:01:26.12 and that is that it is transparent. 00:01:28.19 As you can see, you can peer inside of this worm and see all of the different organs and 00:01:36.23 all of the different cells that make up this animal. 00:01:41.01 And this is true not just in the adult stage, but it's also true at all of the different 00:01:46.02 developmental stages. 00:01:47.23 And this is one of the reasons why Caenorhabditis elegans is a favorite experimental model system 00:01:53.16 for biologists. 00:01:55.01 And many important discoveries in biology were obtained during... 00:02:00.12 using Caenorhabditis elegans as a model system. 00:02:03.18 So, today, we're just gonna talk about experiments that were done in C elegans to understand 00:02:09.26 how the C elegans embryo organizes its head and its tail at different ends of the embryo. 00:02:16.06 So, here is a newly fertilized C elegans egg and you might... so, this is a one-cell stage, 00:02:24.24 and you can see that there are two pronuclei. 00:02:29.02 This is the maternal and paternal pronuclei. 00:02:32.15 These are gonna come together to form the first nucleus of this embryo. 00:02:37.13 And then you're gonna see how quickly cell division ensues to create the many cells that 00:02:44.01 you need to form a worm. 00:02:46.05 So, let's start the video. 00:02:48.04 This video has been sped up -- the entire video would actually take 13 hours to play 00:02:54.02 if we played it at its normal time -- but this is just to show you how the 00:02:59.00 beginning of embryogenesis involves many cell divisions. 00:03:03.01 And then, halfway through embryogenesis, an amazing thing happens, as you can see here, 00:03:08.19 where the embryo starts to become asymmetric. 00:03:12.08 And one end of the embryo is the head and one end of the embryo is the tail. 00:03:16.13 So, how does that happen? 00:03:18.16 So, here, I'm showing you umm... still images from a similar movie, again starting with 00:03:25.16 the fertilized egg and ending with the larva. 00:03:30.18 And here... just to show you different stages of embryogenesis. 00:03:35.18 And here's, again, that stage where you first see an asymmetry, where one side of the embryo 00:03:42.10 is looking different from the other side and that side, here, is the side that will form 00:03:49.02 the tail of the organism. 00:03:51.01 So, we want to know, how does the embryo know where to put its head and its tail? 00:03:56.13 And you can see that, at this stage, there is a clear asymmetry, but maybe you'll notice 00:04:01.10 that at an earlier stage we can already see an asymmetry in this embryo. 00:04:07.05 And I'll just give you a few seconds to look at these pictures and see if you can spot 00:04:13.08 the earliest asymmetry in this series of pictures. 00:04:18.15 And yes, you're right, at this two-cell stage, a two-cell embryo, there's already an asymmetry: 00:04:24.27 one cell is smaller than the other, and that's the cell I was pointing out with the pink arrow. 00:04:31.19 And when C elegans embryos... we just looked at many different embryos growing under the microscope... 00:04:41.20 they noticed that the smaller cell was always formed on the side of the embryo 00:04:47.11 that was going to give rise to the tail. 00:04:49.25 And so it became this idea that this small cell may already know that it's supposed to 00:04:56.13 make posterior structures, and that the bigger cell is fated to make anterior structures. 00:05:03.04 Of course, to demonstrate that, it was important to figure out the entire lineage of C elegans, 00:05:11.14 to follow all of the divisions that the posterior and the anterior cells go through, 00:05:17.11 to link those cells to the adult structures. 00:05:20.25 So, the elucidation of the entire C elegans lineage was a collaboration between many 00:05:29.00 C elegans researchers, led by John Sulston, and the fruit of their labors is shown... 00:05:36.15 is this diagram, where you see the progression from the one-cell stage, at the very top, 00:05:45.18 all the way to all of the cells, the 953 cells that make up the adult worm. 00:05:51.20 So, in this diagram, every cell is represented by a horiz... 00:05:56.26 I mean, a vertical line. 00:05:59.08 And every cell division is represented by a horizontal line. 00:06:03.25 And what John Sulston and his collaborators saw is that, indeed, that posterior, 00:06:10.05 that small cell at the two-cell stage gives rise to posterior structures. 00:06:15.22 So, the question of how the one-cell embryo generates an anterior-posterior axis can be 00:06:24.05 reduced to the simple question of, how does this one-cell embryo divide asymmetrically, 00:06:30.01 into two cells that are already programmed to generate different parts of the worm? 00:06:36.18 Alright. 00:06:37.18 So, to address that question, we need to go back and look again at this one-cell embryo 00:06:43.24 and try to or... understand more about how this one-cell embryo actually came about. 00:06:49.21 How does the process of fertilization happen in C elegans? 00:06:54.07 And so, to do that, we are looking at a picture of, again, this adult hermaphrodite that 00:07:00.24 you saw in the first movie I showed you, but this time we're looking at a still image that is 00:07:07.02 focusing specifically on the gonad of this hermaphrodite. 00:07:11.07 Okay? 00:07:12.07 So, that's the reproductive tissues of this hermaphrodite. 00:07:15.23 And you can see all of the oocytes lined up in the oviduct, and you can see that each 00:07:21.04 of these um... oocytes has a hollow area. 00:07:26.01 That's where the DNA, the maternal DNA, is, so that's the oocytes pronucleus. 00:07:31.03 Then, you can see all the sperm. 00:07:34.24 These are much smaller cells and they're all arranged in the spermatheca. 00:07:40.15 And then you can see the embryos, here, with a newly fertilized egg right here, and then 00:07:46.10 a two-cell embryo, and then a later-stage embryo, here. 00:07:51.24 So, the organization of this gonad is such that when fertilization happens, the sperm 00:07:59.28 enters the oocyte on the side of the oocyte that is opposite where the oocyte pronucleus is, okay? 00:08:08.20 So, that's just the way the gonads are set up, that the sperm enters on that side. 00:08:14.15 But it's interesting to note that that side also ends up being the side where the 00:08:20.27 smaller cell, that cell that's going to form the posterior end of the embryo, resides. 00:08:27.19 And so this led to the possibility that fertilization, maybe, is telling the egg where to form the 00:08:37.26 posterior end of the embryo. 00:08:41.04 So, that's one possibility. 00:08:43.20 But another equally possible... possible option is that, in fact, the sperm is not involved 00:08:50.01 in determining where the posterior end is, but it's the oocyte pronucleus, which is on 00:08:55.17 the other side, which corresponds with where the larger cell is formed. 00:09:01.22 And so maybe the oocyte already knows where the anterior end is. 00:09:07.04 So, those are the two possibilities, and so to distinguish between these two possibilities 00:09:12.23 you can do a simple experiment, which is to change where the sperm enters, okay? 00:09:18.23 So, that's the hypothesis. 00:09:20.21 You know, maybe the sperm induces the posterior end. 00:09:24.10 And, to decide whether that's true or whether the alternative hypothesis, the oocyte pronucleus 00:09:31.15 is determining where the end... the anterior end is, we can do an experiment where we change 00:09:36.17 the position of sperm entry. 00:09:39.06 What would happen if the sperm entered on the same side as the oocyte pronucleus? 00:09:44.23 Would that side become the posterior, as I'm showing here? 00:09:48.26 Or would nothing change? 00:09:50.13 You would still have the anterior on the side of the... the maternal pronucleus. 00:09:55.25 So, this is a simple experiment, theoretically. 00:09:59.14 But actually, in practice, this was a really hard experiment to do, to force the oocyte... 00:10:05.13 the sperm to come in on the wrong side. 00:10:08.20 But this is exactly what Bob Goldstein did. 00:10:12.05 He was able to manipulate the site of sperm entry. 00:10:17.06 And in this figure, here, you can see the result of his experiments. 00:10:22.13 And what he found, to his delight, is that when the sperm comes in on the wrong side, 00:10:28.28 on the side of the oocyte pronucleus, what happens is that the embryo polarity is reversed. 00:10:36.13 Now, you have the smaller cell forming on the side that the sperm entered, which happened 00:10:42.10 to be the same side that the oocyte nucleus was. 00:10:45.24 And this very simple experiment had really a profound implications, because it told us 00:10:51.27 that the oocyte really is like a blank canvas. 00:10:55.20 It doesn't know where the anterior and posterior ends of the embryo are going to arise. 00:11:01.04 The sperm is calling the shots and defining where the posterior end is gonna be. 00:11:08.12 Okay? 00:11:09.22 So, now, we have to understand... well, how does this work? 00:11:12.14 How can the sperm impose polarity to the egg? 00:11:17.25 Okay... so... to do that, we really have to try to understand what are the molecules that 00:11:24.13 exist in this one-cell embryo that are responding... sensing the sperm entry and responding 00:11:31.03 and creating an asymmetry in this one cell, so that it can divide asymmetrically to give 00:11:37.22 two cells with two different fates? 00:11:39.14 So, to identify the molecules involved in this process, the best method is to use genetics. 00:11:46.21 And this is the approach that was taken by two C elegans investigators, 00:11:51.18 Jim Priess and Ken Kemphues. 00:11:54.01 They did genetic screens to look for mutant C elegans that produce embryos that do not 00:12:02.17 polarize properly. 00:12:03.27 These are embryos that divide symmetrically and that die because they can't put their 00:12:09.00 head cells and their tail cells in the right place. 00:12:12.04 So, they did these genetic screens and here are some examples of the mutants that they found. 00:12:18.00 So again, up here, you see the wild-type, normal C elegans embryo that divides asymmetrically. 00:12:24.05 And these are some of the examples of the mutants that they recovered in 00:12:28.24 these genetic screens. 00:12:30.23 And you might appreciate that some of these mutants... they have really symmetric 00:12:36.15 first divisions, where the two cells are almost exactly the same size. 00:12:41.01 So, they call these mutants par mutants, for partitioning-defective, thinking that these 00:12:48.15 genes might be involved in somehow partitioning molecules to create 00:12:54.07 an asymmetry in the one-cell embryo. 00:12:56.22 So, Ken Kemphues went on to clone these genes and identified the molecules that are produced 00:13:04.10 by these genes, the proteins that were produced by these genes. 00:13:07.16 And he also created reagents to see where these proteins are in the one-cell embryo. 00:13:14.06 And, there, he got a really amazing result. 00:13:19.13 This is what he found, that the different PAR proteins sort themselves out in the 00:13:26.08 one-cell stage to the different poles of the embryo. 00:13:30.09 Some of the PAR proteins, depicted in red here, go to the anterior side, and some of 00:13:37.17 the other PAR proteins, in green here, go to the posterior side. 00:13:41.21 So, these PAR proteins somehow know where to go and know how to create different domains 00:13:49.04 in the one-cell embryo. 00:13:51.13 Even more amazingly, these PAR proteins, which Ken Kemphues first cloned from C elegans, 00:13:57.22 turn out to have homologs in all eukaryotic, or most eukaryotic organisms, including man. 00:14:05.19 And we now know that these PAR proteins regulate the polarization of many different cell types 00:14:12.09 in our bodies. 00:14:14.05 But let's go back to what these PAR proteins are actually doing 00:14:17.11 in the one-cell C elegans embryo. 00:14:20.08 So, what do these proteins look like? 00:14:22.25 So, these are little schematics, just to show you the different domains that exist in these proteins. 00:14:29.01 So, again, we can separate the proteins into two groups: the ones that go to the anterior sides, in blue; 00:14:36.05 and the ones that go to the posterior side, in... in... in the pink group. 00:14:41.14 And one of the interesting findings that Ken Kemphues made when he identified all of these 00:14:46.16 proteins is the realization that, in each group, there is a very special type of protein. 00:14:52.11 There is a kinase. 00:14:53.11 So, this is the PKC-3 kinase and the PAR-1 kinase. 00:14:58.00 A kinase is a protein that can phosphorylate other proteins and, as you'll see in a minute, 00:15:03.13 these are very important proteins. 00:15:07.15 Also, we see other proteins in each group that can bind lipids. 00:15:12.11 They have lipid binding domains. 00:15:15.02 And these domains are important to place the PAR proteins at the membrane. 00:15:21.00 That's why these proteins are enriched at the membranes, is because they can bind lipids. 00:15:25.16 Alright. 00:15:26.16 So, another important observation that was made by Ken Kemphues and his colleagues is 00:15:32.27 how these proteins depend on each other for localizing to these different anterior and 00:15:39.07 posterior domains. 00:15:41.00 The way they discovered this is by looking at what happens when you get rid, for example, 00:15:46.02 of the posterior PARs. 00:15:47.25 What happens to the anterior PARs? 00:15:49.15 Well, what happens is that, now, the anterior PARs go all over the membrane and all over 00:15:56.23 the embryo. 00:15:57.23 They just spread everywhere. 00:15:59.23 And the same thing happens when you get rid of the anterior PARs. 00:16:02.18 Now, the posterior PARs go everywhere. 00:16:05.05 So, these experiments said that there's some kind of competition between the anterior and 00:16:11.09 the posterior PARs. 00:16:12.22 They're competing for access to the membrane. 00:16:17.08 And this... subsequently, biochemical experiments suggested the molecular mechanism that underlies 00:16:26.02 this competition. 00:16:27.05 It turns out that this is all dependent on the kinases. 00:16:30.11 So, the PKC-3 kinase phosphorylates both PAR-1 and PAR-2. 00:16:36.20 And, when PAR-1 and PAR-2 are phosphorylated, they can't bind to the membrane anymore. 00:16:42.15 Okay? 00:16:43.20 And then the PAR-1 kinase does the same thing on PAR-3. 00:16:47.17 And when PAR-3 is phosphorylated, it can't access the membrane. 00:16:51.14 And now its partners, PAR-6 and PKC-3, also cannot access the membrane. 00:16:57.28 At the very beginning, when the egg is not polarized yet, the anterior PARs have... 00:17:06.20 are at the membrane, all over the membrane. 00:17:09.17 And they are keeping the posterior PARs off of the membrane by phosphorylating PAR-2 and 00:17:15.26 kicking it off the membrane. 00:17:17.15 So, that's the situation before polarization. 00:17:20.18 And so the question is, well, when the sperm comes in, what happens to help 00:17:28.11 the posterior PARs get on the membrane? 00:17:30.16 So, this question was a question that was addressed by my very first graduate student, 00:17:38.11 Matt Wallenfang, and what Matt decided to do is to look at the different structures 00:17:45.07 that are brought in at fertilization and to see how these structures correlate with the 00:17:51.26 formation of the PAR-2, this posterior domain. 00:17:56.16 And what Matt found is that the... when the sperm fertilizes the egg, it brings along 00:18:03.12 a centrosome, which is a structure that nucleates microtubules. 00:18:10.07 And these microtubules are pictured in red, here. 00:18:13.18 And he saw a very nice spatial correlation between this microtubule aster 00:18:19.24 -- so, this burst of microtubules -- and the formation of the PAR-2 domain. 00:18:26.11 So, this was a correlation. 00:18:29.07 He went one step further and was able to block the formation of this sperm aster and 00:18:35.06 showed that, now, PAR-2 could not get on the membrane. 00:18:37.26 So, that was another clue that maybe there was a cause-and-effect there. 00:18:42.05 But, really, a key insight came from a different experiment, where Matt created mutant embryos 00:18:52.06 that arrest at an earlier stage, before the sperm has a chance to make this microtubule aster. 00:19:02.12 This is a stage where the embryos are... have been fertilized, but the sperm is still dormant, 00:19:09.20 the sperm pronucleus and the sperm centrosome is dormant. 00:19:13.20 And during this time, the oocyte pronucleus is undergoing the meiotic divisions. 00:19:19.17 And to do the meiotic division, the oocyte pronucleus has to elaborate a meiotic spindle, 00:19:27.04 which is also a microtubule-rich structure. 00:19:33.12 And what Matt saw, when he arrested the embryos and forced them to stay at this stage, 00:19:41.11 is that, now, PAR-2 went on the side of the oocyte pronucleus, right where the meiotic spindle 00:19:48.01 was being formed. 00:19:50.03 And so this really suggested to us that it's really not a question of sperm versus oocyte. 00:19:55.25 It's all about microtubules. 00:19:58.00 Whichever structure can form a nice rich microtubule area, that's the place where PAR-2 will go. 00:20:06.16 Now, in a wild-type embryo, the meiotic spindle is just a transient structure and PAR-2 00:20:12.08 goes there for a little while but doesn't stay there, because there's the much bigger sperm asters 00:20:17.03 that form and stay for longer. 00:20:19.08 So, the sperm wins because it's making a stable microtubule aster. 00:20:25.10 Alright. 00:20:26.11 So, microtubules seem to be the key feature here. 00:20:32.06 Okay... so, are microtubules actually the posterior determinant? 00:20:38.03 And so this was a... a... a question and we wondered, well, how could microtubules help 00:20:45.07 PAR-2 get on the membrane? 00:20:48.27 And this question was taken on by another member of my laboratory. 00:20:53.14 This is Fumio Motegi when he did his postdoctoral work in my lab. 00:20:58.11 He asked, what is the connection between the PAR proteins and the microtubules? 00:21:03.20 And he found that one of the PAR proteins, PAR-2, the important posterior PAR protein, 00:21:11.20 actually loves microtubules. 00:21:13.20 It binds to microtubules. 00:21:14.28 So, this is an experiment where Fumio mixed some GFP-tagged PAR-2, so that you can see 00:21:21.16 PAR-2 in green, here, with some rhodamine-labeled microtubules. 00:21:26.11 So, this is an in vitro experiments, so all you have is microtubules that are 00:21:32.14 labeled in red and PAR-2 labeled in green. 00:21:35.15 And you can see that PAR-2 decorates the microtubules. 00:21:38.08 So, that tells us that PAR-2 likes to bind to microtubules. 00:21:42.04 Okay... that was interesting. 00:21:44.06 But then Fumio made an even more interesting observation, which had to do with 00:21:50.17 how PAR-2 gets phosphorylated by PKC-3. 00:21:54.02 So, remember, PAR-2 is being phosphorylated by this anterior kinase that prevents PAR-2 00:22:00.28 from getting on the membrane. 00:22:02.14 So, here's an experiment where Fumio mixed PAR-2 protein with the PKC-3 kinase in vitro, 00:22:11.22 in a test tube, and then he monitors... using radioactivity, the dark signal that you see here... 00:22:17.22 he monitors how PAR-2 gets... is getting phosphorylated by PKC-3. 00:22:22.15 And you can see that, over time, PAR-2 gets more and more phosphorylated by the kinase. 00:22:28.05 Okay. 00:22:29.05 So, that was what we knew. 00:22:31.25 PAR-2 is phosphorylated by PKC-3. 00:22:34.11 But now, watch what happens when, in that reaction, Fumio adds microtubules. 00:22:40.10 So, here's the same reaction, but this time with microtubules. 00:22:44.25 Now you can see that PAR-2 is not phosphorylated as efficiently. 00:22:48.20 Somehow, the microtubules are protecting PAR-2 from phosphorylation by PKC-3. 00:22:56.08 So, that was an interesting observation. 00:22:58.10 So, next, Fumio repeated this experiment, but with a version of PAR-2 that cannot 00:23:05.16 bind to microtubules very well. 00:23:07.19 So, he made mutations in PAR-2 to create a version of PAR-2 that cannot bind microtubules, 00:23:14.13 just by mutating three amino acids. 00:23:17.28 And what he found is that, now, this version can be phosphorylated by PKC-3 very efficiently, 00:23:25.24 even if there are microtubules around. 00:23:29.03 So, this really suggested that, by binding to microtubules, PAR-2 is getting protection 00:23:36.06 from PKC-3. 00:23:39.06 Another experiment that Fumio did is that he took this mutant and put it back into the worm. 00:23:45.05 And what he found is that this PAR-2... defective version of PAR-2 that can't bind microtubules 00:23:51.03 now cannot form a PAR-2 domain. 00:23:54.08 So, putting all of these results together brings us to this very simple model for how 00:24:01.04 the sperm can help PAR-2 get on the membrane. 00:24:05.28 We think that the sperm brings this microtubule-organizing center. 00:24:11.22 And, when all of these microtubules polymerize, they help PAR-2 become protected from PKC-3. 00:24:20.19 Now PAR-2 is not phosphorylated as efficiently by PKC-3 and now PAR-2 can bind to the membrane. 00:24:28.16 It can recruit its partner, PAR-1. 00:24:31.21 Now, remember, PAR-1 is a kinase and PAR-1 phosphorylates PAR-3. 00:24:38.08 When that happens, PAR-3 falls off of the membrane, taking with it its partners, 00:24:45.00 PAR-6 and PKC-3, and that allows more PAR-2 and PAR-1 to get on the membrane. 00:24:51.15 So, this is a simple model for how you can get PAR-2 to at least get on to the membrane 00:25:00.25 that is right next to the microtubules. 00:25:03.09 Okay? 00:25:04.04 So, the sperm brings in microtubules, that helps PAR-2, and PAR-2 recruits PAR-1, 00:25:10.17 and PAR-1 excludes the other PARs. 00:25:13.26 But it turns out that this is not the whole story. 00:25:16.18 You'll notice that, as I showed you, the PAR-2 domain ends up being very large -- it occupies 00:25:24.20 the whole half of... the whole posterior half of the zygote. 00:25:28.12 So, how does it get so big? 00:25:30.02 And, in fact, by the time that we get these really big PAR domains, there are microtubules everywhere. 00:25:37.02 So, how does it really happen? 00:25:39.11 There had to be something else going on. 00:25:41.28 And that something else was figured out by Ed Munro. 00:25:46.14 And what Ed Munro did his he became interested in looking at the actomyosin cytoskeleton 00:25:53.00 that is right underneath the membrane. 00:25:56.27 And here's a little movie of his observation of the actin-myosin cytoskeleton and how this 00:26:06.17 very active cytoskeleton changes upon the formation of the sperm asters. 00:26:16.02 And so, in pink, this little pink dot, here, tells you where the sperm pronucleus is. 00:26:22.14 And watch what happens to the myosin, which is labeled in white in this little movie... 00:26:29.02 watch what happens to it as the polarization process proceeds. 00:26:36.02 You can see that the myosin is getting pushed to one side of the embryo. 00:26:42.08 And what Ed Munro showed is that this actomyosin flow actually carries the anterior PARs with it 00:26:51.12 and helps push them to the anterior side of the embryo. 00:26:57.05 So, this explains why the posterior guys have a chance to occupy a large area, 00:27:05.26 and the anterior PARs are constrained to stay in the anterior side of the embryo. 00:27:13.00 Now, we don't really understand yet how the sperm creates these actomyosin flows. 00:27:20.17 We know the sperm and the sperm aster is somehow involved, but it's not really clear how that happens. 00:27:27.11 So, that is a mystery that remains. 00:27:30.19 So, basically, what I've told you is that the sperm is using two mechanisms to polarize 00:27:39.19 the embryo. 00:27:40.19 One mechanism is a simple mechanism, where the microtubules protect PAR-2 from phosphorylation. 00:27:46.04 And then there is this other more mysterious mechanism, where you have this really remarkable 00:27:52.01 remodeling of the actomyosin cytoskeleton, that flows towards the interior, 00:27:57.27 taking with it the anterior PARs. 00:28:00.05 So, just to summarize what we've learned from these experiments, going back to 00:28:06.21 our original question... 00:28:08.10 what polarizes the embryo? 00:28:09.21 What gives the embryo this body axis that tells it where to put anterior cells and posterior cells? 00:28:16.24 Well, we know that the sperm is responsible. 00:28:20.24 But it's not fertilization per se that is important. 00:28:24.25 It's the organiz... the bringing in of a microtubule-organizing center. 00:28:32.07 And then this structure can protect PAR-2 from phosphorylation and create this posterior domain. 00:28:39.12 And then, at the same time, can create these flows to help propagate this anterior-posterior axis 00:28:46.16 throughout the whole embryo. 00:28:49.02 So, that's how you get the PAR domains. 00:28:53.08 But that's only part of the story. 00:28:55.11 Now, you need to then distribute different molecules in the cytoplasm of the egg, 00:29:03.11 so that some molecules will go to the anterior cell and some molecules will go to the posterior cell. 00:29:09.25 And the question is, how do these PAR domains actually influence what's going on in the cytoplasm 00:29:16.12 to segregate molecules to the two different daughter cells? 00:29:20.13 That is going to be the topic that I will address in the next presentation. 00:29:26.13 Thank you very much for your attention.