Hello and welcome to my first iBiology seminar. My name is Geraldine Seydoux. I'm a professor at Johns Hopkins University and an Investigator with the Howard Hughes Medical Institute. And today I'd like to discuss how embryos create body axes. So, one of the most fascinating questions in biology is, how does a single cell, the fertilized egg, create all of the different cell types that make up an adult organism? And how all these different cell types become organized along the body axis? So that cells that make up the head and cells that make up the tail end up at different ends of the embryo. So, today, I'm gonna go through some of the fundamental experiments that were used to figure out how the body axis of a simple organism, the nematode Caenorhabditis elegans, is created. So, here is C elegans. This is an adult C elegans hermaphrodite. And this short little video shows you one of the most amazing features of this organism and that is that it is transparent. As you can see, you can peer inside of this worm and see all of the different organs and all of the different cells that make up this animal. And this is true not just in the adult stage, but it's also true at all of the different developmental stages. And this is one of the reasons why Caenorhabditis elegans is a favorite experimental model system for biologists. And many important discoveries in biology were obtained during... using Caenorhabditis elegans as a model system. So, today, we're just gonna talk about experiments that were done in C elegans to understand how the C elegans embryo organizes its head and its tail at different ends of the embryo. So, here is a newly fertilized C elegans egg and you might... so, this is a one-cell stage, and you can see that there are two pronuclei. This is the maternal and paternal pronuclei. These are gonna come together to form the first nucleus of this embryo. And then you're gonna see how quickly cell division ensues to create the many cells that you need to form a worm. So, let's start the video. This video has been sped up -- the entire video would actually take 13 hours to play if we played it at its normal time -- but this is just to show you how the beginning of embryogenesis involves many cell divisions. And then, halfway through embryogenesis, an amazing thing happens, as you can see here, where the embryo starts to become asymmetric. And one end of the embryo is the head and one end of the embryo is the tail. So, how does that happen? So, here, I'm showing you umm... still images from a similar movie, again starting with the fertilized egg and ending with the larva. And here... just to show you different stages of embryogenesis. And here's, again, that stage where you first see an asymmetry, where one side of the embryo is looking different from the other side and that side, here, is the side that will form the tail of the organism. So, we want to know, how does the embryo know where to put its head and its tail? And you can see that, at this stage, there is a clear asymmetry, but maybe you'll notice that at an earlier stage we can already see an asymmetry in this embryo. And I'll just give you a few seconds to look at these pictures and see if you can spot the earliest asymmetry in this series of pictures. And yes, you're right, at this two-cell stage, a two-cell embryo, there's already an asymmetry: one cell is smaller than the other, and that's the cell I was pointing out with the pink arrow. And when C elegans embryos... we just looked at many different embryos growing under the microscope... they noticed that the smaller cell was always formed on the side of the embryo that was going to give rise to the tail. And so it became this idea that this small cell may already know that it's supposed to make posterior structures, and that the bigger cell is fated to make anterior structures. Of course, to demonstrate that, it was important to figure out the entire lineage of C elegans, to follow all of the divisions that the posterior and the anterior cells go through, to link those cells to the adult structures. So, the elucidation of the entire C elegans lineage was a collaboration between many C elegans researchers, led by John Sulston, and the fruit of their labors is shown... is this diagram, where you see the progression from the one-cell stage, at the very top, all the way to all of the cells, the 953 cells that make up the adult worm. So, in this diagram, every cell is represented by a horiz... I mean, a vertical line. And every cell division is represented by a horizontal line. And what John Sulston and his collaborators saw is that, indeed, that posterior, that small cell at the two-cell stage gives rise to posterior structures. So, the question of how the one-cell embryo generates an anterior-posterior axis can be reduced to the simple question of, how does this one-cell embryo divide asymmetrically, into two cells that are already programmed to generate different parts of the worm? Alright. So, to address that question, we need to go back and look again at this one-cell embryo and try to or... understand more about how this one-cell embryo actually came about. How does the process of fertilization happen in C elegans? And so, to do that, we are looking at a picture of, again, this adult hermaphrodite that you saw in the first movie I showed you, but this time we're looking at a still image that is focusing specifically on the gonad of this hermaphrodite. Okay? So, that's the reproductive tissues of this hermaphrodite. And you can see all of the oocytes lined up in the oviduct, and you can see that each of these um... oocytes has a hollow area. That's where the DNA, the maternal DNA, is, so that's the oocytes pronucleus. Then, you can see all the sperm. These are much smaller cells and they're all arranged in the spermatheca. And then you can see the embryos, here, with a newly fertilized egg right here, and then a two-cell embryo, and then a later-stage embryo, here. So, the organization of this gonad is such that when fertilization happens, the sperm enters the oocyte on the side of the oocyte that is opposite where the oocyte pronucleus is, okay? So, that's just the way the gonads are set up, that the sperm enters on that side. But it's interesting to note that that side also ends up being the side where the smaller cell, that cell that's going to form the posterior end of the embryo, resides. And so this led to the possibility that fertilization, maybe, is telling the egg where to form the posterior end of the embryo. So, that's one possibility. But another equally possible... possible option is that, in fact, the sperm is not involved in determining where the posterior end is, but it's the oocyte pronucleus, which is on the other side, which corresponds with where the larger cell is formed. And so maybe the oocyte already knows where the anterior end is. So, those are the two possibilities, and so to distinguish between these two possibilities you can do a simple experiment, which is to change where the sperm enters, okay? So, that's the hypothesis. You know, maybe the sperm induces the posterior end. And, to decide whether that's true or whether the alternative hypothesis, the oocyte pronucleus is determining where the end... the anterior end is, we can do an experiment where we change the position of sperm entry. What would happen if the sperm entered on the same side as the oocyte pronucleus? Would that side become the posterior, as I'm showing here? Or would nothing change? You would still have the anterior on the side of the... the maternal pronucleus. So, this is a simple experiment, theoretically. But actually, in practice, this was a really hard experiment to do, to force the oocyte... the sperm to come in on the wrong side. But this is exactly what Bob Goldstein did. He was able to manipulate the site of sperm entry. And in this figure, here, you can see the result of his experiments. And what he found, to his delight, is that when the sperm comes in on the wrong side, on the side of the oocyte pronucleus, what happens is that the embryo polarity is reversed. Now, you have the smaller cell forming on the side that the sperm entered, which happened to be the same side that the oocyte nucleus was. And this very simple experiment had really a profound implications, because it told us that the oocyte really is like a blank canvas. It doesn't know where the anterior and posterior ends of the embryo are going to arise. The sperm is calling the shots and defining where the posterior end is gonna be. Okay? So, now, we have to understand... well, how does this work? How can the sperm impose polarity to the egg? Okay... so... to do that, we really have to try to understand what are the molecules that exist in this one-cell embryo that are responding... sensing the sperm entry and responding and creating an asymmetry in this one cell, so that it can divide asymmetrically to give two cells with two different fates? So, to identify the molecules involved in this process, the best method is to use genetics. And this is the approach that was taken by two C elegans investigators, Jim Priess and Ken Kemphues. They did genetic screens to look for mutant C elegans that produce embryos that do not polarize properly. These are embryos that divide symmetrically and that die because they can't put their head cells and their tail cells in the right place. So, they did these genetic screens and here are some examples of the mutants that they found. So again, up here, you see the wild-type, normal C elegans embryo that divides asymmetrically. And these are some of the examples of the mutants that they recovered in these genetic screens. And you might appreciate that some of these mutants... they have really symmetric first divisions, where the two cells are almost exactly the same size. So, they call these mutants par mutants, for partitioning-defective, thinking that these genes might be involved in somehow partitioning molecules to create an asymmetry in the one-cell embryo. So, Ken Kemphues went on to clone these genes and identified the molecules that are produced by these genes, the proteins that were produced by these genes. And he also created reagents to see where these proteins are in the one-cell embryo. And, there, he got a really amazing result. This is what he found, that the different PAR proteins sort themselves out in the one-cell stage to the different poles of the embryo. Some of the PAR proteins, depicted in red here, go to the anterior side, and some of the other PAR proteins, in green here, go to the posterior side. So, these PAR proteins somehow know where to go and know how to create different domains in the one-cell embryo. Even more amazingly, these PAR proteins, which Ken Kemphues first cloned from C elegans, turn out to have homologs in all eukaryotic, or most eukaryotic organisms, including man. And we now know that these PAR proteins regulate the polarization of many different cell types in our bodies. But let's go back to what these PAR proteins are actually doing in the one-cell C elegans embryo. So, what do these proteins look like? So, these are little schematics, just to show you the different domains that exist in these proteins. So, again, we can separate the proteins into two groups: the ones that go to the anterior sides, in blue; and the ones that go to the posterior side, in... in... in the pink group. And one of the interesting findings that Ken Kemphues made when he identified all of these proteins is the realization that, in each group, there is a very special type of protein. There is a kinase. So, this is the PKC-3 kinase and the PAR-1 kinase. A kinase is a protein that can phosphorylate other proteins and, as you'll see in a minute, these are very important proteins. Also, we see other proteins in each group that can bind lipids. They have lipid binding domains. And these domains are important to place the PAR proteins at the membrane. That's why these proteins are enriched at the membranes, is because they can bind lipids. Alright. So, another important observation that was made by Ken Kemphues and his colleagues is how these proteins depend on each other for localizing to these different anterior and posterior domains. The way they discovered this is by looking at what happens when you get rid, for example, of the posterior PARs. What happens to the anterior PARs? Well, what happens is that, now, the anterior PARs go all over the membrane and all over the embryo. They just spread everywhere. And the same thing happens when you get rid of the anterior PARs. Now, the posterior PARs go everywhere. So, these experiments said that there's some kind of competition between the anterior and the posterior PARs. They're competing for access to the membrane. And this... subsequently, biochemical experiments suggested the molecular mechanism that underlies this competition. It turns out that this is all dependent on the kinases. So, the PKC-3 kinase phosphorylates both PAR-1 and PAR-2. And, when PAR-1 and PAR-2 are phosphorylated, they can't bind to the membrane anymore. Okay? And then the PAR-1 kinase does the same thing on PAR-3. And when PAR-3 is phosphorylated, it can't access the membrane. And now its partners, PAR-6 and PKC-3, also cannot access the membrane. At the very beginning, when the egg is not polarized yet, the anterior PARs have... are at the membrane, all over the membrane. And they are keeping the posterior PARs off of the membrane by phosphorylating PAR-2 and kicking it off the membrane. So, that's the situation before polarization. And so the question is, well, when the sperm comes in, what happens to help the posterior PARs get on the membrane? So, this question was a question that was addressed by my very first graduate student, Matt Wallenfang, and what Matt decided to do is to look at the different structures that are brought in at fertilization and to see how these structures correlate with the formation of the PAR-2, this posterior domain. And what Matt found is that the... when the sperm fertilizes the egg, it brings along a centrosome, which is a structure that nucleates microtubules. And these microtubules are pictured in red, here. And he saw a very nice spatial correlation between this microtubule aster -- so, this burst of microtubules -- and the formation of the PAR-2 domain. So, this was a correlation. He went one step further and was able to block the formation of this sperm aster and showed that, now, PAR-2 could not get on the membrane. So, that was another clue that maybe there was a cause-and-effect there. But, really, a key insight came from a different experiment, where Matt created mutant embryos that arrest at an earlier stage, before the sperm has a chance to make this microtubule aster. This is a stage where the embryos are... have been fertilized, but the sperm is still dormant, the sperm pronucleus and the sperm centrosome is dormant. And during this time, the oocyte pronucleus is undergoing the meiotic divisions. And to do the meiotic division, the oocyte pronucleus has to elaborate a meiotic spindle, which is also a microtubule-rich structure. And what Matt saw, when he arrested the embryos and forced them to stay at this stage, is that, now, PAR-2 went on the side of the oocyte pronucleus, right where the meiotic spindle was being formed. And so this really suggested to us that it's really not a question of sperm versus oocyte. It's all about microtubules. Whichever structure can form a nice rich microtubule area, that's the place where PAR-2 will go. Now, in a wild-type embryo, the meiotic spindle is just a transient structure and PAR-2 goes there for a little while but doesn't stay there, because there's the much bigger sperm asters that form and stay for longer. So, the sperm wins because it's making a stable microtubule aster. Alright. So, microtubules seem to be the key feature here. Okay... so, are microtubules actually the posterior determinant? And so this was a... a... a question and we wondered, well, how could microtubules help PAR-2 get on the membrane? And this question was taken on by another member of my laboratory. This is Fumio Motegi when he did his postdoctoral work in my lab. He asked, what is the connection between the PAR proteins and the microtubules? And he found that one of the PAR proteins, PAR-2, the important posterior PAR protein, actually loves microtubules. It binds to microtubules. So, this is an experiment where Fumio mixed some GFP-tagged PAR-2, so that you can see PAR-2 in green, here, with some rhodamine-labeled microtubules. So, this is an in vitro experiments, so all you have is microtubules that are labeled in red and PAR-2 labeled in green. And you can see that PAR-2 decorates the microtubules. So, that tells us that PAR-2 likes to bind to microtubules. Okay... that was interesting. But then Fumio made an even more interesting observation, which had to do with how PAR-2 gets phosphorylated by PKC-3. So, remember, PAR-2 is being phosphorylated by this anterior kinase that prevents PAR-2 from getting on the membrane. So, here's an experiment where Fumio mixed PAR-2 protein with the PKC-3 kinase in vitro, in a test tube, and then he monitors... using radioactivity, the dark signal that you see here... he monitors how PAR-2 gets... is getting phosphorylated by PKC-3. And you can see that, over time, PAR-2 gets more and more phosphorylated by the kinase. Okay. So, that was what we knew. PAR-2 is phosphorylated by PKC-3. But now, watch what happens when, in that reaction, Fumio adds microtubules. So, here's the same reaction, but this time with microtubules. Now you can see that PAR-2 is not phosphorylated as efficiently. Somehow, the microtubules are protecting PAR-2 from phosphorylation by PKC-3. So, that was an interesting observation. So, next, Fumio repeated this experiment, but with a version of PAR-2 that cannot bind to microtubules very well. So, he made mutations in PAR-2 to create a version of PAR-2 that cannot bind microtubules, just by mutating three amino acids. And what he found is that, now, this version can be phosphorylated by PKC-3 very efficiently, even if there are microtubules around. So, this really suggested that, by binding to microtubules, PAR-2 is getting protection from PKC-3. Another experiment that Fumio did is that he took this mutant and put it back into the worm. And what he found is that this PAR-2... defective version of PAR-2 that can't bind microtubules now cannot form a PAR-2 domain. So, putting all of these results together brings us to this very simple model for how the sperm can help PAR-2 get on the membrane. We think that the sperm brings this microtubule-organizing center. And, when all of these microtubules polymerize, they help PAR-2 become protected from PKC-3. Now PAR-2 is not phosphorylated as efficiently by PKC-3 and now PAR-2 can bind to the membrane. It can recruit its partner, PAR-1. Now, remember, PAR-1 is a kinase and PAR-1 phosphorylates PAR-3. When that happens, PAR-3 falls off of the membrane, taking with it its partners, PAR-6 and PKC-3, and that allows more PAR-2 and PAR-1 to get on the membrane. So, this is a simple model for how you can get PAR-2 to at least get on to the membrane that is right next to the microtubules. Okay? So, the sperm brings in microtubules, that helps PAR-2, and PAR-2 recruits PAR-1, and PAR-1 excludes the other PARs. But it turns out that this is not the whole story. You'll notice that, as I showed you, the PAR-2 domain ends up being very large -- it occupies the whole half of... the whole posterior half of the zygote. So, how does it get so big? And, in fact, by the time that we get these really big PAR domains, there are microtubules everywhere. So, how does it really happen? There had to be something else going on. And that something else was figured out by Ed Munro. And what Ed Munro did his he became interested in looking at the actomyosin cytoskeleton that is right underneath the membrane. And here's a little movie of his observation of the actin-myosin cytoskeleton and how this very active cytoskeleton changes upon the formation of the sperm asters. And so, in pink, this little pink dot, here, tells you where the sperm pronucleus is. And watch what happens to the myosin, which is labeled in white in this little movie... watch what happens to it as the polarization process proceeds. You can see that the myosin is getting pushed to one side of the embryo. And what Ed Munro showed is that this actomyosin flow actually carries the anterior PARs with it and helps push them to the anterior side of the embryo. So, this explains why the posterior guys have a chance to occupy a large area, and the anterior PARs are constrained to stay in the anterior side of the embryo. Now, we don't really understand yet how the sperm creates these actomyosin flows. We know the sperm and the sperm aster is somehow involved, but it's not really clear how that happens. So, that is a mystery that remains. So, basically, what I've told you is that the sperm is using two mechanisms to polarize the embryo. One mechanism is a simple mechanism, where the microtubules protect PAR-2 from phosphorylation. And then there is this other more mysterious mechanism, where you have this really remarkable remodeling of the actomyosin cytoskeleton, that flows towards the interior, taking with it the anterior PARs. So, just to summarize what we've learned from these experiments, going back to our original question... what polarizes the embryo? What gives the embryo this body axis that tells it where to put anterior cells and posterior cells? Well, we know that the sperm is responsible. But it's not fertilization per se that is important. It's the organiz... the bringing in of a microtubule-organizing center. And then this structure can protect PAR-2 from phosphorylation and create this posterior domain. And then, at the same time, can create these flows to help propagate this anterior-posterior axis throughout the whole embryo. So, that's how you get the PAR domains. But that's only part of the story. Now, you need to then distribute different molecules in the cytoplasm of the egg, so that some molecules will go to the anterior cell and some molecules will go to the posterior cell. And the question is, how do these PAR domains actually influence what's going on in the cytoplasm to segregate molecules to the two different daughter cells? That is going to be the topic that I will address in the next presentation. Thank you very much for your attention.
