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Planarian Regeneration

Part 1: Planaria: Scale, Proportion and Organ Regeneration

00:07.1 Hello, everyone.
00:08.2 My name is Alejandro Sánchez Alvarado.
00:10.2 I am an investigator at the Stowers Institute for Medical Research
00:14.2 and the Howard Hughes Medical Institute,
00:16.0 and for the next few minutes
00:18.0 I'm going to tell you a little bit about
00:20.1 how scale, proportion, and organ regeneration
00:23.0 can be studied by investigating and questioning
00:26.1 the biology of the planarian Schmidtea mediterranea.
00:30.1 So, why do we care about scale and proportion?
00:33.0 We care about it because we don't understand why
00:36.1 things are the size they are.
00:37.1 And there are many reasons why we don't understand that,
00:40.2 and that is because for many, many years
00:42.2 most of our interest as developmental biologists
00:46.0 has been to try to understand embryonic development.
00:48.2 We all know that animals grow to a particular size,
00:50.2 we can recognize them as such,
00:52.1 and there's a lot of fossil record indicating that
00:55.2 animals could actually get really, really, really big.
00:58.1 So, what I would like to do is give you an idea
01:00.1 of how we have traditionally thought about scale and proportion
01:03.0 in living organisms.
01:05.1 So, I would like to start by reminding you of Gulliver.
01:09.1 So, Gulliver, as you know,
01:11.2 was a giant in the land of Lilliputians,
01:13.2 and this was a novel written by Jonathan Swift
01:15.2 and in this particular novel what happened here is
01:19.2 he is now a guest in the land of the Lilliputians.
01:21.3 Of course, he's a human being, he needs to eat,
01:24.2 and therefore the Lilliputians were very concerned as to...
01:27.1 how are we going to keep this guy alive?
01:28.2 We need to figure out how much food he needs to eat?
01:30.3 When I was reading this text to my son when he was growing up,
01:34.2 I realized that Jonathan Swift was not just a novelist,
01:36.3 he was actually doing math.
01:38.1 And I'm going to show you in the next passage.
01:40.1 So here, Jonathan Swift tells us
01:43.2 about the logic of how the Lilliputians
01:46.2 arrived to how many rations of food he should eat
01:49.3 to not starve to death,
01:51.0 and I have highlighted here in yellow
01:52.3 the passages that are relevant to us.
01:54.1 So, they concluded that the emperor...
01:59.2 concluded that to feed Gulliver
02:03.1 they needed to provide him with enough food
02:05.3 to feed 1728 Lilliputians.
02:08.2 How did they come up with that number?
02:10.0 Well, the way they came up with that number
02:12.1 is by applying Euclidian geometry.
02:13.2 They took the height of his body, right there,
02:16.3 and then they calculated that it exceeded theirs,
02:20.2 the Lilliputians,
02:22.1 in the proportion of twelve to one.
02:23.2 Already by seeing twelve to one, you realized
02:26.0 we're talking about the British measuring system,
02:27.2 not the metric system,
02:29.0 but the British measuring system.
02:30.2 And then he said that they concluded,
02:32.3 from this similarity of their bodies,
02:34.2 that his body should contain at least 1728.
02:38.3 That's essentially the volume of the number of Lilliputians
02:41.2 that you would have to put inside of Gulliver to fill him up.
02:45.1 And based on that they concluded that
02:48.0 what Gulliver needed to eat was
02:50.2 12 to the cube, or 1728, food rations.
02:55.0 That's a lot of little sheep
02:57.2 and a lot of little things they have to sacrifice just for one meal, okay?
03:00.1 Just for one meal.
03:01.2 The problem is that they used Euclidean geometry for this,
03:04.2 and the way this is done is that you measure the length,
03:07.1 it scales with mass to the cube,
03:09.1 and then as a function of M to the 1/D for a 3-D object,
03:13.3 mass scales to the power of 1/3rd.
03:15.1 That's Euclidean geometry.
03:16.2 So if you take the 1/3 power of 1728,
03:20.0 that gets you 12.
03:21.1 And that's how they calculated this.
03:22.2 So, of course, in the book
03:25.1 they sacrifice all this food and all this food was ingested by Gulliver, presumably,
03:30.1 but food intake and biomass do not really follow this scaling component, alright?
03:34.3 And so they should have done a different type of calculation,
03:39.1 and why is that?
03:40.1 Because we know,
03:42.2 since 1891 by the work of Snell and later by Kleiber in 1932,
03:47.2 that metabolic rate and body mass are really best fit,
03:51.1 not by Euclidean geometry,
03:53.1 but by this particular equation referred to as
03:55.2 the allometric equation.
03:56.2 Now, this equation does not explain anything,
03:58.2 but it fits the data really, really well.
04:00.1 So, essentially what you do is you take the exponential,
04:03.1 take the log on both sides,
04:04.3 and essentially you end up with an equation
04:06.2 that you can use to calculate
04:08.2 these exponentials of metabolic rate and body mass.
04:11.2 So, Kleiber in 1932, Brody in 1934, Benedict in 1938,
04:17.2 and McMahon and others
04:19.2 independently showed that the slope (b) of the
04:22.1 mass: metabolic rate curve
04:24.0 is 0.75 or 3/4 power scaling,
04:26.2 not 1/3, but 3/4 power scaling.
04:29.0 What that means, essentially, is that
04:31.2 Gulliver should have eaten 268 rations, not 1728.
04:36.2 This guy gained weight while he was in Lilliput.
04:39.3 He must have gained weight because he was eating
04:42.0 way more food than his metabolic rate actually needed.
04:44.2 Now, of course, Jonathan Swift did not know about
04:47.1 this allometric equation
04:49.0 and therefore there was no way for him to really
04:50.3 put it into the text.
04:52.0 But why is this?
04:53.3 So, why do we need to scale at 1/3 power as opposed to 1/4 power?
05:01.1 Sorry... scale at 1/4 power instead of 1/3 power?
05:05.0 So, here's where model systems can help us
05:08.1 begin to dissect these very, very complex problems.
05:11.1 I'm going to show you right here the organism that we work with,
05:13.1 this is the planarian Schmidtea mediterranea,
05:15.0 and what I'm showing you here is an animal
05:17.0 that after feeding will grow.
05:18.3 You know, it'll grow from about 1 millimeter
05:21.1 to about 4 millimeters in size,
05:23.0 and that's what you would expect for any living organism
05:25.1 that we are familiar with.
05:28.0 Now, these animals do something really peculiar,
05:30.1 which is that when you starve them,
05:32.0 instead of getting skinnier,
05:33.3 as you would expect,
05:35.2 they'll actually degrow in size
05:37.3 all the way down to 0.5 millimeters again.
05:41.0 And they're doing this not by reducing the size of their cells,
05:44.1 but by eliminating cells.
05:46.1 So, how do you shrink without becoming,
05:49.1 you know, dysfunctional,
05:50.3 a central nervous system, the musculature, the digestive system?
05:55.3 Throughout this process of starvation and degrowth
05:58.0 that you're seeing right here,
05:59.2 these animals never display any abnormal behavior.
06:03.0 So we would like to take advantage of this particular biology
06:06.0 to see whether or not we can understand how,
06:08.1 molecularly and cellularly,
06:10.0 scale and proportion is regulated,
06:12.0 because you see as the animals grow
06:13.3 that the ratios of the sizes of the various organs,
06:16.1 in relation to the size of the animal,
06:17.3 is maintained.
06:19.3 That's what we refer to as an allometric equation or allometric ratio,
06:22.2 or allometry.
06:24.1 So, why doesn't mass scale to the 1/3rd power
06:26.0 as expected for 3-D objects?
06:27.2 This is Euclidean geometry, this is what we expect.
06:30.0 Euclidean geometry is a human invention, alright?
06:31.3 Nature doesn't care about human inventions
06:34.0 so, you know, we approximate nature
06:35.3 by coming up with this way of
06:39.1 understanding volumes and the shapes around us.
06:41.2 So, the problem is that there's no easy answer,
06:44.1 but there's a lot of investigation and research out there
06:47.2 demonstrating that many, many things
06:50.1 are actually scaling to the 1/3 power as opposed to...
06:54.2 that's not scaling to the 1/3 power,
06:56.2 but rather to 1/4 power.
06:58.1 So, for example, the diameters of tree trunks and aortas,
07:01.0 they scale to body mass (M) as 3/8.
07:04.2 Okay, that's 1/4.
07:06.1 You have also situations where cellular metabolism and heart rate...
07:09.2 M to the -1/4.
07:11.1 That's 1/4,
07:12.3 that's not Euclidean geometry.
07:14.2 Times of blood circulation, embryonic growth,
07:16.1 and lifespan
07:18.2 scale also at M to 1/4, not 1/3 but 1/4.
07:22.1 And so there are many, many such examples
07:25.0 of this type of allometric relationship being maintained,
07:28.2 and we really don't understand why
07:31.0 they're all multiples of 1/4.
07:32.3 Why is that living organisms
07:35.2 do not scale in a Euclidean fashion?
07:37.2 They're scaling allometrically,
07:39.2 as I'm showing you in this particular slide.
07:41.2 So, we would like to understand this process
07:43.1 and to understand this process we need to lay down
07:45.2 some fundamental bases to these studies.
07:47.1 So, we took advantage of the fact that our animals
07:50.2 have in their head
07:53.0 a number of cells that you can actually count.
07:55.1 You can see them right there
07:57.0 and on the other side as well, right here, okay?
07:59.0 And so we reasoned that
08:02.2 if the animals are counting cells to control their scale and proportion,
08:05.1 they will be counting these cells as well.
08:07.1 And so what we did is we measured the length of the animal,
08:10.0 just like the emperor in Lilliput would do to Gulliver,
08:12.2 and then we would count the number of cells
08:15.0 that were present in the length,
08:16.1 and then measure those cells as the animals grow and as they degrow,
08:21.0 and then see if they obey these allometric equations.
08:23.0 So we do that.
08:24.2 So, what we did essentially was that
08:27.2 we measured the length of the animal,
08:29.2 this is growth, when the animal is begin fed,
08:31.0 and this is degrowth, when the animal is being starved,
08:32.3 and then on the right, right here,
08:34.2 what you see is the cell counts per size,
08:37.1 and you can see they're all growing in a particular rate
08:41.1 and then degrowing in a particular rate as well.
08:43.2 Alright?
08:44.2 And so what turns out here is that they're adding
08:46.2 approximately two to four of these individual cells
08:49.0 per millimeter of growth.
08:50.3 The animals are counting cells. Alright?
08:53.3 That's... how do they do that?
08:55.1 That's what we need to figure out,
08:57.0 and that's how this scale and proportion is being maintained.
08:59.1 So, do planarians then obey the allometric equation?
09:02.0 Now, they're flat,
09:03.2 so maybe they're maybe not three dimensional,
09:05.0 so maybe they are more Euclidean than not, but...
09:06.3 unlikely, but we went ahead and measured this anyways,
09:09.1 and so we took the equation,
09:10.2 the allometric equation that I introduced to you earlier,
09:12.2 where y is a biological variable,
09:14.2 a is the proportionality coefficient,
09:16.2 M is the body mass
09:18.1 -- things that we can measure --
09:19.2 and b is the body-mass exponent,
09:21.1 which we can calculate from solving the equation with data.
09:23.2 And when we do that, this is what we see.
09:25.2 So, in an animal that's 2 millimeters in length
09:28.0 that weighs this much, about 16.5 micrograms,
09:31.2 we found that the number of observed cells per side
09:36.0 was about 25 per side.
09:37.3 Okay?
09:39.1 And so if we now plug these numbers into this equation right here,
09:41.2 we get a predicted number of 24.5 or 25.
09:46.0 So they are really obeying the allometric equation.
09:48.1 You can also do that by
09:51.1 measuring the size of the animals from 4, 6, and 8 millimeters,
09:55.1 and you can see that the predicted values
09:58.1 are very similar to the observed values,
10:01.2 when we actually measured the number of cells present in these animals.
10:04.1 Well, planarians are great for one particular reason,
10:07.1 which is that they regenerate.
10:08.2 So, we asked the question,
10:10.2 what is counting cells?
10:12.0 Is it the head that is counting cells
10:13.3 or is it the body that is telling the head how to count cells?
10:17.1 So, what we can do with planarians is that
10:19.2 we can cut their heads off, okay,
10:20.3 and then watch the heads regenerate.
10:22.1 But we can do something else,
10:24.1 which is cut the head off,
10:26.1 and then cut the head again, in half,
10:27.3 to produce two heads.
10:29.2 So, they are about half the size of the original head,
10:31.3 but two heads in one animal.
10:33.2 Now, the question is,
10:35.2 is each head going to have half the number of these cells
10:39.1 in relation to the size of the animal,
10:40.3 or will each head have the right number
10:43.2 for an animal of that length?
10:45.1 And when we do that experiment, this is what we see.
10:47.2 We actually see that an animal
10:49.1 that's about 2 millimeters in length
10:51.0 and it has two heads attached to it,
10:53.1 these animals now have the right number of cells
10:56.2 for an animal that's 2 millimeters,
10:58.2 as opposed to half the number per head.
11:00.3 That suggests to us that the information for scaling in these animals
11:05.2 is not being dictated by the tissue that's growing;
11:08.1 it's dictated by the pre-existing tissue.
11:10.1 So that suggests to us that we can now use this biology
11:13.3 to begin to dissect the problem of regeneration
11:16.0 and, as well, the problem of how
11:18.1 the regenerated tissue scales to the appropriate size
11:21.0 so that the animal looks normal
11:22.2 -- it doesn't look like it has a tiny little head
11:24.1 with a huge body
11:26.0 or a tiny little body with a huge head.
11:27.2 This is a very dynamic and active process.
11:33.0 So, to investigate
11:36.0 the basis of this capacity
11:38.0 to restore scale and proportion,
11:39.3 we thought that we would try to simplify the problem even further,
11:42.3 and we would like to use the pharynx,
11:44.1 which is the tube that these animals use to eat,
11:46.2 and you can see that right here
11:48.1 -- it's going to eat this piece of beef liver right here.
11:50.2 This tube is ingesting the food,
11:52.3 and what we like about this tube is that we can use this tube
11:55.1 as a paradigm or an experimental paradigm
11:57.2 to study scale, cell death, and organ regeneration.
12:01.1 I told you the animals shrink as the degrow
12:03.2 and they're doing that by eliminating cells.
12:05.1 Those cells are undergoing cell death,
12:07.1 so now we wanted to know,
12:08.2 how do these tissues scale?
12:10.1 So, the experiment that we chose to do
12:12.1 was to essentially look at
12:15.2 the tissues in the animal
12:18.3 that have cells that are not proliferating,
12:20.2 and the pharynx, the tube that they eat with...
12:23.3 there's no proliferation,
12:25.0 so that allows us to separate proliferation
12:27.2 from cells that are not proliferating.
12:28.3 So, how do you shrink or grow a tissue
12:30.3 that doesn't have proliferating cells,
12:32.2 and if there really is a way in the allometric equation,
12:35.0 it should really scale, right?
12:36.2 So, if you're going to make a tissue that's very big very small,
12:39.1 you have to kill a lot of cells.
12:40.2 And if you want to make a tissue that's very small very big,
12:42.1 you have to add more cells to it.
12:44.0 And the planarian itself, the pharynx,
12:46.2 is devoid of all of these proliferating cells,
12:48.3 which in these organisms are referred to as neoblasts,
12:51.1 which are the stem cells in these animals.
12:53.3 So we thought that...
12:55.2 let's look at the pharynx.
12:56.2 So, what does the pharynx look like?
12:57.3 The pharynx is a nice radially symmetric structure
13:00.1 that's composed of muscle,
13:02.1 it's composed of cilia,
13:03.2 it's composed of neurons,
13:05.0 and it's essentially a well-structured and autonomous system
13:08.2 that will actually seek food
13:11.0 and will allow the animal to eat and feed.
13:12.2 So, it's a complex structure,
13:13.3 so it needs to really control the numbers and types of cells
13:17.0 that come together to provide
13:19.2 this reproducible shape of the pharynx proper.
13:22.2 So, we asked the question,
13:24.2 and this is probably one of my most favorite experiments,
13:27.1 are there cells dying at any given time in the pharynx?
13:30.2 So what we did was to take the pharynx that I just showed you before,
13:33.1 or one like it,
13:34.2 and then we fixed the tissue
13:36.1 and then we sectioned it serially,
13:37.3 and under the electron microscope looked for cells
13:40.2 that looked as if they were dying,
13:42.0 so-called apoptotic cells.
13:43.2 So, I'm going to play the movie so you can see this,
13:45.1 and you'll see some white arrows
13:47.2 showing cells that have all of the characteristics of cell death.
13:50.1 So, even under normal conditions,
13:52.1 the cells of the pharynx...
13:54.1 some of them are turning over,
13:55.3 some of them are dying,
13:57.2 and still the pharynx retains its form and function,
13:59.2 so the animal can eat and the like.
14:01.2 But we can't find in this tissue
14:04.0 any type of proliferative activity.
14:05.2 So we thought, okay, well let's take advantage of the fact
14:08.1 that we can actually measure cell death
14:10.1 and visualize cell death in this tissue.
14:11.3 So, what I would like to tell you now is
14:14.2 an experiment that we designed,
14:16.1 one of my, probably, most favorite experiments we've done in the lab,
14:19.0 to test whether or not we could measure
14:22.1 tissues undergoing this change
14:24.2 that allows them to
14:27.1 acquire the appropriate scale and proportion,
14:30.0 depending on the tissue that they are attached to,
14:32.1 and so we chose to study the pharynx for that purpose.
14:36.0 So, the experiment is actually fairly simple.
14:38.2 We asked the following question:
14:40.1 what would happen to the pharynx
14:42.1 when amputations of the animal
14:44.3 will actually challenge the animal very little
14:47.2 in their scale and proportion
14:48.3 -- this would remove the very tips of the animal,
14:51.1 the cap here and the tail down here --
14:53.3 and so when we remove those,
14:55.1 the animal essentially has to regenerate
14:57.1 very little amounts of tissue
14:59.1 -- very, very few cells --
15:01.0 so we don't expect massive remodeling
15:04.0 of the pre-existing tissue
15:06.2 to allow for the regaining of the scale and proportion of the animal.
15:11.0 Group 2, on the other hand,
15:13.0 that pharynx, after the head and the tail are removed,
15:15.1 is significantly much larger than the actual tissue,
15:18.1 and it does need to shrink,
15:20.2 or this runted tissue needs to grow.
15:22.1 But all of these amputations are being done in water,
15:24.3 there's no external source of carbon,
15:26.2 so there's no other growth.
15:28.0 So the only thing that could happen to this pharynx...
15:29.3 it needs to shrink to assume and acquire
15:33.2 the appropriate size for a fragment this big.
15:35.3 So the prediction would be that
15:37.2 when we amputate and remove these fragments,
15:39.2 that we would expect to see a great deal of cell death in Group 2,
15:43.0 but very little cell death in Group 1,
15:46.1 and when we did that experiment,
15:47.2 what did we see?
15:49.0 Well, what we find is something really astonishing,
15:51.1 which is that that's exactly what happened.
15:53.1 Group 1, which is shown right here,
15:55.1 the little pieces were removed, okay,
15:58.1 and this is what that pharynx looks like.
16:00.1 There's very few apoptotic or TUNEL-positive cells,
16:04.0 which are essentially comparable to animals
16:06.3 that were not amputated.
16:08.0 So this is the normal rate of death
16:10.0 that you see in a normal tissue at any given time.
16:12.3 But Group 2, on the other hand,
16:14.2 is just lighting up like a Christmas tree, lights everywhere,
16:19.0 I mean, TUNEL-positive nuclei everywhere.
16:20.2 And you would think that
16:24.1 something this massive would result in
16:26.1 a complete loss of function,
16:27.3 complete malfunction of the pharynx proper.
16:29.2 So, what good is it to shrink the tissue
16:31.3 if it's going to lose its function?
16:33.1 The thing is that that's not what happens.
16:36.1 This tissue, even though it's falling apart,
16:38.2 it's not falling apart in a chaotic fashion.
16:41.1 It's probably falling apart in a very coordinated and organized way
16:44.2 that we don't understand
16:46.2 that allows it to remove cells out of its anatomy
16:50.0 while retaining its proper function.
16:52.2 And that's shown right here.
16:54.0 Here's a pharynx that was removed from the animal
16:57.1 and the pharynx itself is
16:59.2 probably is undergoing massive apoptosis,
17:01.3 but it is actually working.
17:03.1 It's actually seeking food,
17:05.0 you can see the little tissue that just went it...
17:07.0 it's going to come out at its other end,
17:08.2 and it continues to do this.
17:09.3 So even though it's losing cells,
17:11.2 its ability to contract,
17:14.0 its ability to sense the environment,
17:15.2 its ability to seek food,
17:16.3 they're all still there,
17:18.1 and so that suggests to us that, as a paradigm,
17:20.1 as an experimental paradigm,
17:21.3 we should be able to perturb the process
17:24.1 to allow us to identify genes that are associated
17:26.2 with the normal function of these events.
17:30.1 So, we like the pharynx
17:33.2 as a way to study this problem
17:35.2 because, as I showed you,
17:37.3 I can take the pharynx out of the animal.
17:39.0 It doesn't have any metabolic activity,
17:40.3 we know it's obeying allometric equations and proportions,
17:44.3 but now we want to know, can...
17:47.1 after we pull the pharynx,
17:48.2 can it actually grow back?
17:50.1 And when it does grow back,
17:51.3 does it grow back to the appropriate size or not?
17:53.1 And so what I would like to tell you in the next few slides
17:56.1 is an approach that we undertook in the laboratory
17:58.1 to try to identify the processes by which
18:01.0 this organ regeneration is being deployed in planarians.
18:05.1 And so for that, we took advantage of the fact that
18:08.2 you can expose these animals
18:10.3 to small amounts of sodium azide
18:12.3 and the pharynx in the animals is ejected,
18:15.2 just like the co-pilot seat of a 007 car.
18:19.1 The pharynx comes out, you know, it will decay,
18:22.2 and now the animal has the job of
18:24.3 regenerating the pharynx completely.
18:26.1 So now we can observe the whole process of regeneration
18:28.2 after the pharynx has been ejected.
18:30.1 And the way we know the pharynx has been regenerated
18:33.1 and it's actually working is that
18:35.1 we can put food in a petri dish of animals that don't have a pharynx at all,
18:38.1 and then once the pharynx regenerates
18:40.1 they'll actually seek the food and ingest it.
18:42.0 And we label the food with a dye
18:43.2 so we can see whether or not they ate,
18:44.3 and that's the screen.
18:46.1 So if we now perturb genes in...
18:48.3 the functions of genes in these animals
18:50.2 and this process is affected,
18:52.2 then what should happen is they should not be able to eat,
18:55.0 or if they eat they eat in a weird way.
18:57.1 Here's a control situation.
18:58.1 These are animals that were unperturbed,
19:00.0 the pharynx was removed,
19:01.1 essentially by 7 days they were eating again
19:03.2 and they can get, you know,
19:06.1 the nutrients and can continue to grow.
19:07.2 So it takes about 7 days for these animals
19:09.2 to regenerate a pharynx.
19:11.0 That's an incredibly fast process
19:12.1 and it also requires that new tissue
19:14.0 to be functionally integrated to the pre-existing tissue.
19:16.3 So, we tested several hundred genes
19:21.2 for their capacity to perturb this process of regeneration,
19:23.2 and what we essentially found is that
19:26.0 they fall into three categories.
19:27.1 This is what we measured:
19:28.3 the ability of the animals to take food,
19:30.3 the length of the pharynx
19:32.1 -- is it allometrically properly formed? --
19:34.2 and of course how much proliferation is taking place
19:36.2 in the tissue that surrounds the pharynx,
19:37.2 so as the new tissue, the new pharynx,
19:40.1 can be formed.
19:41.1 And we find genes that are associated with regulating the proliferating cells,
19:44.3 which are shown right here and they're shown right here.
19:47.1 You can see that there's no pharyngeal growth
19:49.1 when these genes are eliminated,
19:50.2 and as you can see the number of mitoses,
19:52.0 in some cases, are affected,
19:53.2 and in others are not.
19:54.3 We need to understand what those relationships are,
19:56.1 that even though cells are dividing
19:58.1 you still don't get a pharynx,
19:59.1 as you can see right here.
20:00.3 There's another collection of genes, in the middle,
20:02.2 that actually prevent the animals from eating effectively,
20:07.0 the pharynxes... or pharynges, sorry, that are formed,
20:10.2 are of different sizes,
20:12.1 and the proliferative activity that you see right here
20:14.0 does not really correlate quite nicely.
20:15.2 So there are situations where there is a lot of proliferation,
20:18.3 but a very small pharynx,
20:20.1 or there's a lot of proliferation and pharynx
20:22.2 and then inability to feed, etc, etc,
20:24.2 telling us that this collection of genes
20:26.2 is probably very important in maturing
20:28.2 and establishing the pharynx as a functional organ.
20:30.2 But the third group of genes,
20:32.2 which is actually very interesting...
20:34.2 these particular genes right here,
20:37.1 they actually allow the pharynx to grow,
20:40.0 the animals can eat, shown right here,
20:42.1 and you can see that proliferation is essentially not affected,
20:45.1 it's comparable to control.
20:47.0 And so what happens to these animals is that,
20:50.0 in this assay,
20:51.2 they could not eat.
20:52.2 So they form a perfectly normal pharynx,
20:54.3 but the animal doesn't know it has a pharynx,
20:56.2 so it can not go and eat,
20:58.0 so this provides us with a way,
21:00.0 an experimental way,
21:01.3 to begin to really dissect these problems.
21:03.2 How do you regenerate a specific organ,
21:05.2 how do you allow it to grow to the proper scale and proportion,
21:08.2 and then after all that is accomplished,
21:10.1 how do you allow that tissue to functionally integrate
21:12.3 to the pre-existing tissue so that the animal regains
21:15.1 all of its proper functions.
21:16.3 Well, we're going to focus for the next few minutes
21:18.2 on one particular gene,
21:19.3 because it's a deeply conserved gene across most metazoans,
21:23.0 which is this gene in the middle.
21:24.3 So, this gene actually prevented
21:27.1 the regeneration of the pharynx, significantly,
21:29.2 and you can see here that
21:32.0 they essentially don't eat at all,
21:33.1 and it does not affect proliferation per se.
21:36.1 And this gene is referred to as FoxA.
21:38.3 And this gene was identified initially in C. elegans,
21:41.3 the roundworm nematode right here,
21:43.1 that when it's eliminated
21:45.2 the entire pharynx of the animal
21:46.3 -- the way they eat, just like the pharynx of the planarian --
21:48.2 is not formed at all.
21:49.2 It's gone.
21:51.0 When you look at this gene in mammals,
21:52.2 in this case, mouse embryos,
21:54.0 it also plays a fundamental role
21:57.0 in regulating the differentiation of the endoderm,
21:59.1 the gut of these animals.
22:01.1 So, in planarians, what this gene does, essentially,
22:03.1 is that it actually prevents pharyngeal regeneration,
22:05.3 just as you would expect based on
22:08.1 what we know about other systems,
22:09.2 but what is cool about this is that
22:10.2 this is not an embryonic animal,
22:12.0 this is an adult animal.
22:13.2 So that suggests that
22:15.2 the genetic cascades that are required
22:17.1 to establish the pharynx for the first time in an embryo
22:19.3 somehow need to be reactivated and redeployed in an adult system
22:23.0 such that that tissue can be regenerated,
22:24.2 and that's the kind of thing in the lab
22:26.2 that we want to try to elucidate.
22:29.1 So, here's an example: here's the control,
22:32.0 that's the pharynx regenerating,
22:33.2 quite nice,
22:35.3 here is the neurons and the epithelium is present in there,
22:37.0 you can see them merging with the muscle,
22:38.3 a beautiful pharynx beginning to develop.
22:41.0 If you now get rid of this gene FoxA by RNAi
22:44.0 and its function is abrogated,
22:45.2 you can see essentially that there is no regeneration of the pharynx.
22:48.2 So it's an incredibly robust,
22:50.2 very penetrant effect that allows us, now,
22:53.2 to ask the question,
22:55.2 what do we need to tell a stem cell
22:58.1 to become a pharyngeal tissue?
23:00.1 And so here's where the gene is expressed,
23:02.2 so these little blue dots that you see right here
23:04.2 are the cells that are expressing this particular gene,
23:08.1 those are the cells that will eventually be...
23:10.1 we suggest...
23:11.3 that are going to be part of the pharynx proper.
23:13.2 And so when you look at the regeneration of the pharynx
23:17.1 in an animal that has not been irradiated,
23:19.1 this is a wild type control,
23:20.2 you can see the pocket where the pharynx resides,
23:22.2 it's expressing this gene, FoxA.
23:24.2 By day 1, you see accumulation of signal at the base,
23:27.1 by day 3, something that looks like a pharynx,
23:30.1 and by day 7, you essentially have something
23:32.1 that's a perfectly normal pharynx.
23:33.3 Now, we can get rid of these cells,
23:35.1 because these are the neoblasts,
23:36.3 the stem cells that are dividing right here surrounding the pharynx,
23:39.1 and now we can ask the question,
23:40.2 do we need cell proliferation for a pharynx to form?
23:43.2 And when we do that experiment,
23:46.1 elimination of the neoblasts results also
23:48.3 in elimination of the pharynx proper.
23:51.2 And that's the expression of FoxA,
23:53.1 it's essentially gone in the tissue
23:55.1 that surrounds the pharynx,
23:56.2 so we now know that we need the stem cells,
23:57.3 that this tissue is formed by proliferation of stem cells,
24:02.1 and that in the absence of those cells
24:04.1 there is no FoxA expression
24:06.2 associated with the process of regeneration.
24:09.2 So now we can ask the question,
24:11.1 is FoxA really expressed in stem cells?
24:12.3 So, here are the cells in blue,
24:14.3 here is the signal of the genes expressing...
24:17.2 of the chromosomes expressing FoxA in these cells.
24:20.3 Here's a marker of stem cells,
24:22.3 and then we can superpose the two
24:24.1 and then see whether or not the stem cells are expressing FoxA,
24:26.3 and they in fact are,
24:28.3 but if you actually amputate the tissue
24:30.2 what you see is that there is a huge increase
24:32.2 in the number of cells that are now expressing FoxA,
24:35.1 and those cells are also neoblasts.
24:37.1 So, amputation is now redirecting
24:41.2 cells that would normally give rise to other tissues
24:43.0 to now give rise to the pharynx proper.
24:45.1 So, how is that all regulated?
24:46.3 And I think that one of the ways by which this happens
24:49.0 is by the activation of this gene, FoxA, in planarians,
24:53.0 which is going to tell us a great deal about how to regenerate
24:55.2 endodermal tissues in ourselves and other organisms.
24:59.2 So, this is just a quantitation showing that
25:01.2 there is a significant increase
25:03.2 in the number of stem cells that are now expressing this marker.
25:06.1 And so we now think that organ regeneration decisions,
25:09.1 how you make a new tissue from scratch,
25:11.3 is actually taking place in the stem cells themselves,
25:14.3 as opposed to anywhere else.
25:16.2 It is the stem cells that are making a decision,
25:18.2 so to speak,
25:20.0 to become endodermally fated,
25:22.0 to give rise to the pharynx proper.
25:24.0 Okay.
25:26.1 So what happens if you get rid of FoxA?
25:28.1 If you get rid of FoxA and you don't get a pharynx...
25:30.2 I showed you earlier that it's a burst of proliferation,
25:32.3 the cells, the neoblasts are sensing
25:35.1 that there is a tissue missing,
25:37.0 so they make more and more cells,
25:39.0 but in the absence of FoxA
25:40.2 that surplus of proliferating cells
25:42.2 cannot give rise to the pharynx.
25:43.2 So what happens to those cells?
25:45.0 Do they undergo apoptosis, do they die?
25:47.1 Or do they go on to do something else?
25:48.2 Well, what we see is that
25:51.0 they actually give rise to these bumps in these animals,
25:52.2 and when we look at the tissues that are arising in these animals,
25:55.3 they are essentially tissues that are associated
25:58.1 with some aspects of the pharynx,
26:00.0 neurons, this is a neurotransmitter,
26:03.1 and of course in FoxA we see no detection of FoxA,
26:06.0 but these really abnormal growths.
26:08.1 So, what happens now is that that surplus of cells
26:11.1 don't know what to do and they differentiate primarily into neurons and muscle,
26:15.0 and they give these bumps right here
26:17.1 that don't look anything like a pharynx proper.
26:19.0 So, FoxA RNAi animals
26:21.2 really forming this spike is actually...
26:23.1 in the center of the animal,
26:24.3 is a consequence of abnormal differentiation of the neoblasts
26:29.0 to regenerate the pharynx proper.
26:31.0 So what we see now is that
26:33.1 this outgrowth is enriched for disorganized neurons and muscle,
26:36.0 as I just mentioned to you earlier.
26:38.2 So, here's a control,
26:39.3 and you can see now the affected animals
26:41.2 essentially showing that these spikes are full of neurons
26:44.1 and also full of muscle,
26:45.3 suggesting then that the entire organization
26:49.2 of all of the cells to provide
26:52.0 the structure of the pharynx
26:53.3 and to scale the pharynx properly
26:55.1 is seemingly dependent, in its majority,
26:59.1 on the expression of the gene FoxA,
27:01.0 which is really a remarkable finding
27:04.1 because it allows us, now,
27:07.0 to use that as a point of departure
27:08.3 to dissect this problem much more specifically.
27:11.2 So, this allows us, then, to set up a series of genetic cascades
27:14.1 of gene exfamilial melanomapression
27:16.1 to explain how these different cells
27:18.2 get co-opted and channeled to give rise
27:22.0 to the pharynx proper.
27:23.1 So, the genes that are expressing in Phase 1
27:24.2 are the genes that we think are required
27:26.2 for the proliferation of the neoblasts.
27:28.0 The genes that we find expressed in Phase 2,
27:30.0 these are genes that actually
27:32.1 are giving rise to committed progenitors,
27:33.2 so they give rise to the cells that will now become pharynx.
27:36.0 And then of course the genes in Phase 3
27:38.0 are the ones that take these progenitor cells
27:40.1 and differentiate them into the appropriate form and function.
27:43.1 And so that allows us then to draw this map,
27:46.0 where a single cell, a stem cell,
27:48.1 expressing these genes, okay,
27:50.1 that I showed you earlier in the process,
27:52.1 somehow come together to activate
27:54.2 this other cohort of genes
27:56.1 that will now fate the progeny of these neoblasts
27:58.2 to give rise to pharyngeal progenitors,
28:01.0 and these pharyngeal progenitors,
28:02.3 through mechanisms we don't understand,
28:04.1 come together and begin to give rise to the pharynx proper,
28:07.1 which is entirely regulated by this gene FoxA.
28:10.2 So, as you can see,
28:12.0 the complexity of the process diminishes
28:13.2 the more and more committed the cells become.
28:15.1 So we can now work our way
28:17.3 forwards and backwards in trying to understand
28:20.0 how all these structures acquired their fates,
28:22.2 how they come together,
28:24.1 and how they scale and functionally integrate
28:26.2 into the pre-existing tissue
28:28.2 to allow for full restoration of function and activity
28:31.0 in animals that were injured by the removal of their pharynges.
28:35.0 So, what's happening at the organismal level?
28:37.2 How are these decisions made?
28:39.1 What are the signals that allow stem cells
28:41.3 to produce the right type
28:43.2 and the right number of cells
28:45.0 such that the animals maintain their proper form and function?
28:48.1 And this is what we don't know,
28:49.3 but we suggest the possibility that
28:52.0 once the pharynx reaches a particular size,
28:54.0 it sends some kind of signal back to the stem cells proper
28:56.3 that prevents them from making more of this tissue,
28:59.1 so we don't end up with extra long pharynges
29:01.1 or extra short pharynges.
29:02.3 And those kinds of mechanisms are the ones
29:05.2 that we're going to have to uncover in the years ahead
29:07.2 to try to understand how the scale and proportion come together.
29:10.2 So, we don't know what the signals are,
29:13.3 and that's the fun part of this business
29:15.2 is that we can now begin to really
29:18.0 dissect this at a very high level of granularity
29:20.2 and a very detailed mechanistic description of the process.
29:25.2 So, in conclusion,
29:28.0 it is now possible to experimentally interrogate aspects
29:31.0 of scale and proportion
29:32.2 using planarians as a model system,
29:33.3 and how these organisms
29:36.0 regulate these processes to allow
29:39.2 the animals to assume and acquire their appropriate
29:41.2 allometric proportions.
29:43.1 Secondly, the genes that are associated
29:45.2 with specific organ systems
29:47.2 and their regeneration can be identified
29:49.1 and we can dissect them.
29:50.2 So, we can no longer just describe,
29:52.1 we can actually perturb and extract from that information
29:54.3 a mechanistic understanding
29:57.2 of how these processes unfold in time and space.
30:00.2 And then, finally, that planarians
30:03.2 really now open a door for us
30:05.2 to do a systematic analysis of how regenerated organs
30:08.3 scale and are functionally integrated
30:10.2 to pre-existing tissues.
30:12.1 And so I hope that this was informative to you and...
30:17.0 thank you very much.

Part 2: Planarian Stem Cells

00:07.1 Hello everyone.
00:08.2 My name is Alejandro Sánchez Alvarado.
00:10.0 I am an investigator with the Stowers Institute
00:12.2 for Medical Research
00:14.2 and the Howard Hughes Medical Institute,
00:16.0 and for the next few minutes
00:17.1 I'm going to give you a summary
00:19.0 of what we have learned in the past few years
00:21.1 about the planarian stem cells
00:23.1 also known as neoblasts.
00:25.2 So, the term neoblast was coined
00:27.2 by a graduate student in Bryn Mawr College in 1893
00:30.2 to refer to these undifferentiated cells
00:33.1 that are found distributed throughout the body plan
00:36.0 of the planarian anatomy.
00:38.0 And these cells are responsible, in great part,
00:40.2 for the immense capacity to regenerate missing body parts
00:44.2 after amputation in these animals.
00:46.1 So, what you see here is essentially
00:48.2 a seven-day progression of an animal
00:50.2 that was decapitated
00:52.2 and you can watch how the tissues that begin to form
00:55.1 in front of the plane of amputation
00:57.0 begin to differentiate into something
00:59.0 that looks like a planarian head.
01:00.2 These cells are broadly distributed
01:02.3 throughout the anatomy of the organism,
01:04.3 they are labeled here in green,
01:06.2 and you can see, for example,
01:07.3 that they are in essentially every part of the animal,
01:12.1 with the exception of the tip of the head of the animal, per se,
01:15.2 and the pharynx proper.
01:17.1 And these are the only two tissues that,
01:19.0 when amputated from the animal,
01:20.2 will not regenerate a complete animal,
01:22.2 and we know this from the work of Morgan, T.H. Morgan,
01:26.2 in 1898 and 1901.
01:27.3 But the rest of tissue,
01:29.1 as long as it has these green cells, these neoblasts,
01:31.2 they'll actually launch a regenerative response
01:33.2 to restore the form and function of that fragment
01:36.2 into a complete animal.
01:38.2 So, what do these cells look like?
01:40.2 These cells are highly undifferentiated,
01:43.2 they're relatively abundant,
01:45.0 you can see here the nucleus in pink,
01:47.0 there are very few electron dense materials by the electron micrograph,
01:51.2 suggesting that most of the DNA is in a relaxed state.
01:54.2 It's not condensed,
01:56.1 which means that that DNA is probably open for business.
01:58.2 Transcription should be taking place
02:00.2 in a variety of ways
02:02.3 to allow these cells to differentiate
02:04.2 into the 40 to 50 different cell types
02:06.2 that make up the anatomy of this organism.
02:08.2 The cytoplasm is highly basophilic,
02:10.2 so it's filled with RNAs
02:13.2 -- mitochondrial RNA, ribosomal RNA,
02:15.1 messenger RNA,
02:16.3 and probably long non-coding RNAs
02:18.2 and microRNAs
02:20.2 and the entire population and types of RNAs in these animals.
02:23.1 What is remarkable about these cells is that
02:26.3 they are not rare;
02:28.1 they're actually very abundant,
02:29.2 which you can readily see by just looking at this organism
02:31.1 where the neoblasts were labeled.
02:33.2 So, these neoblasts correspond by anatomy alone,
02:35.2 by morphology of the cells,
02:36.3 to about 25% of all of the cells in this animal.
02:40.1 That is a gross exaggeration of a particular cell type,
02:43.0 particularly an undifferentiated cell type,
02:45.1 that can potentially turn into any tissue type in these animals.
02:49.0 Secondly, these are the only dividing cells in planarians.
02:52.1 Again, another exaggeration,
02:54.0 normally that's not the way it works,
02:55.2 but in these animals you have stem cells,
02:57.1 which are the only ones that divide
02:59.2 in these asexually reproducing animals,
03:00.3 and then the other cells,
03:02.1 which are the differentiated cells,
03:03.2 which are entirely post-mitotic.
03:05.2 They will not divide any more.
03:07.1 And then we also now know that
03:09.2 they are not only collectively totipotent...
03:11.1 and what do I mean by that?
03:12.2 What I mean by that is that
03:14.1 a collection of these cells,
03:15.1 when you remove this fragment from the animal, for example,
03:18.1 then this little square can regenerate a complete animal
03:21.2 and then those few neoblasts that are present in there
03:24.3 will divide, proliferate, and differentiate
03:27.0 into all the tissue types that are missing.
03:28.2 But more recently,
03:30.3 a prior postdoctoral scientist in my lab,
03:32.2 who is now running his own laboratory
03:34.2 at the Whitehead Institute at MIT,
03:36.2 demonstrated that you can remove
03:39.1 an individual cell from these animals,
03:41.0 but them back into an animal
03:43.0 in which all the neoblasts were eliminated
03:44.1 and, with some frequency,
03:46.1 that individual cell is capable of
03:50.0 restoring all of the cells and all of the tissues in these animals,
03:53.1 settling the suspicion, the long-held suspicion,
03:56.0 that these cells really contain the capacity to be,
03:58.2 if not toti-, at least pluripotent,
04:01.0 and capable of restoring and producing
04:03.0 all the cell types in these animals.
04:04.2 So that makes these cells particularly interesting,
04:07.0 because what it tells us is that
04:10.0 they figured out a way
04:12.0 to regulate their genomic output
04:13.3 into very specific products
04:17.0 to give rise to muscle,
04:18.1 or in other instance neurons,
04:19.2 or in other instances epithelium,
04:21.0 and so forth and so on.
04:22.1 And we need to understand how these cells are
04:25.2 managing all this information to produce tissues
04:27.2 of the right number
04:29.1 and of the right type
04:31.1 to allow the animal to be recognizable as a planarian.
04:33.1 So, we know that amputation
04:36.2 is capable of activating the proliferation of these cells,
04:39.1 so at any given time that these cells are dividing,
04:41.3 at any given time, in fact,
04:44.1 2% of all neoblasts are entering the cell cycle,
04:46.2 which is a really fast and furious activity,
04:48.3 but amputation actually increases that rate
04:51.1 to even higher numbers,
04:52.3 and I'm going to show you that right here.
04:54.2 You can see here a head regenerating a tail.
04:57.1 So, this is the head regenerating a tail, so this is the head fragment.
05:00.2 You can see now a trunk that is now going to be regenerating
05:02.2 a head and a tail.
05:04.0 And then you can see a tail fragment,
05:05.1 which is essentially going to eventually
05:06.3 regenerate a head, right here, okay?
05:09.1 But what you see here in this third column in the middle
05:12.2 is the number of cells that are undergoing cell division,
05:15.0 so we're using an antibody against
05:17.1 a phosphorylated form of histone H3
05:20.0 serine number 10, in these animals,
05:23.1 in these proteins,
05:24.2 where that's the time when the cell is traversing from G2 into mitosis,
05:27.1 so it allows us to see what cells are dividing.
05:29.1 We can quantify this process,
05:30.2 and now we know that amputation
05:32.3 initially causes a big burst of proliferation.
05:35.0 So, amputation alone can activate these cells
05:37.2 to hyperproliferate.
05:39.0 So, what we would like to do is understand
05:41.1 how these cells respond to the environment,
05:42.2 and by not responding in such a way
05:44.2 that they would produce tumors
05:46.3 or produce some amorphous structures,
05:48.1 but essentially regenerate the right tissue types
05:51.2 and the right structures that are missing after amputations.
05:54.1 So, what I would like to do in the next few minutes
05:56.1 is to show you the way in which,
05:57.3 for the past five to six years,
05:59.1 maybe a little longer than that,
06:01.0 we've been dissecting neoblast biology.
06:02.3 And what I hope to accomplish in this presentation
06:04.3 is to go over four things:
06:07.0 how we went about identifying
06:09.2 markers that are associated with the neoblasts;
06:11.0 how we identified markers that
06:14.1 allowed us to distinguish the neoblasts from the progenitors,
06:16.3 the cells that were produced after cell division;
06:19.1 we'd also like to understand how these cells behave
06:22.2 -- we have no understanding,
06:25.0 or deep understanding, rather,
06:26.3 of how these cells are behaving under normal homeostatic conditions
06:30.2 or under regenerative conditions;
06:32.2 and then finally a little bit about
06:35.0 how these cells actually do cell division,
06:37.1 which was a surprise for us
06:40.1 at the end of the day.
06:42.0 So, let's start with the neoblast markers.
06:44.1 So, these neoblast markers
06:46.1 are genes or proteins
06:48.3 that are being made specifically by the stem cells
06:50.2 that will allow us to now distinguish them molecularly
06:53.1 from their cohorts, the surrounding cohorts.
06:55.1 So, how do we go about identifying the specific genes?
06:58.3 Well, I told you earlier that
07:00.3 these are the only cells that are undergoing cell division,
07:04.0 so we took advantage of an experiment that was executed
07:07.0 in the early 1900s by a scientist, [unknown],
07:10.1 that allowed him to remove all the neoblasts from planarians
07:14.2 by exposing them to large dosages of irradiation.
07:17.3 And irradiation kills only dividing cells,
07:20.0 because the chromatin gets broken up into pieces,
07:22.1 when cells try to divide
07:24.2 they cannot pair their chromosomes,
07:25.2 and they undergo apoptosis, complete cell death.
07:28.1 So, here's the situation:
07:30.0 we take an animal, like so, in green are the stem cells,
07:32.2 we expose these animals to irradiation,
07:34.2 it could be gamma irradiation or X-ray irradiation,
07:37.1 and then after a period of time
07:38.3 all of these green cells will be gone.
07:40.2 The animal, however,
07:42.0 will survive on the virtue
07:43.3 of its differentiated cells
07:45.2 for as long as 3-4 weeks,
07:47.1 which gives us plenty of time
07:49.2 to look at the behavior of these post-mitotic cells.
07:52.0 So, the idea is essentially as follows:
07:54.2 can we compare these two states,
07:56.1 the wild type animal and the irradiated animal,
07:58.2 and see what differences pop up?
08:00.2 Those differences should be associated
08:02.2 with the tissues that are missing,
08:04.1 and the tissues that are missing in this case
08:06.1 are going to be the neoblasts.
08:07.1 So, that's the idea.
08:08.1 We take samples one day after irradiation,
08:11.1 seven days after irradiation,
08:13.2 and the hope is that by profiling RNA expression
08:16.3 we can identify genes that are exclusively associated
08:19.2 with the stem cells and their progeny,
08:22.0 and I'll explain that in more detail
08:24.1 in just the next slide.
08:25.2 So, what we did was to
08:27.2 measure differences in gene expression
08:30.0 between the wild type and the irradiated animal,
08:32.3 and what we noticed is that there's a group of genes
08:36.2 that essentially disappear, that's the columns in red, here,
08:38.3 24 hours post-irradiation.
08:40.2 These genes are probably associated with the neoblasts,
08:43.2 because the neoblasts were physically removed
08:45.3 from the whole animal.
08:47.3 Everything else is there expect for the neoblasts,
08:49.0 so if we see genes disappear or expression of genes disappear,
08:51.0 that's because the neoblasts are gone,
08:52.2 so we assume that these genes are associated with those cells.
08:55.2 This is how we went about identifying
08:57.3 genes associated with the neoblasts.
08:59.2 And the gene that was most severely affected by irradiation,
09:03.0 it's this molecule referred to as Smedwi-2.
09:06.1 Smedwi-2 is a member of the Argonaute family of proteins,
09:10.1 initially discovered in plants,
09:11.3 which we know from plant biology
09:13.2 to be intrinsically associated
09:15.1 with the regulation of stem cells in plants.
09:17.2 So, if animals and plants share a common ancestor,
09:20.2 this molecule was there, probably regulating stem cells,
09:24.1 in the ancestor that gave rise to both plants and animals.
09:26.3 So this is a very old molecule
09:28.2 and it's surprising and delightful
09:30.3 to find a molecule like this expressed in neoblasts in planarians.
09:33.2 So, now we know that perhaps this molecule
09:36.1 really is very, very important
09:38.1 in regulating stem cell function.
09:39.1 And so when we look at where in the animal
09:42.3 this gene is expressed,
09:44.1 we find it expressed essentially in the neoblasts.
09:46.1 All these little green dots
09:48.1 are all of the stem cells that exist
09:50.1 in an asexually reproducing animal
09:52.1 at any given time.
09:53.1 And in magenta you see the cells
09:55.1 that are undergoing cell division, okay?
09:57.0 So we can now take advantage of
10:01.0 RNA-mediated genetic interference, or RNAi,
10:03.1 and perturb the function of this molecule
10:06.1 and then see what consequences that has
10:08.1 on the function of the neoblasts themselves.
10:10.2 And so we did that
10:14.1 in order to determine the function,
10:17.1 but to confirm first whether or not these are
10:20.1 neoblast-specific genes,
10:21.2 we took advantage of a methodology
10:23.1 that was developed by our colleagues in Japan,
10:24.3 Kiyokazu Agata and his lab,
10:27.3 that by sorting out these cells...
10:29.1 essentially taking these animals,
10:30.3 you make a soup of cells,
10:32.1 and then you measure DNA content...
10:33.2 and DNA content increases as the cells divide,
10:36.0 so these are the neoblasts, alright?
10:37.3 And the cells whose DNA content is not changing will be down here.
10:43.0 Now we can take this profile
10:45.0 and apply it again to an animal
10:47.1 where the neoblasts were removed by irradiation,
10:49.2 run the same profile,
10:50.3 and what do we see?
10:52.1 Well, the cells that are dividing are gone,
10:53.3 alright, so now we know that all the neoblasts
10:55.3 are going to be here,
10:57.0 but there's another window of cells
10:58.3 called the X2 population, right here,
11:00.1 that are also gone.
11:01.2 So we think that the cells are jumping between these two windows,
11:03.2 but that's where our neoblast population is.
11:05.2 And there's a group of cells that were not altered
11:07.2 by the irradiation
11:09.0 and we call this the post-mitotic division progeny.
11:11.0 So we now purified cells from each of these windows
11:14.0 and we tried to measure the expression
11:17.1 of these molecules, Smedwi-2 in these cells, that's what we see.
11:21.1 That only the X1 and the X2 windows
11:24.2 are expressing PIWI or Smedwi-2,
11:26.2 suggesting then that this is a very specific marker
11:29.1 to the neoblasts themselves.
11:30.3 So, to go back to what I was saying earlier,
11:33.1 when we disrupt the function of these genes,
11:35.2 what we essentially see is the following:
11:37.2 we were able to essentially recapitulate, or copy,
11:41.1 the phenotype that we see of animals
11:43.2 that were subjected to irradiation.
11:45.1 They're incapable of mounting a regenerative response,
11:47.2 so no head or tails are formed,
11:49.1 and you can see they form this little taco shell shape, which we refer...
11:54.1 we call ventral curling,
11:55.2 and the smedwi-2 RNAi gives exactly the same phenotype.
11:58.1 But why did it give us exactly the same phenotype?
11:59.3 And so... because we know that
12:03.1 the cells that are expressing PIWI essentially
12:05.0 are only the stem cells,
12:06.1 they're not present differentiated cells,
12:07.2 and yet the consequence is
12:09.2 only detectable in the differentiated cells.
12:11.2 So we followed these cells in time
12:13.3 to see what would happen to the neoblasts themselves
12:15.3 after we eliminate the molecule,
12:20.1 we noticed that the neoblasts were still there,
12:21.3 and yet they were not exerting their function,
12:23.1 and the reason why they were not exerting their function
12:25.1 was because the neoblasts themselves
12:27.3 could not differentiate into the appropriate cell type.
12:30.2 So you're looking here at the epidermis of the animal,
12:32.3 these are referred to as columnar epithelial cells,
12:34.3 the nuclei were labeled with BrdU
12:37.0 to allow us to follow the cells through time,
12:39.1 and this is what happens under normal conditions,
12:42.0 this is what happens when the molecule PIWI is perturbed by RNAi.
12:45.1 The cells are made,
12:47.1 they migrate to the proper tissue,
12:48.2 but when they get there they don't differentiate properly.
12:51.0 So what we're seeing here is a consequence of a gene
12:53.2 that's expressed in the neoblasts,
12:55.2 no longer expressed in the progeny,
12:57.0 but it has consequences on the ability of that cell,
12:59.2 removed by many hours and by many microns of distance away from the source,
13:04.3 causing a serious phenotypic defect
13:06.3 that is incompatible with life.
13:08.2 So now we know that this molecule is actually very, very important,
13:11.3 not for the maintenance of neoblasts,
13:13.1 but for actually telling the neoblasts
13:15.0 what to become later on
13:17.0 as they become post-mitotic.
13:18.2 So, this was actually
13:21.1 something that was discovered in our laboratory in 2005,
13:24.0 and then a few months later
13:26.0 it became apparent that the reason
13:28.0 why this was happening
13:29.2 is because it was a new type of microRNA,
13:31.2 it's referred to as piRNAs,
13:33.1 that this molecule was regulating.
13:34.2 So, we didn't know that was happening,
13:36.1 but that's probably why we see this phenotype.
13:38.0 These piRNAs are not being made.
13:40.1 So, we thought that...
13:42.2 now we have a handle on markers
13:46.2 that are associated with the neoblasts...
13:47.2 can we identify markers associated
13:50.1 with their post-mitotic progeny,
13:52.0 follow the same process?
13:53.1 So that's what we're going to do in
13:55.1 the next part of the presentation,
13:56.3 these neoblast progeny markers,
13:58.2 and so what we thought we would do is that...
14:01.1 if these genes disappeared 24 hours later, okay,
14:04.1 they would be gone after a week,
14:06.1 but then there's these genes that did not disappear 24 hours later,
14:09.1 but they were gone 7 days later.
14:11.1 So we think that those are the cells
14:13.2 that are no longer being made by neoblasts,
14:15.1 and those are transitory cells
14:17.1 on their way to terminal differentiation.
14:19.1 And so what we thought we would do is
14:21.1 we'll look at these particular cells
14:22.2 and then see whether or not
14:24.3 they are expressing these genes at different locations.
14:27.2 And so we referred to this as
14:29.3 potential markers of the progenitor population,
14:32.1 okay?
14:33.2 And so this is the experiment that we did,
14:35.0 these are the genes that are expressed in the neoblasts,
14:37.3 these are the genes that were still present,
14:40.3 there 24 hours post-irradiation,
14:42.1 you can see them right here,
14:43.2 but by day 2 they're gone, by day 4
14:46.1 they're essentially gone again.
14:47.2 If you look at category 3 genes,
14:49.1 they are present there on day 1,
14:51.1 they're present there on day 2,
14:53.0 and they're gone by day 4.
14:54.2 So that suggested that these cells
14:56.1 were actually changing their expression profiles
14:59.1 as time was progressing and the cells were differentiating,
15:00.3 and so what we did was to essentially label the cells,
15:03.2 permanently, with BrdU,
15:04.1 and then we followed these cells
15:06.2 as time progresses.
15:07.2 So these cells now are double-labeled for BrdU,
15:10.1 which permanently labels their DNA.
15:12.3 The gene is expressed in these neoblasts
15:14.3 and now we ask, what happens to the BrdU,
15:16.1 which is completely independent of gene expression,
15:18.3 will we see it now in the next post-mitotic cells,
15:22.0 but expressing the genes that we think are expressed in the progenitors?
15:24.3 And what we see essentially is that,
15:27.0 yes, the BrdU-positive cells
15:28.3 which were yesterday expressing PIWI,
15:30.2 now are not expressing PIWI,
15:32.0 and are expressing these other early progeny markers.
15:35.0 You can follow these cells through time
15:36.2 and you essentially see the same thing,
15:38.1 that after 96 hours this gene is shut off
15:41.1 and then a new set of genes turn on,
15:43.1 and it's double-labeled by BrdU.
15:44.2 This is a permanent and a single marker
15:47.0 that allows us to follow these cells through time.
15:49.3 So, that allows us to identify and define
15:53.0 the first lineage of stem cells in planarians,
15:55.2 which we essentially just
16:00.1 show here in diagrammatic form.
16:01.2 You have molecules associated with the stem cells,
16:03.2 molecules associated with the post-mitotic division progeny...
16:06.2 this is the early, 24 hours after irradiation,
16:09.0 and these are the markers that appear
16:12.2 after 24 hours,
16:14.1 as the cells begin to go on in their march to differentiation
16:16.0 to give rise to either skin, or epidermis, rather,
16:19.1 muscle, neurons, and so forth.
16:21.3 So, using these tools and using this approach,
16:24.0 we have begun to decorate
16:26.2 the diversity of fates and the diversity of cell types
16:29.3 that are present in this organism.
16:32.0 More recently, however,
16:34.0 new technology has become available
16:36.0 to actually do this at a much higher level of resolution,
16:38.2 and that's shown right here,
16:40.0 this is work from a past postdoctoral scientist in the lab,
16:42.2 Peter Reddien,
16:43.3 where they essentially looked at 96 genes and 96 cells,
16:48.1 which are essentially shown right here,
16:49.2 and what they see when they test these individuals cells
16:52.3 against these 96 genes,
16:54.1 they identify subpopulations of neoblasts
16:57.1 that were expressing specific genes
17:00.0 at any given time,
17:01.2 suggesting that even though all of the neoblasts
17:03.2 are expressing PIWI,
17:04.3 there are subpopulations within that PIWI-positive cell population
17:09.0 that are expressing other specific markers
17:11.3 that probably are going to dictate the fate
17:14.2 that these cells are going to take,
17:16.2 under regular homeostasis,
17:17.2 to give rise to appropriate structures.
17:20.2 So, I think that, in the next few years, we're going to see a boon in our understanding
17:23.1 of how these different cell types
17:25.2 are specified and regulated
17:28.0 to produce the right type and right number of cells
17:30.1 under both homeostatic conditions
17:32.1 and regenerative conditions in planarians.
17:34.1 And by doing so, inform how stem cells
17:37.1 do what they do in the animal kingdom,
17:41.0 and perhaps even the plant kingdom,
17:43.0 but that remains to be determined.
17:45.0 So, what do these cells do?
17:47.2 You know, they just sit there,
17:49.2 we see them essentially just sitting in tissue,
17:52.0 and so we wanted to know,
17:54.0 what kinds of behaviors do they display?
17:55.2 And to do that we took advantage of
17:57.2 a really, really interesting experimental paradigm
17:59.3 that was devised, believe it or not,
18:02.1 in 1945-1946 in Paris, after World War II,
18:05.3 by Étienne Wolf and François Dubois,
18:08.2 and I'm gonna walk you through this process.
18:10.3 This is what they did.
18:12.1 This experiment is absolutely beautiful.
18:13.3 It's a PhD thesis, essentially.
18:15.2 They took animals that are wild type,
18:17.3 they took animals that were irradiated,
18:19.2 they don't have any neoblasts,
18:21.0 they removed tissue from the donor,
18:22.2 transplanted to the wild type, and asked the question,
18:27.2 does this tissue that has neoblasts,
18:29.1 can it actually rescue the irradiated animal or not?
18:31.2 Okay?
18:33.0 So, when you do that,
18:34.1 if you transplant irradiated tissue into an irradiated animal,
18:36.1 the animal dies.
18:37.2 No problem, I mean, this is what you would expect.
18:39.1 There are no neoblasts in there.
18:40.2 However, if you now transplant
18:43.2 wild type tissue into an irradiated animal,
18:46.0 yes, the animals get smaller because they're degrowing,
18:48.0 but it actually rescues the animal,
18:50.1 such that after 32 days they're still alive,
18:52.3 they have a nice looking head,
18:54.1 they have a pharynx,
18:55.2 they look perfectly recovered,
18:57.1 just by the transplantation,
18:58.2 suggesting that the tissue that's been transplanted
19:00.2 and the neoblasts within them
19:02.1 can actually repopulated the irradiated tissue
19:04.0 and rescue the animal.
19:06.0 If you now do the experiment in such a way that
19:08.2 you now transplant tissue from the sexually reproducing animal
19:11.3 into the asexually reproducing animal,
19:14.1 can the sexually reproducing animal
19:17.1 transform the asexual animal into a sexual animal, okay?
19:19.3 And so, we can tell the differences
19:22.0 by looking at the chromosomes,
19:24.3 and essentially what we do is that
19:26.2 we take an irradiated asexual animal as a host
19:28.2 and we take a sexual wild type animal as a donor
19:32.1 for the graft,
19:34.0 and what we find essentially is that not only does it rescue the asexually reproducing animal,
19:38.2 it actually transforms them into the hermaphroditic, sexual animal.
19:42.1 So that's really important for us to know
19:44.2 because that tells us that the stem cells, the neoblasts,
19:46.2 can give rise not only to the somatic tissues,
19:49.1 but also to the germline,
19:51.0 suggesting that these cells are as close to a totipotent stem cell
19:54.0 as you can possibly get in the animal kingdom,
19:56.3 and makes them even more interesting to try to understand
19:59.1 because it might help us understand and dissect
20:02.2 the processes by which the cells control their potency.
20:05.1 Okay?
20:06.1 So, how do they populate the tissue?
20:08.1 Alright?
20:09.2 I mean, you're transplanting them into an animal and the question is...
20:12.0 they're undifferentiated,
20:13.3 so do they move, do they just migrate
20:16.2 through a passive proliferation,
20:18.1 or what are they doing?
20:19.2 So, this is the work of a graduate student in the lab at the time,
20:21.1 Otto Guedelhoefer,
20:22.2 that essentially used the tools that we identified
20:25.1 with those gene expression profiles
20:28.0 to label the neoblasts and their progeny,
20:29.3 and then we can follow the fate of these cells in time.
20:34.0 So, here's an irradiated animal that received a graft,
20:35.2 here's the graft with the normal wild type tissue,
20:37.1 all the blue dots are stem cells with the PIWI marker,
20:40.1 and now we ask the question,
20:41.3 what happens through time?
20:43.1 3 days, 4 days, all the way to 12 days post-transplantation or PT?
20:47.1 And what you can see here is that,
20:48.2 like an epicenter,
20:50.2 the cells begin to migrate radially
20:52.3 to repopulate the animal.
20:54.0 So the cells can move, okay?
20:55.2 And that's how they rescue the tissue,
20:57.2 which is really important for us to know,
20:59.2 that even when undifferentiated they appear to be moving at some rate,
21:02.2 not very fast,
21:04.3 but they are moving at some rate.
21:06.2 So, now we can ask the question,
21:08.0 do they move and then proliferate,
21:09.2 or do they move as they proliferate?
21:11.1 So we used this marker that labels mitotic activity,
21:13.0 here's the animal with the graft,
21:15.2 here's the activity of proliferation,
21:18.1 and as you can see,
21:19.3 what happens is that the cells are migrating out of the tissue,
21:22.1 and you can see the tissue here in the center,
21:24.0 you can see positive cells in the pre-existing irradiated tissue,
21:26.3 and those are cells that are dividing.
21:29.0 So they're essentially just expanding and repopulating the organism.
21:32.2 And here comes the clincher, okay,
21:35.0 this is the great experiment that Étienne Wolf and François Dubois
21:39.2 came up with.
21:40.1 Now we can ask the question, as follows:
21:41.2 Do they really migrate? Okay?
21:43.1 Can we come up with an experimental paradigm
21:45.2 that would allow us to measure this?
21:46.2 And this is the brilliance of the experiment.
21:48.1 What they decided to do was to take a planarian, okay,
21:51.0 and then put a little shield of lead on top of it,
21:53.3 and then expose the head and the tail to lethal irradiation.
21:57.2 That should essentially eliminate all of the neoblasts
22:00.1 in the head and the tail,
22:01.3 and now you ask the question,
22:03.0 do the protected cells in the center of the animal,
22:05.1 will they now travel anteriorly and posteriorly
22:08.2 to repopulate those tissues and rescue them?
22:10.1 So, that's the situation.
22:12.1 These are undifferentiated cells,
22:14.2 they are protected by the lead,
22:16.0 what do they do?
22:18.0 So, we repeated the experiments,
22:19.2 we made these little bridges of lead, and so now...
22:22.1 here are the animals,
22:23.3 they are subjected to irradiation,
22:25.2 the anterior and posterior ends were subjected to irradiation,
22:27.0 the center was protected by the lead.
22:29.1 As you can see already,
22:31.2 tissues like the head, for example, in this organism right here
22:35.1 are beginning to resorb.
22:36.2 They're not being rescued by the neoblasts.
22:38.2 So now we can ask the question with the markers,
22:40.2 what are they doing?
22:42.0 And you can see here the protected tissue, the neoblasts are there,
22:44.3 the head and the tail are not labeled,
22:46.3 and what happens through time is that
22:49.0 the cells don't migrate at all.
22:50.2 The tissues begin to fall apart
22:52.2 and even though the tissues are falling apart,
22:54.0 these neoblasts are stationary.
22:56.1 They're not migrating at all.
22:57.2 Then we asked the question, well, what happens
23:00.0 if you actually cut the animal?
23:01.2 What happens if we now take these animals
23:03.1 that are protected in the middle
23:04.2 and decapitate them?
23:05.3 Would decapitation now force these cells
23:09.1 to mobilize and repopulate the animal?
23:10.2 And when we do that,
23:12.2 by decapitating the animal, this is what we see.
23:14.2 So, here's no amputation,
23:16.1 here's the wavefront of the neoblasts,
23:18.2 and when we remove the head,
23:20.2 now we can see cells essentially expressing...
23:23.0 or present right near the plane of amputation,
23:26.0 suggesting that they were migrating.
23:28.0 Not only are they migrating,
23:29.1 they're migrating directionally,
23:30.3 because if you look at the tail,
23:32.1 we see no migration of these cells towards the tail.
23:34.1 So the neoblasts can actually be mobilized,
23:36.2 and this is an attribute of stem cells in planarians
23:39.1 which we were not aware of,
23:41.1 and it's really important for us to be aware of
23:44.0 in order for us to understand what these cells are sensing in the environment
23:46.2 to allow them to mount a regenerative response.
23:49.1 So, these studies then allow us to conclude that
23:53.2 neoblasts migrate when animals are wounded,
23:55.2 when there's a bridge,
23:57.1 a through-and-through bridge of structural integrity,
23:59.1 and that migration of those cells appears to be directional,
24:02.1 so it's not just a random walk,
24:03.3 it is directional.
24:05.1 That suggests that the plane of amputation
24:06.3 is sending a very powerful and specific signal
24:08.2 for the cells to mobilize to that particular location.
24:12.1 And the final thing that I want to share with you
24:15.1 about neoblasts
24:17.1 is a really surprising discovery
24:18.3 that we were not anticipating to make.
24:20.1 We wanted to really perturb cell division
24:22.0 because we know that these are the only cells that are dividing,
24:24.0 and so we though that, well, why not?
24:26.0 If we modulate this,
24:27.2 we should modulate the ability of these cells
24:29.1 to make different tissue types.
24:30.3 And so this was a collaboration between the laboratories...
24:33.2 my laboratory and the laboratory of Wallace Marshall at UCSF,
24:36.2 where a very talented postdoctoral scientist,
24:39.1 Juliette Azimzadeh,
24:40.3 wanted to find out how
24:43.2 the cilia in organisms is constructed and regulated.
24:47.1 So, we know, as I told you earlier,
24:49.2 that these are the only cells that divide in planarians
24:51.2 -- this is a neoblast in telophase --
24:54.1 and to divide you need to form these centriolar structures
24:57.3 that connect to asters
25:01.1 to separate the chromosomes from each other.
25:03.1 And we know from the literature
25:05.3 that that's what happens.
25:07.1 You can see the centrioles in red,
25:08.2 this is a movie I borrowed from Jordan Raff at Oxford University,
25:11.1 and then these are the... the uhhh...
25:13.3 the centrioles dividing.
25:16.0 In green is the actual DNA
25:18.1 and in red are the asters
25:20.2 separating the cells and producing two daughter cells from one cell.
25:24.1 And so for many years we've thought that
25:26.2 the centrioles are absolutely essential for the orientation of the cells
25:29.0 and the separation of the chromosomes,
25:30.2 and that all animals actually utilize centrioles
25:33.1 to execute this function.
25:35.1 So, we thought, okay fine,
25:38.1 let's clone all the centriolar proteins
25:39.2 and then see what we find.
25:41.1 And so we did that and we were surprised that
25:44.0 it didn't affect cell division,
25:45.2 and so we though, well, why is that?
25:47.1 So we looked a little deeper
25:49.2 and then we realized that there are at least two types of cell division:
25:51.2 so-called centrosomal and acentrosomal.
25:54.0 This is the type of cell division that you see in the germ cells,
25:56.1 the ones that produce gametes.
25:58.0 They don't required centrioles at the tips of the asters
26:00.2 for separation to proceed.
26:02.2 And when we look at planarians, essentially,
26:05.1 we see that there are centrioles,
26:06.1 but they are associated with the cilia,
26:07.2 the ventral cilia of the animals,
26:09.0 but they are not really required, at all,
26:12.1 for the proliferation of these animals.
26:14.1 So if you look at these components right her,
26:16.1 sas-4 and plk-4,
26:17.3 neoblasts have regenerated,
26:21.0 but genes that affect the cell cycle
26:22.3 prevent the same regeneration,
26:24.3 and where you end up by testing the presence or absence of the the centrioles
26:27.3 using an antibody against this protein,
26:30.2 we realized that the neoblasts don't have centrioles.
26:33.2 So, this is an example, for the first time ever,
26:36.1 of an animal that does cell division,
26:38.2 mitotic cell division,
26:40.1 without centrioles at all, completely unsuspected.
26:43.2 And when we look at electron microscopy
26:45.1 we can see the chromosomes,
26:47.1 we can see the spindle pole,
26:49.1 you can see this pericentriolar matrix,
26:50.3 but there are no centrioles in this cell division.
26:53.1 So, as I said, this is the first demonstration
26:55.1 that centrioles are not essential for animal mitosis
26:57.0 and I would like to suggest that
26:59.0 it's probably gonna be true for other organisms as well,
27:01.0 and that this acentriolar cell division
27:03.1 is common to meiosis but not mitosis,
27:05.2 even though planarians are really just doing mitosis
27:08.2 as far as we can tell.
27:10.0 Alright, so, how are neoblasts then
27:13.1 regulated to produce the right type
27:16.0 and number of cells
27:17.2 to maintain animal form and function?
27:18.3 So, I've been telling you all along
27:20.2 that when you cut these animals into three fragments...
27:22.1 the head fragment has no pharynx,
27:23.3 it has to take some neoblasts,
27:25.2 co-opt them in a way that can produce the pharynx
27:28.1 such that you can complete regeneration of a complete animal.
27:31.1 The tail is the same way.
27:32.2 You have to generate a head,
27:34.2 generate a pharynx,
27:36.1 and allow the animal to regenerate into a complete organism.
27:38.1 And the same thing is true for the trunk, okay?
27:40.2 So, how does it do it?
27:42.2 It's not just the types of...
27:44.2 the location and the position of these tissues,
27:47.1 but we also know that there's signaling events
27:48.3 at the anterior end and the posterior end
27:50.1 which are not inherited, so to speak,
27:53.0 by the amputated fragments.
27:54.0 So somehow this fragment, now,
27:55.2 needs to activate this posterior signal
27:57.2 to allow for the animal to have both anterior and posterior signals.
28:01.2 It's not clear to us how this is done,
28:04.0 and so we would like to suggest that
28:07.1 this type of regulation is essentially going to be based
28:10.2 on what state of determination or differentiation
28:14.2 these neoblasts, which reside in the middle of the animal,
28:16.3 may or may not be.
28:18.3 And right now the popular hypothesis is that
28:22.1 these neoblasts are essentially assuming, or acquiring,
28:25.1 specific fates.
28:26.0 It's what we refer to as a deterministic model,
28:28.1 where the pluripotent stem cell
28:30.1 produces specialized progenitors,
28:31.1 and these specialized progenitors will go on
28:33.2 to produce the necessary cells
28:36.1 after injury-induced plasticity,
28:38.1 and then eventually, once all that is repaired,
28:40.1 it returns to homeostasis again,
28:42.1 right before amputation.
28:43.2 This is really hard to
28:48.1 use as an explanation
28:50.1 for how fragments of animals
28:53.1 that don't have either the anterior or the posterior end,
28:55.0 or any of the signals,
28:56.2 resetting themselves, if these cells are already committed
28:59.0 to this particular homeostatic state.
29:00.2 So what we're favoring in the lab right now
29:03.1 is an alternative model,
29:04.3 which we refer to as the probabilistic model,
29:06.1 which means that at no time are the neoblasts really
29:09.3 committed to one particular fate,
29:11.1 but they're essentially entering and exiting
29:13.2 a particular state probabilistically... probabilistically.
29:16.2 And that allows for a tremendous degree of plasticity
29:19.0 such that, depending on the context,
29:21.0 if an animal is amputated
29:23.0 and that cell was in this particular trough,
29:25.1 it can actually come out of it and then,
29:27.0 through the signals of amputation,
29:28.2 be channeled into a specific way.
29:30.0 So they're not really all into specific channels
29:32.1 that are predetermined and not interchangeable,
29:35.1 but it's a probabilistic model
29:36.3 where the cells are coming in and out.
29:38.1 So, we would like to, in the near future,
29:39.2 try to visualize this by seeing
29:42.1 whether or not the same neoblast can actually turn on and turn off
29:45.3 different genes associated with different fates.
29:47.1 And it that is the case then the probabilistic model will...
29:52.1 umm... we'll have evidence to support that.
29:55.0 So that's what we have learned
29:58.3 in a nutshell about the neoblasts.
30:00.2 There's much more that we need to figure out.
30:03.0 Essentially, how do you control the number of neoblasts in the animal?
30:06.2 How do you control the fates of these cells
30:08.2 under both homeostatic and regeneration conditions?
30:11.1 And I think the years ahead will provide us
30:14.0 with a really clear understanding
30:15.2 of how stem cells actually work, as it were,
30:18.1 in an adult organism in vivo.

Part 3: Expanding the Number of Model Systems is Essential

00:07.1 Hello.
00:08.2 My name is Alejandro Sánchez Alvarado.
00:10.0 I am an investigator with the Stowers Institute for Medical Research
00:13.2 as well as the Howard Hughes Medical Institute,
00:17.0 and what I would like to share with you today
00:19.2 in the next few slides
00:22.0 is a way to make the argument
00:24.1 as to why we need to expand
00:26.3 the numbers of model systems
00:28.2 in order for us to understand not just life,
00:31.2 but also human biology and the diseases that afflict us.
00:34.1 And so, to do that,
00:36.0 I'm going to tell you a little bit about
00:40.3 this idea of the origin and diversity of animals.
00:42.3 We all know that there's an immense diversity of organisms out there,
00:46.2 we all know about the principles of evolution
00:49.0 and how we are related to each other
00:51.1 by very, very deep associations
00:53.1 with the origins of particular genes and the like.
00:55.2 But even though we know all this,
00:57.1 we know that it's possible,
00:59.1 we really don't know what is possible in nature
01:02.1 as it is right now.
01:04.1 And the reason for that is because
01:05.3 the nature of biomedical research
01:07.2 is not really allowing us to actually look.
01:10.1 We have to actually do the work with the systems
01:12.2 that are available to us
01:14.1 in order to ask questions
01:15.2 that we think are relevant to understanding our own lives as human beings
01:19.1 as well as life in general as a phenomenon
01:21.3 that happens on this planet.
01:22.3 So, I'm going to give you an example
01:25.1 of what I mean by this.
01:27.2 So, we have studied very, very species thus far
01:30.0 of the immense diversity of organisms
01:32.2 that are present in the globe.
01:34.1 And the main organisms that we've used,
01:35.3 and that the NIH and other funding agencies
01:37.3 have invested a huge amount of resources for us to understand
01:41.0 and use as vessels
01:43.1 to generate knowledge and understanding of biological processes
01:45.2 are represented right here.
01:47.0 These are incredibly powerful model systems
01:49.1 that have, you know,
01:51.2 ushered a really profound understanding of biological systems
01:54.3 and biological processes,
01:56.1 starting from humans and mice,
01:58.0 frogs and chicks,
01:59.3 and of course the zebrafish
02:02.0 as the representatives of the vertebrate world.
02:04.1 And they are all being used in large numbers of laboratories,
02:07.2 not just in the U.S.,
02:09.1 but across the world,
02:10.2 to ask the questions, essentially, of:
02:11.3 What is life?
02:13.1 How does it arise?
02:14.3 And what happens when it goes wrong?
02:16.3 And we understand those mechanisms.
02:18.2 Part of the process in trying to understand life
02:21.2 has also involved the utilization and understanding
02:25.2 of invertebrate model systems,
02:27.0 which I'm showing you right here,
02:28.2 C. elegans and Drosophila,
02:30.0 that are an incredible set of organisms
02:34.1 to ask genetic questions associated with
02:36.2 complex biological processes.
02:38.1 So, what we've learned from these animals
02:40.0 is immense,
02:41.1 or at least we feel that way
02:43.0 because we have now reached a level of understanding
02:44.3 of certain biological processes
02:46.2 that our predecessors
02:49.2 would just envy and would not be able to rival
02:51.1 by any amount of experimentation at the time
02:55.1 and observation at the time.
02:58.1 But... that's what we know, those are the systems we use,
03:01.3 it's about seven model systems in general,
03:03.3 give or take a couple more,
03:05.1 but it would be interesting to know
03:07.2 and to get an appreciation
03:09.2 of what is already possible out there
03:12.1 by just simply studying nature,
03:14.0 by doing what I refer to as inductive inquiry,
03:16.0 which is not necessarily what we do in the lab today,
03:18.1 which is mostly hypothesis-driven research.
03:20.3 And inductive inquiry is essentially...
03:23.2 it gets called fishing expeditions
03:25.1 and a few other monikers
03:27.0 that don't make it very exciting
03:28.1 and of course don't make it very fundable,
03:29.2 because it's exploration,
03:31.1 it's curiosity-driven perhaps,
03:32.2 but it's essentially not being buttressed
03:35.0 by some hypothesis that will allow you to
03:37.1 explore this diversity in nature.
03:39.2 So, I'm going to start by giving you an example,
03:42.0 which are protists,
03:43.0 which are unicellular organisms, okay,
03:45.1 that are nowhere near similar to us, they're unicellular.
03:50.2 And the one that's most famous, of course, is Tetrahymena,
03:52.2 which is about 40-48 microns in diameter, okay.
03:55.1 These organisms were selected
03:58.1 for investigation many years ago
04:00.1 because they display some really interesting biological attributes
04:02.2 that made them vulnerable to experimentation
04:05.0 and for us to understand major principles of life.
04:07.3 And so from a single protist organism, okay,
04:11.0 we were able to extract the following information:
04:13.2 ribozymes were discovered in protists,
04:16.0 this actually resulted in a Nobel Prize in 1989;
04:19.2 telomeres, by Greider, Blackburn, Szostak, and others,
04:23.1 also resulted in a Nobel Prize...
04:25.1 a single unicellular organism
04:27.2 producing two Nobel Prizes already
04:29.2 and promising many more to comes;
04:31.2 because the first molecular motor was discovered in Tetrahymena as well
04:36.1 by Gibbons and Rowe in 1965;
04:38.1 and then of course histone modifications and epigenetics,
04:41.1 the first demonstration that this was happening,
04:43.2 by Allis in 1979,
04:45.0 also took place in this organism right here.
04:47.2 What is interesting though,
04:49.1 just to put it in context,
04:51.2 is that you'd think that this is really esoteric science, right?
04:53.2 But all of this research was actually funded, initially,
04:57.3 by the NIH --
05:01.1 the National Institute of General Medical Sciences.
05:02.3 Every single one of these,
05:06.0 you know, bold proposals
05:07.3 to do inductive inquiry in an organism
05:10.0 that doesn't have hair,
05:11.2 it's not a mammal,
05:13.0 in fact it's a single protist,
05:14.2 resulted in funding that provided us with
05:17.2 tremendous understanding of biological first principles.
05:20.1 Okay.
05:22.0 So, what that suggests to me, essentially, is that
05:25.1 things have changed a little bit,
05:26.2 because if you were to come up with a single organism like this
05:28.3 and propose to investigate this research,
05:31.1 the likelihood of success right now
05:33.2 would be pretty low,
05:35.3 I mean, that's been the experience of many of my colleagues,
05:38.2 primarily because we think that
05:42.1 we have enough model systems available to us
05:43.2 to ask all the questions we need to ask.
05:45.2 Okay, fair...
05:47.2 we can assume that that is the case.
05:49.1 But what if I were to tell you that
05:51.2 this is what we think protists look like, right?
05:54.0 We know what is possible in the protist world
05:56.0 -- they're small things, microscopic,
05:58.3 ranging in size from 40-48 microns.
06:01.0 And what if I show you this other organism,
06:03.1 which is about 20 centimeters in size
06:05.1 and is also a protist.
06:08.0 This is Syringammina fragilissima.
06:10.2 So, the phenotypic/genotypic space
06:12.2 that a single-celled organism can occupy
06:14.2 goes essentially...
06:17.1 25,000-fold difference in scale.
06:19.2 If I didn't tell you that this was a protist,
06:21.2 a unicellular organism, you wouldn't know it,
06:23.2 and so this space that's being occupied
06:26.1 by the single genome of these single organisms is huge.
06:29.2 So what is possible in nature,
06:32.2 we don't appreciate,
06:34.0 because we're not looking.
06:35.1 Now, it would be fair if I told you this single...
06:37.2 this protist, Syringammina fragilissima,
06:40.0 was discovered recently.
06:41.2 This thing was discovered in the late 1800s
06:43.1 off the coast of Scotland, okay,
06:45.1 and we don't know anything about it today.
06:49.0 And my guess is that, if Tetrahymena
06:51.2 had all these secrets to teach us about,
06:53.3 you know, telomeres, ribozymes, molecular motors...
06:57.3 what other secrets is this organism holding
07:01.1 that we should really learn about?
07:04.2 And we don't know.
07:05.2 We don't know because we're not looking, alright?
07:07.2 So, if you now take a more objective approach
07:09.2 to what this means and you now look
07:12.2 at the metazoan tree of life,
07:14.1 which I'm listing right here,
07:16.3 okay, what you see essentially is that the seven model systems that we utilize,
07:19.0 these five vertebrates, okay, essentially are vertebrates.
07:23.2 All these other groups are not represented
07:27.1 to the same degree of abundance
07:31.1 in the biomedical milieu, okay?
07:33.2 If you look at the other two, you know,
07:37.1 workhorses of genetics in the invertebrate world,
07:39.0 and I would venture to say in all of animal genetics,
07:41.1 C. elegans and Drosophila,
07:43.1 we're talking again about two phyla,
07:45.1 the nematodes and the arthropods,
07:47.2 and these other phyla are not being studied.
07:50.0 If you now count how many phyla
07:52.2 make up the phylogenetic tree,
07:54.1 which is essentially rooted in a single cohort of organisms, down here,
07:57.2 we're talking about 29 different phyla.
08:00.1 Now, I ask you, okay,
08:02.1 what is the statistical probability
08:05.2 that 7 organisms such as these
08:08.2 will encompass the immense diversity of biological activities
08:11.0 that are present in the metazoan tree of life?
08:13.3 Just do the math...
08:16.1 that's 9x10^-5 percent of all animal species
08:19.0 that are represented by these 7 organisms.
08:21.1 Is that statistically significant?
08:23.2 Of course it's not.
08:25.1 So what is the likelihood that every single biological problem
08:28.0 that we really want to understand
08:29.3 is going to be represented in these animals?
08:31.3 Very low, that's the bottom line,
08:34.0 it's very low.
08:35.2 And if you were to tell me that it's not possible
08:38.1 to do investigations in all these other animals
08:40.0 because they're different, they're weird,
08:41.2 they're hard to access, and so forth and so on,
08:43.1 that's a good argument 20, 50, 100 years ago,
08:46.3 but now, with the technologies we have accessible to us today,
08:50.1 well, we can actually bring science to nature
08:52.2 as opposed to the other way around.
08:54.1 Well, we have to bring nature, domesticate it,
08:56.1 and hammer it in the lab.
08:58.0 I think we can do the opposite.
08:59.1 We should be able to do that nowadays.
09:01.3 So, why do I say that?
09:03.1 Because there's a cohort of phyla
09:07.0 for which we know so little
09:09.2 relative for what we know for these 7 organisms.
09:12.1 So the likelihood of us
09:15.2 not making equivalent discoveries,
09:17.3 and significant and insightful,
09:21.0 in the next 10 years if we were to dedicate ourselves
09:23.0 to some of these animals in relation to what we have studied,
09:26.1 it's zero.
09:27.2 This is awaiting... okay,
09:29.3 this is a mine of new discoveries waiting to be picked
09:31.2 by people willing to actually do inductive inquiry,
09:34.3 buttressed of course by the scientific method.
09:37.1 Alright?
09:38.2 So, normally the argument is as follows:
09:40.1 you know, we have this really high resolution
09:42.2 of established model systems,
09:44.3 we have genomes,
09:46.1 we have ways to edit their genomes,
09:47.2 we have genetics,
09:49.0 we have tools,
09:50.1 we can modify every single nucleotide in the genome,
09:53.1 and that's great.
09:54.2 That's the high resolution of established model systems,
09:56.2 but you're essentially limiting yourself
09:58.2 to the biological attributes represented
10:00.1 in that very small cohort of animals.
10:02.2 Whereas, the low resolution of emerging model systems
10:05.0 is seen as a deficiency
10:07.2 because, well, they're hard to bring to the lab,
10:09.1 they don't have all the tools,
10:10.3 maybe the genomes are not ready,
10:12.0 and so forth and so on.
10:13.1 Well, I liken this rationale as saying that
10:17.0 the high resolution of established model systems,
10:18.2 that's like mapping already discovered continents.
10:21.0 We know more or less what the biological activities are going to be
10:24.2 in the systems that we have studied for so long
10:27.1 and in such great detail
10:29.1 in the present time.
10:30.2 Whereas, working with these other systems
10:32.3 allows us to discover new biology
10:34.2 and therefore discover new continents,
10:36.0 and that is the value of being able to bring
10:38.0 other model systems into the milieu
10:41.1 of biomedical research.
10:42.3 Alright?
10:44.1 So, I think there is merit to try to develop
10:45.3 the necessary granularity and tools
10:47.3 to exploit all this new biology
10:50.1 that we're not even aware is probably happening
10:52.1 right there presently, as we look out the window.
10:55.2 We don't look.
10:57.1 We're not encouraged to look. Alright?
10:59.1 We're encouraged to do other things.
11:01.0 And for good reasons, obviously,
11:02.2 but we're encouraged to do other things,
11:04.0 and I think there should be room for both.
11:05.2 Okay, so what I'd like to do is give an example
11:08.3 of why looking at weird animals
11:10.3 might actually be a perfectly viable and useful proposition
11:14.3 to understand, you know, our own biology, okay?
11:17.2 So, I want to give you a small,
11:19.2 a short story of a discovery
11:22.1 that was done by looking at the excretory system
11:24.2 of an animal that doesn't look anything like us,
11:26.1 which are these planarians that I'm going to tell you about
11:28.2 in just a few minutes,
11:30.1 and what I would like to do is to walk you through
11:32.1 what we think we understand
11:34.1 about the evolution of the excretory system,
11:35.3 the kidneys and the corresponding or attending diseases
11:41.0 when things go bad. Okay?
11:42.1 So, what do we know the kidney?
11:44.1 Well, a lot has been learned in the past 30 years,
11:48.0 but for some reason the kidney has not really
11:51.0 attracted as much attention as say the heart or the brain,
11:52.3 and there's a really interesting...
11:55.0 when you start looking at the old literature,
11:56.2 and by old I mean just 1960s,
11:58.1 I don't mean like early 1900s, just 1960s.
12:01.0 So, for example we know that if you take a urine sample of a human
12:04.3 at any given time,
12:06.2 there's a huge amount of daily shedding
12:08.1 of renal tubule epithelial cells.
12:10.1 If this is constantly happening
12:12.0 and you're not replacing it,
12:13.2 we would be dead.
12:15.0 Our excretory system would be completely spent.
12:16.3 So that means that that tissue is being reconstructed
12:19.0 on a daily if not hourly basis
12:21.1 to allow us to keep that system functioning
12:23.1 for the 80 years or so that we might actually live for,
12:25.2 if we're lucky enough.
12:27.3 Secondly, we know that there is
12:30.0 compensatory renal growth following nephrectomy,
12:32.2 and this has been discovered and reported in 1975
12:34.2 -- one kidney is removed for transplantation,
12:36.3 the other kidney will grow to compensate
12:39.0 for the function of the missing kidney.
12:40.3 So, if those tissues were really static
12:44.0 and there was no growth,
12:45.1 this would not be possible.
12:46.3 So, we already know it's happening,
12:48.0 we just don't know how it is happening.
12:49.2 Third, we know that in cases, in pediatric cases,
12:53.2 where either a solitary or a single functioning kidney exists,
12:56.1 we also see renal growth,
12:58.2 suggesting again that these tissues can add cells
13:01.2 and can add size to the structures as well.
13:04.0 So it's not a static system,
13:05.2 it's a dynamic system that is constantly undergoing change.
13:08.3 So... great...
13:12.1 so, where is that change coming from?
13:13.2 Is it that you have stem cells residing in there,
13:15.2 and if they're there why can't we regenerate these things?
13:18.2 So, the most recent discovery was published in 2014
13:21.0 by Rinkevich in Irv Weissman's lab,
13:23.1 showing that by doing clonal analysis in mice,
13:26.1 there's constant repair and growth of the renal tubules.
13:29.1 They were able to identify, by clonal mapping,
13:31.1 individual cells that are dividing,
13:33.2 producing new cells and replacing those cells
13:36.0 that are shedding as we urinate on a regular basis,
13:38.0 suggesting the tissues are really constantly rebuilding themselves.
13:41.2 Okay.
13:43.1 So that means essentially that the kidney
13:45.2 really is a dynamic system, developmentally speaking, okay?
13:48.2 So, then, what do we know about the evolution of kidneys, alright?
13:52.0 So, if you look at the papers that are cited in the literature
13:54.3 about the evolution of kidneys,
13:56.1 you always see a reference for this paper,
13:58.3 from Howard Smith, 1943.
14:01.1 And if you look at the evolution of this tree of life,
14:03.2 what you see is essentially that
14:05.2 this is only the lineage that gives rise to us,
14:07.1 this is the so-called deuterostomes.
14:09.3 Most of the non-deuterostome invertebrates
14:11.3 are absent from this tree.
14:13.2 Why?
14:14.2 Because we're assuming that our kidneys
14:16.2 are so different from everybody else's
14:18.1 that we invented kidneys,
14:20.1 that deuterostomes invented kidneys,
14:22.2 and the other systems are doing something completely different,
14:24.2 analogous but not homologous to our excretory system.
14:27.0 And so it stops right here, somewhere down here,
14:31.0 as showing that...
14:33.0 what the structure of the nephridia, or the excretory tubules,
14:35.2 are in this system.
14:36.3 And there is good reason to, you know,
14:38.3 believe that that might be the case,
14:40.0 and all you have to do is just look at
14:43.0 diagrams of the kidneys, which I'm going to show you.
14:44.1 But before I do that,
14:46.0 what I would like to do is work from the following hypothesis,
14:48.1 and we hypothesize that
14:51.1 the kidney's ability for growth and repair,
14:52.3 rather than being of vertebrate origin,
14:55.1 is evolutionarily conserved
14:58.0 and dates back to the evolution of the excretory system itself.
15:00.2 And I'm hoping that I'm going to be able to provide you with
15:02.3 sufficient evidence to support this hypothesis.
15:05.2 So, why do we think that
15:07.1 the kidney was invented by vertebrates?
15:09.2 Well, you have to look at the unit,
15:11.0 at the functional unit of the kidney,
15:13.1 in this case, the nephron, okay?
15:14.2 So, what you see essentially is that
15:17.0 here is where the circulation goes,
15:18.1 it goes through this filtration apparatus referred to as podocytes,
15:21.1 it goes into the proximal tubule, the distal tubule,
15:24.1 urine is made, and it gets excreted.
15:26.2 And that's what this structure looks likes.
15:28.2 Okay, great.
15:29.2 We need an invertebrate model system for gene discovery.
15:32.1 How does Drosophila do it?
15:34.1 Well, there is no separation between the circulatory system
15:37.2 and the excretory system,
15:38.3 so they're not really homologous.
15:41.0 They're sort of analogous,
15:42.1 and so our nephron
15:46.1 is way more complex that the fly's,
15:47.3 so there's only a few thing we'd be able to study in the flies, okay?
15:52.2 Okay, what about C. elegans?
15:54.0 C. elegans must have an excretory system.
15:55.1 Yes, they do, but it's a single cell.
15:58.1 A single cell, alright?
15:59.3 So, if you look at this evidence, you say,
16:01.3 oh, all invertebrates, you know,
16:03.2 none of their excretory systems look anything like ours,
16:06.0 then you say, great,
16:07.2 vertebrates invented the kidney, right?
16:09.2 And so you look at the ancestor of the entire tree of life,
16:13.1 let's say cnidarians, okay,
16:15.2 well, they don't have an excretory system.
16:17.1 So that lends to the idea that the excretory system
16:19.2 was invented recently in evolution,
16:20.3 and you can build you arguments and say,
16:23.1 you know, vertebrates are the way to go,
16:25.2 and so therefore we need to only study the kidney in the vertebrates,
16:28.2 and the other organisms might provide us with some insights,
16:31.0 but not necessarily the insights required
16:34.0 to dissect diseases, functions, and malfunctions
16:37.2 and the like.
16:39.0 But if you look now at a close relative of the vertebrates,
16:41.2 in this case, amphioxus, okay,
16:43.1 what you see here is that, yes,
16:45.1 you have a circulatory system, you have this filtration system,
16:47.1 and these cells have cilia that beat
16:49.2 to make the fluid flow into the... uhh...
16:53.1 into the tubule so that it can be urinated.
16:55.0 Now, this group of animals,
16:57.2 which is highly understudied,
16:59.1 and contains the largest collection of body plans on the planet,
17:02.2 and for which we know almost nothing,
17:04.1 referred to as the Lophotrochozoans,
17:06.1 have very interesting excretory systems.
17:08.2 So, you can see a planarian, which is the one that I'm going to tell you about,
17:12.1 that structure is very similar to the amphioxus
17:14.1 in that it has a filtration apparatus,
17:17.1 it has cilia that allows the flow
17:20.1 to go into these tubules and then be excreted.
17:22.0 So, we asked the question:
17:23.2 how similar is this system,
17:25.2 this excretory system in planarians,
17:27.2 to the system that is found in the deuterostomes?
17:29.3 Do they share a common ancestor or not?
17:32.1 Or did they evolve independently?
17:35.0 So, here's what the excretory system looks like in planarians.
17:37.0 There's no singular kidney, associated in one organ.
17:40.2 The excretory system is distributed throughout the body of the animal,
17:43.1 and you can see all of these little white dots right there,
17:46.1 those are those cilia that beat,
17:48.0 so-called flame cells
17:50.1 that actually move the fluid that is going to be excreted
17:52.0 into the tubules for excretion from the animal.
17:54.1 They are distributed throughout the body of the animal,
17:56.3 you can see the head right here
17:58.1 and the tail of the animal,
17:59.2 these have different shapes or distributions along this tissue,
18:02.1 and what is nice about this tissue
18:04.1 is that not only does it have a proper distribution
18:06.0 along the anterior-posterior axis,
18:07.1 but also the dorsal-ventral axis.
18:09.1 And you can see here a depth pseudocoloring
18:12.0 of where the flame cells, the cells with cilia,
18:14.2 and the tubules are in this organism.
18:16.2 We can visualize these things.
18:18.1 Alright.
18:19.2 So, we refer to these structures as protonephridia,
18:21.3 and that's how the literature refers to them,
18:23.2 and it's part of the excretory system of these organisms.
18:26.3 So, this is what they look like.
18:28.0 So, we can now begin to visualize them
18:30.2 in three dimensions, and what we see is that
18:33.0 the fluid will enter through here,
18:34.2 go through these proximal tubules,
18:36.1 empty into these distal tubules,
18:37.2 and then these distal tubules enter
18:40.0 into the exit tubule
18:42.2 where the excretion occurs in these animals, okay?
18:46.0 So, it's a complex system, we can visualize it.
18:47.3 If we perturb it, we should be able to see
18:50.0 a specific perturbation.
18:51.2 So, the first thing that we did,
18:53.1 particularly a graduate student in my lab, Hanh Vu,
18:54.3 was to essentially clone all of the genes
18:58.1 that are known to be solute carriers in this animal.
19:01.2 And to do that, we looked at all of the solute carrier proteins
19:04.1 that are found in mammals
19:05.3 and we cloned all the homologs in planarians,
19:09.0 and then ask,
19:10.2 are the expressed in the protonephridia or not?
19:12.1 And it turns out that they are,
19:13.2 but they're not only expressed in a particular...
19:15.1 you know, in a random location,
19:17.1 they're actually expressed in very precise locations
19:20.1 along the proximal, distal, and collecting duct
19:23.2 of both the planarians
19:28.1 and homologous to the situation in mammals,
19:29.2 suggesting then that it's not just gene expression,
19:32.1 but it's structural along the length of the tubule,
19:35.1 providing the tubules specific identities and functions
19:37.3 along the route by which this fluid is going to be excreted.
19:41.1 So, solutes can be brought in,
19:43.1 others can be emptied out.
19:44.1 So, we tested that by looking at
19:47.1 the genes associated with controlling pH,
19:49.2 carbonic anhydrases,
19:51.2 these solute carriers.
19:53.0 And so we looked and found the homologs in both mice and
19:57.3 vertebrates... sorry...
19:59.1 and umm... planarians,
20:01.0 and what we see essentially is that when we perturb these genes by RNAi,
20:03.0 we used a pH sensitive dye,
20:05.2 we see that essentially the pH in the animal changes.
20:08.2 And these genes are located in an anatomical position
20:12.2 that is homologous to the anatomical position
20:15.1 of the mammalian nephrons.
20:16.2 That's millions of years of evolution
20:18.1 in a non-deuterostome invertebrate... coincidence?
20:21.2 Yes, it could be coincidence,
20:23.1 so we thought, how much of a coincidence is it?
20:25.0 Can we actually go further, so...
20:27.0 the function tells us already that it's functionally conserved.
20:31.2 So, now we can ask another question, which is:
20:35.1 do these homologies that we began to find between the nephrons
20:38.1 and the protonephridia
20:40.0 extend to common pathologies?
20:41.2 Because if the diseases are conserved, molecularly,
20:43.2 then it's no longer really a coincidence, alright?
20:46.2 So we asked the following question...
20:48.1 we know that, in the mammalian kidney,
20:50.1 mutations in these two molecules, NPHS1 and NEPH1,
20:53.2 cause what's called slit diaphragm loss,
20:56.0 which results in massive edema in humans.
20:58.3 Those kidneys look awful.
21:00.1 So, here's an electron micrograph of what I mean.
21:02.1 So, here's the... uhh...
21:04.2 the cell that makes the filtration,
21:06.1 this region right here referred to as podocytes,
21:08.1 and you can see those right here.
21:09.1 This is where the fluids are being filtered
21:11.1 from the circulation back into the excretory system.
21:14.3 We now take electron micrographs of our planarians
21:17.0 in the same location of the excretory system,
21:19.1 the so-called flame cells,
21:21.2 and what we noticed was that, yes, there's cilia there,
21:24.2 which is absent in here,
21:26.0 but we saw these beads along the edge of the animal
21:28.2 that resemble, significantly,
21:30.1 the structure of these podocytes.
21:32.1 It turns out that when we do in situ hybridizations of these molecules,
21:34.3 that's exactly where they're expressed,
21:36.2 which is exactly where they are expressed in the vertebrate condition.
21:39.2 Alright.
21:40.2 So, what does that mean?
21:42.0 It means that if we now perturb it, we should expect to see edemas, alright?
21:46.0 So we go ahead and silence those genes by RNAi
21:48.1 and that's exactly what we get:
21:50.1 planarians that now cannot excrete and they get edematous
21:53.1 and they eventually begin to blow up
21:55.1 and then just die.
21:56.2 If we take an electron micrograph of these animals after RNAi,
22:01.2 this is what we find.
22:03.1 We find that those podocytes,
22:05.0 which is here in the normal condition
22:07.1 -- you can see those really nice filtration sites --
22:09.2 it's gone in both of these.
22:11.3 And this is indistinguishable from the vertebrate condition.
22:15.1 When those genes are absent,
22:16.3 the podocytes are not made, edema occurs,
22:19.1 and there's renal malfunction.
22:21.2 So that's already another line of evidence
22:23.3 to suggest that it's not just the genes that are expressed
22:26.0 in the right location
22:27.1 and that the structures are homologous,
22:28.3 but the pathologies are also conserved.
22:31.2 So, we can go one step further
22:33.1 and look at other pathologies
22:35.1 that are found in the kidney,
22:36.2 for which there are no invertebrate model systems
22:38.3 for us to compare.
22:40.2 So, the cystic kidney disease or diseases, so to speak...
22:46.2 they can only be modeled in mammals, so far,
22:49.1 and so we've asked the question,
22:51.1 well, can we find model molecules associated with cystic kidney disease
22:53.2 in the planarian genome,
22:55.1 and what do they do in planarians?
22:56.2 So, things like the nephrocystins, shown right here,
22:59.1 which actually cause diseases like nephronophthisis,
23:01.2 and when we look at the genome
23:04.1 what we find is that we find a lot of the genes
23:06.1 that are known in humans to be defective
23:08.1 to cause these diseases
23:10.0 and, interestingly enough,
23:11.3 these genes are missing in Drosophila and C. elegans.
23:14.2 So that's probably why we cannot model these diseases
23:16.2 in these organisms because,
23:18.1 well, they're lacking the genes.
23:19.2 But planarians have those genes, so what does it mean?
23:22.2 Well, it means that Drosophila and C. elegans lost those genes,
23:25.1 planarians have them, humans have them,
23:28.1 the ancestor should have had both.
23:30.2 Because they couldn't have invented both of these genes independently,
23:33.2 suggesting that the origin of the excretory system
23:36.0 is really conserved.
23:37.1 They share a common ancestor.
23:39.2 So now we can ask the question:
23:41.3 if that is true and we go ahead
23:44.1 and perturb the functions of these molecules,
23:46.0 will we get cystic kidney disease or not,
23:50.2 in planarians?
23:52.0 So, that's what we did, and so we can see here
23:54.3 in the Nephrocystin 8 [RNAi], what you can see essentially is
23:59.1 the solute carrier in purple is much more...
24:01.1 sorry, in red...
24:02.3 is much more abundant than in the control, right here,
24:05.2 and when you superpose with tubules
24:07.3 you can see that you actually have cysts in there.
24:09.2 This is very similar to the situation that you see in mammals, okay?
24:13.1 You can now reconstruct this whole image three-dimensionally,
24:16.1 and you can now clearly see where the cysts are
24:18.3 along the protonephridia of these organisms,
24:21.2 suggesting then that it's not just that the molecules are shared,
24:23.0 also the pathologies are shared.
24:26.1 We can take this even further and ask the question
24:29.1 that is associated with cystic kidney disease,
24:30.2 which is that the kidney somehow senses the rate of flow,
24:34.3 and when that is perturbed that also causes
24:38.1 cystic kidney disease in humans.
24:39.2 So now we can ask the question:
24:40.3 if we perturb flow in planarians,
24:42.2 in the protonephridia,
24:44.0 will we get cystic kidney-like disease in planarians or not?
24:47.3 So, we do that.
24:49.0 So, we look at the cilia, here's the control,
24:50.2 the cilia are beating just fine.
24:52.1 We get rid of a molecule that is associated with the flagellar motility,
24:57.0 and you can see that the cilia are not beating normally,
24:59.1 so this will actually affect the flow rate of the organism,
25:02.3 and you can look at the other molecule
25:05.1 that's involved in making the cilia, LRRC50,
25:07.2 and there's no movement either.
25:09.2 And when we do that, just by perturbing the flow,
25:12.0 by targeting these molecules with RNAi,
25:14.1 we actually were able to produce cysts,
25:16.3 or cyst-like structures, in planarians.
25:19.1 So it's not just the genes but it's also the function,
25:21.2 the sensing of the flow,
25:23.1 suggesting that this is way too much of a coincidence
25:27.0 and that, in fact, the ancestor to the vertebrate kidney
25:30.1 was shared by both the protostomes
25:32.3 and the deuterostomes.
25:34.1 That then provides us with a novel system
25:37.2 for us to actually do gene discovery.
25:39.1 We don't know why sensing of flow malfunction
25:43.1 results in cysts in mammals
25:45.3 -- we just don't know why that is.
25:47.2 It's difficult to do those experiments in mammals
25:49.2 for very good reasons,
25:51.1 but if you have a model that actually shares structure,
25:53.2 it shares genes,
25:54.3 and it shares malfunctions,
25:56.3 that is a good a model as you're possibly going to get
25:58.3 to be able to do gene discovery
26:00.1 and try to identify the root cause
26:02.2 of some of these diseases that occur in mammals.
26:05.2 So we think that it's not just
26:08.1 the function and the structural conservation,
26:10.0 but also the fact that
26:12.1 you can get cystic tubules by just perturbing flow.
26:15.0 And that that disease arises from
26:18.2 the misregulation and misproliferation
26:21.1 of the stem cells that probably reside in the tubules
26:23.1 to allow for this overgrowth to take place.
26:26.2 And so I think that this system will allow us, now,
26:29.0 to uncover the molecular and perhaps cellular basis
26:31.1 of the early ontogenetic processes
26:33.1 that result in the production of some of these diseases,
26:35.2 which are very, very difficult to sort out in mammals
26:38.2 because it's expensive to do those experiments.
26:41.1 So, I hope that I have convinced you that
26:45.1 there is benefit in looking at organisms
26:47.2 that are not necessarily similar to us,
26:49.3 because I think that if you make the argument
26:54.2 that an organism that has a particular biological attribute is weird,
26:58.1 but it's... oh yeah, that only happens in planarians,
27:00.2 or that only happens in protists, okay...
27:03.1 but it's not in mammals...
27:05.0 when you make that argument, you're essentially denying evolution.
27:07.3 It's the equivalent of being a climate change denier,
27:10.0 because you're saying that because it only happens in planarians,
27:13.1 it's only specific to planarians,
27:14.3 it's only specific to those organisms,
27:16.2 when that is not the case.
27:17.3 That is the power of evolution
27:20.1 trying to help us understand the ontogeny
27:22.2 and the development of these diseases
27:24.1 across the phylogenetic tree of life,
27:26.2 and all of these organisms will have specific attributes that are exaggerated,
27:30.2 that we should be able to exploit to be able to inform our own biology.
27:34.3 So I think we need more of these types of paradigms.
27:37.1 So, we think that bilaterian secretory system
27:39.2 shares a common ancestor.
27:41.0 We think that these complex topological and molecular similarities
27:44.1 are inescapable, they are there,
27:47.1 they are verifiable,
27:48.2 and therefore suggest some conservation.
27:50.3 We also think that the excretory system genes
27:54.1 that are shared with vertebrates
27:56.1 clearly have been lost in Drosophila and C. elegans,
27:57.2 so there is yet a need for identifying new model systems
28:01.3 to fill in those gaps.
28:03.2 And then the shared pathologies, you know,
28:06.2 the podocyte-deficient edema and the CKDs,
28:10.0 really suggest that planarians and perhaps other organisms like them
28:13.3 will allow us to identify orthologous invertebrate paradigms
28:18.1 in which to model vertebrate kidney development and disease.
28:23.1 Thank you.

Videos in this Talk
  • Part 1: Planaria: Scale, Proportion and Organ Regeneration
    Part 1: Planaria: Scale, Proportion and Organ Regeneration
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 2: Planarian Stem Cells
    Part 2: Planarian Stem Cells
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
  • Part 3: Expanding the Number of Model Systems is Essential
    Part 3: Expanding the Number of Model Systems is Essential
    Audience:
    • Student
    • Researcher
    • Educators of H. School / Intro Undergrad
    • Educators of Adv. Undergrad / Grad
Speaker: Alejandro Sánchez Alvarado
Total Duration: 1:29:57
Recorded: July 2015
All Talks in Development and Stem Cells

Talk Overview

Planarians are free-living flatworms best known for their amazing ability to regenerate. They also “shrink”, by losing cells, during starvation. In his first lecture, Dr. Sánchez Alvarado discusses the intrinsic problems of scale and proportion that face planarians during growth and regeneration. How do these organisms increase and decrease their cell number without perturbing the functionality of the different tissues?  How do they regenerate a specific organ and how does it integrate and function properly? By performing an RNAi screen of the planarian pharynx, Sánchez Alvarado and his lab were able to identify genes required for different steps in these complex processes.

In his second video, Sánchez Alvarado focuses on planarian stem cells called neoblasts.  By identifying genetic markers for each stage of stem cell differentiation (pre, early, late and terminal differentiation), Sánchez Alvarado’s lab was able to show that neoblasts are truly totipotent, giving rise to all cell types.  Surprisingly, they also demonstrated for the first time that planarian neoblasts undergo acentriolar mitosis, a process previously known to occur only during meiotic cell division in animals.

Scientists, influenced by grants and resources, are focusing their studies on a small number of model organisms. Sánchez Alvarado argues that the diverse array of understudied organisms could provide us with insights into previously unknown biological processes.  By comparing excretory pathways, Sánchez Alvarado has demonstrated extensive similarities between vertebrate kidneys and planarian protonephridia.  In fact, mutations that cause human polycystic kidney disease induce similar pathologies in planarians. These findings reflect on the ability of non-traditional model organisms to provide insight into biology and human disease.

Speaker Bio

Alejandro Sánchez Alvarado

Alejandro Sanchez-Alvarado

Dr. Alejandro Sánchez Alvarado is an Investigator at the Stowers Institute for Medical Research in Kansas City, MO and the Howard Hughes Medical Institute.  He is also co-director of the summer course on embryology at the Marine Biological Laboratory in Woods Hole, MA. Sánchez Alvarado moved from Venezuela and received his Bachelor’s degree in Molecular… Continue Reading

Playlist: Stem Cells

Related Resources

Carolyn E. Adler and Alejandro Sánchez Alvarado (2015). Types or States? Cellular Dynamics and Regenerative Potential. Trends in Cell Biology, 25: 687–696. https://www.ncbi.nlm.nih.gov/pubmed/26437587

Carolyn E. Adler, Chris W. Seidel, Sean McKinney and Alejandro Sánchez Alvarado (2014). Selective amputation of the pharynx identifies a FoxA-dependent regeneration program in planaria. eLife 3.  https://www.ncbi.nlm.nih.gov/pubmed/24737865

Hanh Thi-Kim Vu, Jochen C. Rink, Sean A. McKinney, Melainia McClain, Naharajan Lakshmanaperumal, Richard Alexander and Alejandro Sánchez Alvarado (2015). Stem cells and fluid flow drive cyst formation in an invertebrate excretory organ. eLife 4. https://www.ncbi.nlm.nih.gov/pubmed/26057828

Alejandro Sánchez Alvarado and Shinya Yamanaka (2014). Rethinking differentiation: stem cells, regeneration, and plasticity.  Cell, 157:110-9. https://www.ncbi.nlm.nih.gov/pubmed/24679530

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