Control of Cell Growth in Animal Development
Transcript of Part 2: Cell Number Control
00:00:03.09 So we've talked about control of cell size and cell growth, and using Schwann cells, 00:00:10.17 the myelinating glial cells of the peripheral nervous system, to study the problem, 00:00:14.27 and now I'm going to switch gears and talk about the other aspect of growth control, 00:00:20.19 which is cell number control. And I'm going to switch from talking about the 00:00:26.19 myelinating glial cell of the peripheral nervous system, the Schwann cells, and talk about 00:00:30.14 the myelinating glial cells of the central nervous system, the oligodendrocytes, 00:00:34.05 and in particular, the precursor cells that give rise to them. 00:00:39.01 As I said, cell number control is much more important than cell size control in 00:00:47.14 determining the size of an organ or an animal, at least when dealing with mammals, as I said, 00:00:54.01 humans are about 3,000 times the size of a mouse and they have about 3,000 times more cells. 00:00:59.02 So what one really needs to understand, is how do you control cell number, and 00:01:04.14 there is two processes that are important here, one is cell division, which creates more cells, 00:01:10.00 and the other is cell death, that gets rid of cells. 00:01:13.02 I'm going to start by talking about cell death, but first let me introduce the oligodendrocyte and its precursors. 00:01:24.01 So oligodendrocytes make myelin in the central nervous system and they derive from precursor cells, 00:01:31.17 appropriately called oligodendrocyte precursor cells, or OPCs, 00:01:37.00 and the oligodendrocyte precursors divide rapidly then they stop dividing 00:01:42.00 and differentiate into oligodendrocytes which then myelinate axons. 00:01:47.02 We study these cells in the optic nerve, and we do that because the optic nerve is 00:01:53.22 one of the simplest bits of the mammalian central nervous system, which is enormously complex, 00:01:58.05 and it's simple because it doesn't contain any nerve cells. 00:02:02.01 It contains the axons shown here in blue from the retinal ganglion cells 00:02:07.23 that project from the eye back to the brain carrying visual information. 00:02:12.01 But in the nerve itself there are no nerve cells but there are glial or supporting cells: 00:02:17.19 there are astrocytes and there are oligodendrocytes. 00:02:20.01 The astrocytes have many functions, many of which are not known, 00:02:24.12 the oligodendrocytes mainly myelinate axons in the nerve and as I said they derive from dividing oligodendrocytes precursors, 00:02:32.14 or OPCs, and these cells in the optic nerve migrate into the nerve 00:02:37.06 early in development. They divide a limited number of times, and then they stop and terminally differentiate. 00:02:44.13 They don't divide again once they differentiate, and they myelinate axons. 00:02:48.02 So the number of oligodendrocytes in the optic nerve of a rat, of an adult rat, is about 350,000 00:02:59.05 and we want to know why. How is that number determined, why isn't it 100,000, why isn't it 500,000. 00:03:07.00 From what I've already told you, you will see that that number is going to depend on at least three things. 00:03:13.00 One is, how many precursors migrate into the nerve during development, 00:03:17.22 and we don't know that number, but I suspect its small. 00:03:20.01 Secondly, how much cell death occurs in the lineage, either in the precursors or in the oligodendrocytes, 00:03:26.07 and third, how many times the precursors divide once they are in the nerve 00:03:30.03 before they stop, differentiate, and start myelinating axons. 00:03:36.01 So I'm going to start by talking about the contribution of cell death to controlling cell number, 00:03:43.00 so this is the control of cell survival and death of oligodendrocytes, and you'll see it makes a major contribution 00:03:49.26 to getting the numbers of oligodendrocytes in the adult nerve right. 00:03:54.02 So here's an experiment done by a graduate student, Ian Hart, many years ago. 00:04:00.01 People in the United States had argued, that the oligodendrocyte precursor is 00:04:05.26 induced to differentiate into an oligodendrocyte by IGF-1, our old friend, Insulin-like Growth Factor-1, 00:04:12.03 because they found in culture, it increased the number of oligodendrocytes that developed. 00:04:17.02 So Ian, we had a different view, our view was if you remove the mitogen for oligodendrocytes precursors, 00:04:24.26 which is mainly platelet derived growth factor PDGF, that the cells 00:04:28.24 automatically differentiated into oligodendrocytes by default. 00:04:32.02 So to try to address this controversy, Ian took single oligodendrocyte precursors, 00:04:39.12 put them in a microwell, either without any signaling molecules or with IGF-1 00:04:45.29 or supernatants of cultures of its neighboring cells such as astrocytes. 00:04:50.01 What he found was, when there were no signals at all, the cell very quickly died. It died within a day, 00:04:57.10 and the morphology of the death was apoptosis, which was already known 00:05:02.24 to be a suicide program that the cell can activate to kill itself neatly and quickly. 00:05:07.02 Whereas when IGF-1 was present with a single cell, that was sufficient to allow the 00:05:13.24 cell to survive and not undergo apoptosis and signals from neighbors including IGF-1, 00:05:19.10 but others could also promote the survival of these cells. 00:05:23.02 And so we asked ourselves, why should it be, so we by the way concluded that IGF-1 isn't a differentiation 00:05:32.16 inducer in this system, it's simply allowing the cell to survive so that it can differentiate. 00:05:38.01 But the question is, why should the oligodendrocyte precursor die when there are no signals 00:05:44.02 there because we're giving it the nutrients and the vitamins and all the other things that cells need to live, 00:05:49.06 and so we propose that maybe all cells in an animal depend on 00:05:55.26 signals from other cells in order to keep this suicide apoptotic program off, 00:06:02.02 and this was a so-called "death by default" mechanism. 00:06:07.01 Now this would have advantages in building a complex animal because it would 00:06:11.11 ensure that any cell that didn't develop appropriately on the right time schedule or ended up in the wrong place, 00:06:18.07 would automatically kill itself because it wouldn't get the signals it needs to survive. 00:06:22.28 So it could be a very useful way of ensuring that cells only survive where and when they're needed. 00:06:29.02 But in addition, it provides a very powerful mechanism for regulating cell number. 00:06:34.21 As had already been shown in the nervous system, in the so-called neurotrophic hypothesis. 00:06:40.03 There it was shown years earlier that neurons are overproduced, many classes of neurons are overproduced, 00:06:50.09 they send out their axons, there long processes, contact the target cells 00:06:55.04 that they are going to innervate, there they compete for limiting amounts of the survival signal, the neurotrophic factors 00:07:02.00 released by the target cells only about half of them get enough to survive, and the other half kill themselves 00:07:08.26 by undergoing apoptosis and that is a very powerful mechanism 00:07:13.15 of adjusting the number of nerve cells to the number of target cells they innervate. 00:07:18.00 To carry this kind of hypothesis through to oligodendrocyte development, 00:07:24.09 Ben Barres, who was a post-doctoral fellow at the time, now in his own laboratory at Stanford, 00:07:29.29 showed that during normal development in the rat optic nerve, at least 50% of the newly formed oligodendrocytes 00:07:37.29 died by apoptosis, they killed themselves. So that led him to propose 00:07:42.11 this model that oligodendrocyte precursor cells divide rapidly, they stop dividing, 00:07:49.15 they differentiate into oligodendrocytes and at this point, the survival signals 00:07:54.16 that the newly formed oligodendrocytes need changes. 00:07:58.02 They now need to find an axon in order to live, and about half of those cells find an axon and live, 00:08:05.13 and myelinate the axon, the other half fail to find an axon and kill themselves, 00:08:10.19 and that would be a way of matching the number of oligodendrocytes to the number 00:08:15.01 and length of axons that needed myelination. So Ben and others then tested this hypothesis and 00:08:21.13 overwhelming evidence was rapidly provided that this was actually the way it works in 00:08:27.03 controlling oligodendrocyte survival and controlling their number. 00:08:30.02 Ben first showed that if you cut the optic nerve just behind the eye, 00:08:35.06 the axons distally along the nerve very rapidly degenerate in a day or two and what Ben showed is that 00:08:41.27 virtually all the oligodendrocytes then undergo apoptosis, they kill themselves, 00:08:46.21 consistent with the idea that they need a signal from axons to avoid apoptosis. 00:08:51.03 Then Julia Burne when she was a graduate student in the lab, made use of a transgenic mouse 00:08:57.05 that Jean-Claude Martinou had made which expresses a Bcl-2 gene, 00:09:03.08 which is anti-apoptotic, it produces a protein that blocks apoptosis, in neurons in the retina, and because retinal neurons 00:09:12.18 die during neuronal development, when you overexpress this transgene that death is markedly decreased 00:09:18.01 and you end up with double the number of axons in the optic nerve. 00:09:21.01 As a result, there's double the amount of survival signal, fewer oligodendrocytes die, and their number increases 00:09:28.29 to match the increased number of axons. You could see why evolution would have chosen to do it this way. 00:09:35.06 Overproduce the cells and then death sculpts the number down to the number that are appropriate. 00:09:41.02 Bill Richardson, a colleague at University College, showed that if you overexpress platelet derived growth factor 00:09:48.15 in the optic nerve, then you greatly stimulate the proliferation in the oligo precursors, 00:09:53.14 and you get many more oligodendrocytes produced than normal. 00:09:57.00 But because the number of axons hasn't changed, all those extra oligodendrocytes kill themselves, 00:10:02.25 and you end up with a normal number of oligodendrocytes as you would predict 00:10:06.14 from the model that Barres had proposed. Charles Ffrench-Constant who was a post-doc at the time, 00:10:13.10 showed in his own laboratory in Cambridge that if you decrease oligodendrocyte 00:10:19.11 production in a way that I'm not going to describe, then again, 00:10:23.26 fewer oligodendrocytes die because there is less competition for the survival signals that the axon provides, 00:10:33.01 and so the number again adjusts to be normal. So what is the survival signal, 00:10:40.19 or what are the survival signals that the axon provides? Pierre-Alain Fernandez, 00:10:47.26 who was then a postdoctoral fellow, provided strong evidence that our old friend glial growth factor, 00:10:53.26 the neuregulin, is an important survival signal that the axon provides for the newly formed axon. 00:11:01.01 First he showed as shown here that when you take oligodendrocytes in culture without signals, 00:11:06.11 they very quickly undergo apoptosis, whereas if you add glial growth factor, you promote their survival potently. 00:11:14.02 Then he showed that neurons from the dorsal root ganglion, a sensory ganglion beside the spinal cord, 00:11:22.08 will also promote the survival of oligodendrocytes in the absence of other signals. 00:11:27.03 And if you add the dorsal root ganglion neurons and additionally add antibody against glial growth factor, 00:11:35.07 that decreases the survival promoting effect of the axons, suggestion the axons 00:11:42.00 are promoting survival of oligodendrocytes through GGF. 00:11:46.02 And finally, if you add GGF with the antibody, which neutralizes the antibody, 00:11:51.15 you bring back the survival promoting effects to the normal level. 00:11:55.00 The critical experiments that Pierre-Alain did was to show that neuregulin, 00:12:00.11 the glial growth factor, operates in the animal as well to promote survival of oligodendrocytes. 00:12:07.01 So here what he's done is he takes a cell line COS cells, a monkey cell line, 00:12:14.00 and puts in various genes that enables those cells to secrete various types of signaling proteins. 00:12:21.01 So if he takes COS cells and he transfects them with a gene that encodes glial growth factor 00:12:28.17 and then transplants those COS cells into the brain of a developing rat, then you see that the apoptosis of 00:12:37.11 oligodendrocytes is greatly decreased so that adding exogenous GGF 00:12:42.15 to a developing rat brain, an optic nerve, decreases the normal death of oligodendrocytes in the optic nerve. 00:12:51.02 Then in this critical experiment he shows that if you transfect the COS cells 00:12:56.26 with a construct that encodes a receptor for glial growth factor 00:13:02.01 fused to the Fc region of an immunoglobulin molecule, and this molecule is secreted by 00:13:07.08 the COS cells and now transfer those cells into the brain of a developing rat, then you greatly increase apoptosis. 00:13:15.01 So you are neutralizing the endogenous glial growth factor 00:13:19.04 presumably provided by the axons and now death of oligodendrocytes go up. 00:13:23.02 So all of that together strongly suggests that the axons in the optic nerve are promoting survival 00:13:32.06 in newly formed oligodendrocytes at least in part by glial growth factor on the surface of the axon where it is known to be. 00:13:41.02 So now I want to move from cell survival and death control as a process that helps control 00:13:49.09 the number of oligodendrocytes in the developing optic nerve, to proliferation control. 00:13:54.03 And again, looking at the optic nerve here, I remind you that early in development the oligodendrocyte 00:14:05.10 precursors, the OPCs, migrate into the nerve, they divide rapidly 00:14:10.05 a limited number of times and then they stop and differentiate into oligodendrocytes. 00:14:16.00 The number of divisions is going to greatly influence the number of oligodendrocytes that develop. 00:14:23.00 So the question we addressed is why do oligodendrocyte precursors stop dividing and differentiate when they do? 00:14:31.01 And I should say that most of the cells in your body work in that way, they are developed from precursor cells 00:14:38.07 that divide a limited number of times, and then they stop and differentiate, 00:14:42.26 that is true for the skin, for the gut, for most cells in the brain and the muscle and most blood cells and so on. 00:14:49.00 Yet there isn't a single case where we understand why the cells stop dividing and differentiate 00:14:55.03 when they do, and we wanted to try to address that question for the oligodendrocyte precursor. 00:15:01.02 So the first thing we did was to get this dropping out of division and differentiation to occur 00:15:09.16 in a dissociated cell culture of the optic nerve. So if you look at the optic nerve, in blue 00:15:15.10 you see the development of oligodendrocytes in the intact rat optic nerve. 00:15:20.00 So the precursors migrate into the nerve a few days before birth, they divide there, 00:15:25.27 the first precursors stop dividing and become oligodendrocytes on the day of birth, 00:15:30.20 which is embryonic day 21 around in the rat, and new precursors drop out of division and differentiate 00:15:37.27 for the next six weeks postnatally in the optic nerve of a rat. 00:15:41.17 If you take the cells out of the optic nerve three days before birth, dissociate them, put them in culture 00:15:48.05 in the presence of serum, these cells will divide. The first oligodendrocyte precursor stop dividing 00:15:54.27 and differentiate after 3 days, the equivalent of the day of birth, and new ones will do so for the next weeks in culture. 00:16:01.01 So we've now confined to a culture dish, the process that we want to study. That is 00:16:06.26 when the cells stop dividing and differentiate seems to happen just as it does in the nerve in this culture dish. 00:16:14.01 This is a pretty complicated culture. There is about six or seven cell types 00:16:18.16 in the optic nerve and in this culture there is fetal calf serum present. 00:16:23.01 So a big step forward was taken by Ben Barres as I said earlier now in his own lab in Stanford, 00:16:32.03 he purified the oligodendrocyte precursors from the rat optic nerve. We had antibodies that distinguished 00:16:38.27 the precursors from the differentiated oligodendrocytes and from the astrocytes and so using 00:16:43.09 sequentially immunopanning he was able to purify the oligodendrocyte precursors to homogeneity. 00:16:50.02 And he showed that you take these precursors, put them in culture 00:16:53.23 with the major mitogen PDGF, platelet derived growth factor, 00:16:58.09 that Bill Richardson and Mark Noble had previously shown as the main mitogen for the oligodendrocyte precursors. 00:17:04.02 If you do that, these cells will divide in culture, they will migrate in culture 00:17:09.07 as they do in the animal, and they will stop dividing and differentiate after a limited number of divisions. 00:17:15.02 So in this relatively simple culture system the cells seem to behave in the way they do in the animal. 00:17:22.02 And here is what the cells look like in a time-lapse video recording. So you can see that the 00:17:29.10 cells are migrating, these are purified oligodendrocyte precursors in PDGF, they are migrating, 00:17:34.25 they are dividing, and here is a cell that has stopped dividing, stopped migrating, 00:17:38.02 becoming an oligodendrocyte. Here's a cell that's doing the same, stops migrating, stops dividing, 00:17:43.25 and becoming an oligo, and after seven or eight days these cells will have all stop dividing and differentiate. 00:17:50.01 And the reason the cells do it at different times even though they come from the same optic nerve, 00:17:55.04 we think is because they are at different stages of maturation. 00:17:59.02 Cause if you go earlier, if this is a post-natal day seven optic nerve, where these cells were isolated, 00:18:04.05 if you go to the embryo then the cells go through more divisions before they stop and differentiate 00:18:09.11 so it seems likely that this different number of divisions depends on the maturation of the cells and that video 00:18:15.12 was taken by Nathalie Billon when she was a post-doctoral fellow, now in her own laboratory in Nice in France. 00:18:22.00 Okay, so the next step forward was taken by Fen-Biao Gao at the Gladstone Institute in San Francisco, 00:18:30.21 where he purified the oligodendrocyte precursors using 00:18:34.15 the sequential immunopanning procedure that Barres had developed 00:18:37.03 from embryonic day 18 rat optic nerve. Now that was no mean feat because these cells are less than 0.1% 00:18:44.16 of the cells in the nerve, but he was able to do it. And if you culture those cells in PDGF 00:18:49.04 without serum, now those cells will behave just as they do in the nerve. 00:18:53.24 They will divide, the first cells will stop dividing and differentiating at the equivalent of the day of birth, 00:18:58.02 and new cells will do so for the next days or weeks in culture. So that critical experiment says that the 00:19:08.06 timing of when the cells stop dividing and differentiate is either built into the population of the cells, 00:19:14.09 because during this experiment the environment is kept constant, there is no other cell types present, 00:19:20.10 so it is either built into the population of precursors or its built in the individual cells. 00:19:25.01 The evidence that it might be built in individual cells came earlier from a graduate student Sally Temple, 00:19:32.24 who is now in her own laboratory in Albany in New York. What she did was to micromanipulate a single oligodendrocyte precursor 00:19:40.15 and put it on a monolayer of astrocytes in a microwell. The astrocytes make 00:19:46.06 platelet derived growth factor and the survival signals that these cells need to survive 00:19:50.28 and so the cell will now divide and differentiate and form a clone. 00:19:56.09 What she found is that in blue are the number of oligodendrocyte precursors 00:20:02.21 within this typical clone, and the cell in this case divides 00:20:06.21 four times to give eight cells, and at this point, 00:20:09.28 well it gives 16 cells rather, and at this point all of the progeny of this single cell 00:20:15.28 stopped dividing and differentiate to form oligodendrocytes at the same time. 00:20:21.18 So that strongly argued that the cell has some built in mechanism to in fact control when it stops dividing. 00:20:30.06 But Sally actually proved that in this way. She put the astrocytes in the side of the microwell, 00:20:36.06 put a single precursor on the base, let the cell divide once and then transfered 00:20:41.25 the sibling cells to two separate astrocyte monolayers, and showed that if one sibling divided three times 00:20:49.17 for three days and then stopped so the other sibling tended to do as well. 00:20:55.01 So that established beyond any doubt that built into each individual precursor cell is a mechanism 00:21:02.09 that’s determining or helping to determine when the cell stops dividing and differentiate. 00:21:09.01 Could it be that the cell is simply counting divisions. In Sally's experiment using postnatal day seven optic 00:21:15.16 nerve precursors, the maximum number of divisions she saw was eight. Is it possible that the cells are 00:21:21.18 just counting and when they get to eight they just stop and differentiate. 00:21:24.01 Here's another experiment done by Fen-Biao Gao that suggests that isn't the way this thing is working. 00:21:30.09 He dropped the temperature in the culture dish to 33 degrees and compared it to cells cultured at the usual 37 degrees. 00:21:39.01 Now this is an artifact, in an animal, in a mammal, the temperature never gets to 33 degrees if the animal is alive. 00:21:45.01 However, as you'd expect, at the low temperature the cell cycle time is increased. 00:21:52.22 The cell cycle is slow, that's what you'd expect at the low temperature. 00:21:56.01 What you might not have expected is that at the low temperature the cells stop dividing, 00:22:01.03 and differentiate sooner after fewer divisions at the lower temperature so you dissociated cell division 00:22:09.13 counting and timing when start dividing and differentiate, and so we now call this thing an intracellular timer, 00:22:16.01 because it seems to measure time in some way that doesn't depend on counting cell divisions. 00:22:22.02 Although this timer is built into the individual precursor cell, it doesn't act autonomously. 00:22:31.02 Cells in animals never act autonomously, every process in the cell, almost, is regulated by signals 00:22:39.20 from other cells, and that is true too of this timer. As Ben Barres showed when he was a post-doc, 00:22:45.27 first if you take PDGF out of this culture of purified precursors, the cells immediately stop dividing and differentiate. 00:22:55.01 So for the timer to work normally, you need PDGF to keep the cells dividing. 00:23:01.01 If there is no thyroid hormone here, then the cells just continue to divide, they don't stop dividing 00:23:09.03 and differentiate when they normally would. But if you let the cells divide in PDGF without thyroid 00:23:14.11 hormone for eight days and now add back thyroid, now the cells very quickly stop dividing and differentiate, 00:23:24.05 and if the cells are in PDGF and thyroid hormone together, then they stop after seven or eight days and differentiate. 00:23:32.00 Some doing it earlier, some doing it later. So the simple interpretation of this experiment is that there is 00:23:40.00 an intracellular timer, the timer itself does not depend on thyroid hormone, but when the timer says 00:23:47.18 now, stop dividing and differentiating, if there is no thyroid hormone there the cells continue to 00:23:52.06 proliferate and don't differentiate. So the timer has at least two components, something that is 00:23:57.05 keeping time independent of thyroid hormone and an effector mechanism that depends on 00:24:02.05 thyroid hormone that allows the cell to stop and differentiate. So now we wanted to understand, what's 00:24:09.07 the molecular mechanism of this intracellular timer, and it turns out that it's quite complex. 00:24:16.02 I'm going to give you two examples of proteins that change in their concentration in the cell as the precursors 00:24:24.23 are proliferating that make sense and are components of the timer. One of these is the P27 member 00:24:34.29 of the CIP/KIP family of cyclin-dependent kinase inhibitors. These proteins act as a brake on 00:24:43.16 the cyclin E/cyclin A Cdk2 complexes that are important for driving the cell through G1 and into S phase. 00:24:51.02 So if it's part of the timer you would expect it to increase as the cells proliferate to help take the cells 00:24:58.04 out of division after a certain period of time. And that is indeed what happens, and that was shown by 00:25:04.13 Bea Durand when she was a post-doctoral fellow, now in her own laboratory at the Pasteur Institute in Paris. 00:25:10.02 So here's she's taken purified oligodendrocyte precursors from the post-natal day seven 00:25:15.13 rat optic nerve, cultured them in, in this case, without thyroid hormone, but in the presence of PDGF. 00:25:24.02 So these cells in the course of this experiment are going to continue to divide, 00:25:28.20 they are not going to stop and differentiate because there is no thyroid hormone, but if you look at what happens 00:25:33.15 to the level of P27 in the nucleus, asses quantitatively by 00:25:38.03 confocal fluorescent microscopy, early on the levels are low, late on the levels are high. 00:25:45.02 If you plot the levels in the population they start off low, increase, and then plateau right around the time 00:25:52.00 the cells would have stopped dividing, so that's exactly the behavior you'd expect if P27 00:25:57.09 was playing a role in helping to take the cells out of division. And I should say that these large bars 00:26:03.26 here are not error of standard deviations, they are the extremes within the population. 00:26:08.23 And remember, that at this point there's a heterogeneity of cells in terms of how long they will divide for 00:26:14.17 before they differentiate. Some will stop now, other will go for another four, five, six days. 00:26:19.24 If you look at a clone of cells, then the level of P27 is the same in all the cells in the clone. 00:26:27.01 Here is one explanation for why the timer works faster at the low temperature. 00:26:37.26 If you look at the level of the P27 protein in oligodendrocyte precursors dividing in culture at 33 degrees, 00:26:47.00 when the timer ran faster, P27 levels rise faster. So presumably this is one reason why the timer is accelerated 00:26:57.10 at the low temperature. It's probably not the only reason, but it's one reason. So here's a gain of function experiment 00:27:03.25 to ask if you overexpress P27 in purified oligodendrocyte precursors proliferating in culture, 00:27:10.27 in the presence of both in this case PDGF and thyroid hormone, what happens to the timing of when 00:27:19.19 the cells stop and differentiate. The answer is if you use a retrovirus to express either green 00:27:28.04 fluorescent protein or green fluorescent protein and P27, you find that green fluorescent protein 00:27:36.03 has no effect on the time. The cells will divide, stop dividing, and differentiate, most of them 00:27:40.24 within seven to eight days. But if you overexpress P27 so that the levels rise artificially fast, 00:27:47.26 now you accelerate the timer and cells stop dividing and differentiate sooner. That's what you'd 00:27:54.20 expect if P27 was part of this timer. Here's a loss of function experiment, Bea Durand collaborated 00:28:02.19 with one of the three labs that knocked out the gene encoding P27 in the mouse, 00:28:09.16 and this was Jim Roberts in Seattle. And then she got the mouse from Jim Roberts, isolated the optic nerve cells, 00:28:17.26 cultured them in the presence of PDGF and thyroid hormone so that the timer should operate normally, 00:28:25.01 and then counted the number of oligodendrocytes in clones to ask how many divisions 00:28:31.00 do the cells go through before they stopped and differentiated. And what they found was, what she found, 00:28:36.15 was in the wildtype animal the maximum number of divisions was six. Now interestingly in rats of the same age 00:28:44.02 that number was eight. And whether one reason a rat is bigger than a mouse is that precursors go 00:28:51.14 through more divisions before they stop could well be one reason the rat is bigger than the mouse in this case. 00:28:57.02 But the important experiment's this one, where you have only one copy of P27 gene or no copies, 00:29:05.10 then some of the cells proliferate for longer, and you get extra divisions before the cells stop dividing. 00:29:11.02 Cell cycle time is unchanged, the experiment argues strongly, in fact these experiments taken together 00:29:17.18 argue strongly, that P27 is a component of the timer. It's a minor component because when you take it away, 00:29:24.21 the cells still stop and differentiate, but they don't do so accurately. 00:29:29.01 They go through another division or two abnormally before they stop and differentiate. 00:29:34.01 Now the generality here is that P27 knockout mice that have no P27 at all, are 30% larger than wildtype 00:29:43.19 normal mice, and the reason they are larger is that they have more cells in every organ that's been looked at. 00:29:48.02 And they have more cells because there is extra proliferation, not because there is less cell death. 00:29:53.24 So it seemed likely that P27 plays a role in helping to take cells out of division at the right time 00:30:00.19 in many cell lineages, not just the oligodendrocyte lineage. 00:30:04.01 Even more generally, there are P27 homologs in both C. elegans worms and in Drosophila. 00:30:13.02 One gene in each of those organisms, and when you inactivate those genes you get an extra division 00:30:20.03 or two in multiple lineages, both in the worm and in the fly. So it argues that CDK inhibitor proteins 00:30:26.14 such as P27 probably play a role in timing when cells come out of division in all animals. 00:30:33.02 Now to identify a component, a protein component of the timer, doesn't actually tell you how the timer 00:30:42.20 works. You need to know why P27 protein accumulates over time as the oligodendrocyte precursors proliferate. 00:30:51.02 Well we don't know why it accumulates but we do know that the mechanisms are post-transcriptional 00:30:57.05 thanks to the work of Yasu Tokumoto when he was a post-doctoral fellow, now back in Tokyo. 00:31:03.03 He showed using RT-PCR, or real time PCR, that as the protein in OPCs increases over time as they 00:31:13.23 proliferate, messenger RNA levels for P27 remain flat. So whatever the mechanisms are, they are 00:31:22.18 post-transcriptional. Recently, a talented post-doctoral fellow in Ben Barres lab at Stanford, Jason Dugan, 00:31:32.02 has identified P57, which is another member of the CIP/KIP family of CDK inhibitors 00:31:38.00 as an important component of the timer. He's shown, just like in the case of P27, 00:31:42.23 it increases over time and plateaus, and in this case he's shown that the messenger RNA 00:31:48.01 and the protein increase together, arguing that its likely 00:31:52.29 transcriptional control. So I now want to turn to a second or third protein 00:31:58.25 that works in the opposite way of the CDK inhibitors. These are the inhibitors of differentiation 00:32:07.00 proteins. The so-called Id proteins. These proteins block the basic helix-loop-helix proteins that are 00:32:17.08 known to be required for differentiation in most lineages. They bind to them, block their activity, 00:32:23.15 inhibit differentiation and thereby promote proliferation. So if the Id proteins are going to play a part 00:32:30.29 in this timer, their concentration should fall over time as these cells proliferate. 00:32:36.02 That is indeed what happens as was shown by Toru Kondo, a very talented post-doctoral fellow 00:32:42.21 now in his own laboratory in Kobe, Japan. So he showed that four Id proteins, or at least messenger RNAs, 00:32:52.08 are expressed in oligodendrocyte precursors but only one of them decreases over time 00:32:57.24 as these cells proliferate, suggesting its a component of the timer. And in this case, both the 00:33:04.06 messenger RNA and the protein fall progressively both in vitro as shown here and in vivo 00:33:12.03 message and protein fall progressively as the precursors divide. Here's a gain of function experiment 00:33:20.07 where Toru's used a retrovirus to put Id-4 protein into oligodendrocyte precursors 00:33:28.10 together with green fluorescent protein. And now he induces differentiation by removing platelet derived 00:33:34.16 growth factor and shows that the cells that are transfected and overexpressed Id-4 fail to drop out of 00:33:41.21 division and do not differentiate, whereas overexpressing Id-1 in these cells with GFP doesn't have this effect. 00:33:49.01 So it's some specificity for Id-4 over the other Id proteins, and the effect here is quantified in this graph. 00:33:57.10 So overexpression of Id-4 blocks differentiation and promotes proliferation. 00:34:03.01 Finally here's a loss of function experiment where he's collaborated with Fred Zablitsky in Nottingham who 00:34:09.26 has made an Id-4 knockout mouse. Toru has taken neural stem cells from the brains of these knocked 00:34:17.29 out mice, puts them in culture with PDGF, thyroid hormone, and another signaling protein 00:34:23.07 sonic hedgehog, that’s required to get oligodendrocyte development on schedule. 00:34:28.02 In the Id-4 knockout mouse, you get oligodendrocytes days prematurely compared to the 00:34:36.15 wild-type neural stem cells, suggestion that Id-4 really is a component of the timer and helps oligodendrocyte 00:34:44.03 precursors to differentiate at the right time. So let me finish by going back to the initial question. 00:34:54.02 Why is it that we grow to be larger than mice? 00:34:59.15 I've already told you that the reason we are bigger than mice is that we have more cells than mice. 00:35:05.02 The reason we have more cells is that on average, human cells divide more before they stop than do mouse cells. 00:35:14.02 The question is, why do they divide more times before they stop? 00:35:19.18 Is it because the intracellular timers that I've been talking about that help determine how long the cells divide 00:35:27.09 before they stop are set differently, are programmed differently so that cells divide much longer 00:35:33.04 in humans before they stop because of an intrinsic timer mechanism set in this way. 00:35:38.03 Or is it that the signals, the mitogens, the growth factors that are responsible for driving the growth 00:35:46.25 and proliferation of human cells are around longer than they are in the mouse. 00:35:53.14 My guess is that both of these things will turn out to be true, but the fact is we don't know and 00:35:59.12 we don't know largely because this problem hasn't been studied in this way. 00:36:05.03 So what I have ignored is the fact that most of us don't look like mice. Not only are we larger 00:36:13.16 than mice but we don't look like mice. And the reason we don't look like mice is because these 00:36:19.21 local controls on growth, so that you get a nose and you get ears of this shape and so on, 00:36:25.14 are controlled differently in a mouse from in a human. And we know very little about the local controls 00:36:34.07 that pattern growth control such that humans have bigger heads than mice and 00:36:40.24 they don't have the same facial appearance and so on. 00:36:45.01 Anyway, this is a really important problem in development, growth control, and it needs more 00:36:51.27 people working on it. So it just remains for me to thank the people who I think I've mentioned 00:36:59.05 throughout this talk, who have made all this work possible, and I'm grateful to them, 00:37:05.18 they are an outstanding group of people without them I wouldn't be here today so thanks.