• Skip to primary navigation
  • Skip to main content
  • Skip to footer

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.

This material is based upon work supported by the National Science Foundation and the National Institute of General Medical Sciences under Grant No. 2122350 and 1 R25 GM139147. Any opinion, finding, conclusion, or recommendation expressed in these videos are solely those of the speakers and do not necessarily represent the views of the Science Communication Lab/iBiology, the National Science Foundation, the National Institutes of Health, or other Science Communication Lab funders.

© 2023 - 2006 iBiology · All content under CC BY-NC-ND 3.0 license · Privacy Policy · Terms of Use · Usage Policy
 

Power by iBiology