Session 7: Evolution of Vertebrates

00:00:07.18 Hi, my name is Melina Hale.
00:00:09.01 I'm a professor at the University of Chicago.
00:00:11.14 My lab works on
00:00:12.25 neurobiology, biomechanics,
00:00:14.09 and evolution.
00:00:16.18 In a previous video,
00:00:17.28 I gave an introduction to evolution,
00:00:20.08 and now I want to give you
00:00:22.08 an example of how we study evolution
00:00:24.02 in our lab,
00:00:25.14 and one of the things we look at is
00:00:27.20 the evolution of neural circuits.
00:00:28.27 So I'll talk about one of the systems we study,
00:00:31.28 which is the startle response,
00:00:33.12 its neural circuits,
00:00:34.20 and how we think they're evolving
00:00:36.22 over time.
00:00:37.26 So, the example on the screen here is...
00:00:41.02 you might be able to tell it's a fish.
00:00:42.07 It's actually a larval zebrafish.
00:00:44.09 It's about five days old
00:00:45.26 and three millimeters long,
00:00:47.19 and the probe you see
00:00:50.18 coming in from the left side of the screen
00:00:53.20 is a little injector and it has dye in it,
00:00:55.20 and one of the ways we get fish to startle,
00:00:58.14 to study this behavior,
00:01:00.20 is through puffing a little bit of dye at their body
00:01:02.29 and then watching their response.
00:01:04.22 So, you'll see its response.
00:01:06.13 It's analogous to a startle in humans,
00:01:09.28 which might be if you're at a scary movie
00:01:11.16 and something surprising happens,
00:01:13.08 we jump.
00:01:15.05 This is the fish equivalent of that.
00:01:16.27 It looks a bit different.
00:01:18.18 Let me show you the video
00:01:20.22 and we can talk about it a little bit.
00:01:22.12 So, that's the startle.
00:01:23.28 So, you might have been able to see the dye
00:01:25.22 hit the side of the head of the animal,
00:01:27.12 and then you see this big turn to one side,
00:01:30.04 and the animal swims away.
00:01:31.26 This is the behavior and circuit
00:01:33.24 we'll mainly be talking about today.
00:01:40.14 So, let's back up for just a minute, though,
00:01:43.09 and talk about how we study the brain.
00:01:45.29 A lot of different researchers
00:01:48.02 are working on brain function and structure,
00:01:50.16 and have been for many, many years,
00:01:52.04 and there's a lot of beautiful work going on.
00:01:54.14 Some of it tries to
00:01:56.26 identify how regions of the brain are functioning.
00:01:59.10 So, here we see half of the brain, only,
00:02:02.00 in a translucent skull,
00:02:04.13 and in red is one little part of the brain
00:02:06.29 that's called Broca's area,
00:02:08.13 and you'll see it when it comes back around,
00:02:10.02 right there.
00:02:11.12 This is actually part of the brain
00:02:13.05 that's really active when we are speaking,
00:02:15.13 so as I'm talking to you,
00:02:17.20 that part of my brain
00:02:19.20 is really, really active.
00:02:21.13 And people go and study
00:02:23.02 all these different regions of the brain
00:02:24.13 to understand when they're active
00:02:26.20 in relationship to behavior
00:02:28.28 and how they interact.
00:02:30.11 So, in addition to understanding
00:02:32.24 the cells that are in these locations,
00:02:34.12 like in Broca's area,
00:02:37.02 researchers are also trying to understand
00:02:39.15 how these cells connect.
00:02:40.26 So, the cells in the nervous system
00:02:43.13 are not just little balls
00:02:45.01 like you might see in some typical pictures of cells.
00:02:48.09 They have long processes on them
00:02:50.09 and those processes connect between cells,
00:02:53.01 from cell to cell,
00:02:54.10 and among regions of the brain.
00:02:55.24 So, what we're looking at on this image
00:02:58.01 is lots of different processes,
00:03:00.01 from regions of the brain,
00:03:01.15 from particular neurons
00:03:03.10 that are traveling to other parts of the brain
00:03:04.25 to integrate information,
00:03:06.19 to transmit information,
00:03:08.12 and ultimately to generate
00:03:11.05 some sort of function,
00:03:12.09 whether it's a behavior
00:03:13.26 or a stored memory...
00:03:15.13 various things like that.
00:03:19.21 Another way we've been studying the brain,
00:03:22.05 in addition to doing experiments
00:03:26.01 that try to look at
00:03:27.25 connections and regions,
00:03:29.25 is through wonderful neurology work
00:03:32.17 that's examined how injuries
00:03:35.03 are associated with changes
00:03:36.26 in brain function and behavior.
00:03:38.12 And that work started a very, very long time ago.
00:03:42.02 These are pages from the
00:03:43.13 Edwin Smith papyrus,
00:03:45.09 which is from about 1500 BC,
00:03:48.08 and is the first, the earliest documented
00:03:52.16 incident of people
00:03:55.15 actually relating an injury
00:03:57.18 to the nervous system,
00:03:58.26 or to the body,
00:04:00.17 to a behavior,
00:04:02.06 so seeing that there's an injury
00:04:04.03 to a part of the spinal cord or brain
00:04:05.25 and how that affects
00:04:07.21 the movements of an animal
00:04:09.08 or the behavior of a human,
00:04:11.13 in particular in this.
00:04:13.11 In my lab, we're interested in understanding
00:04:16.13 how particular neurons, nerve cells,
00:04:19.01 connect to one another,
00:04:20.11 and how those cells
00:04:22.02 and the simple circuits they're a part of
00:04:24.15 generate movements,
00:04:25.26 the startle movement in particular
00:04:27.19 that I showed you earlier.
00:04:29.08 So, to understand circuits and how they function,
00:04:32.15 a lot of us use simple model systems
00:04:35.10 for that work.
00:04:36.24 They're much simpler,
00:04:38.09 less complicated than the human brain
00:04:40.06 or large brains,
00:04:42.01 but yet they let us get at basic principles
00:04:44.15 of how the nervous system functions.
00:04:46.19 One of the fantastic models
00:04:48.16 that's been used for neural circuit work
00:04:50.23 is the nematode C. elegans.
00:04:54.16 The nematode is pictured right down here.
00:04:56.27 It's a tiny little worm,
00:04:58.26 really, really small.
00:05:00.15 It has only a small number of neurons
00:05:03.00 in its nervous system,
00:05:04.12 a few over 200,
00:05:06.19 and folks working on C. elegans
00:05:08.25 have been able to map
00:05:10.27 how those neurons connect to one another.
00:05:12.22 So, what you're seeing in these little blips
00:05:14.28 to the left of the nematode
00:05:16.20 are different neurons and their pathways,
00:05:19.03 the connections from one to the other.
00:05:23.05 Now, there are a number of other
00:05:25.08 simple model systems that have been
00:05:27.07 used productively in neuroscience
00:05:29.09 -- the sea hare on the left,
00:05:31.02 lobsters, lampreys,
00:05:32.25 and bees are just a few examples of those --
00:05:35.15 and they've provided an understanding of
00:05:39.07 foundational principles of connectivity and function,
00:05:42.14 really important things that
00:05:44.17 have influenced a lot of work in neuroscience,
00:05:46.04 for example the work in learning and memory
00:05:48.23 in the sea hare on the left.
00:05:53.10 When you're interested in evolution, though,
00:05:55.22 as we are,
00:05:57.13 we still have some issues here.
00:05:58.23 So, we can have all of these simple models
00:06:01.03 spread across the phylogeny,
00:06:02.27 but because there's so few of them,
00:06:04.16 and they're so far apart,
00:06:06.13 trying to reconstruct
00:06:08.12 the evolutionary history of a neural circuit
00:06:11.13 is really difficult,
00:06:13.03 and we're just at the beginning stages
00:06:14.21 of being able to do that
00:06:16.16 in organisms.
00:06:19.05 The startle behavior,
00:06:21.22 for several reasons that I'll talk about
00:06:23.15 in a few minutes,
00:06:24.26 is one of the best systems
00:06:26.28 for understanding how neural circuits evolve.
00:06:29.27 Part of the reason that it's so useful
00:06:32.09 is that because the startle behavior
00:06:34.10 occurs in so many animals
00:06:36.12 across the phylogeny,
00:06:37.28 it's a key predator avoidance behavior,
00:06:40.04 so you might imagine, evolutionarily,
00:06:42.06 it's going to be very important and highly conserved.
00:06:45.17 What we're looking at here is
00:06:48.05 a pike, a northern pike,
00:06:50.23 the image on the right,
00:06:52.10 and what you're seeing on the left is a still from a video
00:06:54.11 that I'll show you in a second.
00:06:55.28 Just to orient you to that video,
00:06:57.12 we're actually looking at the belly of the fish,
00:06:59.19 the ventral view,
00:07:01.15 and that gray shadow above it
00:07:03.12 is my hand coming over,
00:07:05.05 and I'm going to startle this animal.
00:07:07.22 So, if we look at the movie of that behavior,
00:07:10.16 you'll see how the pike startles,
00:07:14.12 so let's... it's going...
00:07:16.24 there, I've touched the fish on the head
00:07:18.28 and you can see it do that really fast movement away,
00:07:21.22 like the larval zebrafish
00:07:23.26 in the first image that I showed you.
00:07:26.13 Now, this type of startle,
00:07:28.07 this bend to one side and swim away,
00:07:31.10 is characteristic of many, many species of fish.
00:07:34.17 If we look at in just a set of silhouettes,
00:07:38.10 we can really break down what that behavior is.
00:07:42.08 Generally, these fish start
00:07:44.02 in a straight position,
00:07:45.20 so as they're sitting in the water
00:07:47.08 not expecting a predator to come along,
00:07:49.01 they're just hanging out and their body is straight.
00:07:51.08 When the sense a predator coming from one side,
00:07:53.20 as shown in image three,
00:07:55.11 they do this massive turn and bend,
00:07:58.13 they orient their head away from the predator,
00:08:01.22 and then they use a number of swimming strokes
00:08:04.09 to get out of there as fast as they can.
00:08:06.29 This is a typical response, as I said,
00:08:09.16 of a number of fish including zebrafish,
00:08:11.20 and pike, and many others.
00:08:15.18 Now, what's driving that behavior? Let me tell you a little bit about the
00:08:19.17 neural circuit that's important
00:08:22.02 for that response.
00:08:24.11 The circuit is highly conserved,
00:08:26.07 so we see some of the same neurons
00:08:28.22 in pike, and in zebrafish,
00:08:30.10 and all the other species,
00:08:31.21 even though they're hundreds of millions of years
00:08:34.27 separated in evolutionary time,
00:08:37.11 in terms of when they diverged.
00:08:39.19 So, this is...
00:08:41.29 you might not be able to tell right away,
00:08:43.25 but this is the brain of the zebrafish,
00:08:46.02 with certain neurons labeled.
00:08:48.00 So, just to orient you...
00:08:49.19 here are the eyes and the ears of the brain,
00:08:53.00 the otic capsules,
00:08:54.23 and all those white blips
00:08:56.17 are different nerve cells
00:08:58.14 that are in the brain
00:08:59.26 and are sending those processes,
00:09:01.15 what are called axons,
00:09:02.29 down into the spinal cord of the animal.
00:09:04.20 So, they're sending signals
00:09:06.11 down to the body
00:09:07.26 to get the fish to do something.
00:09:09.16 This is a larval zebrafish,
00:09:11.20 so the head is only a millimeter or so across.
00:09:14.07 Now, these, here, are really important cells
00:09:18.13 for the startle response.
00:09:20.05 They're called the Mauthner neurons
00:09:21.23 and there are only two of them.
00:09:23.17 They're giant neurons that are in
00:09:26.07 all of these fish
00:09:27.23 that do this type of startle that I've shown you,
00:09:30.12 and they're located in the hindbrain of the animal.
00:09:34.13 Up here are a different set of neurons
00:09:36.23 we won't be talking about,
00:09:38.04 but they're neurons in the midbrain
00:09:39.21 that control that rhythmic swimming
00:09:41.17 that you might have seen at the end of the videos
00:09:44.00 that I showed you.
00:09:45.19 So we're going to focus on the Mauthner cell
00:09:47.21 and the Mauthner cell circuit.
00:09:50.08 So, this is a simplified
00:09:52.08 Mauthner cell circuit,
00:09:53.23 so the Mauthner cell
00:09:55.10 and the neurons that it connects to.
00:09:56.28 I just wanted to show you a little bit about
00:09:59.09 how it works in the organism.
00:10:01.09 So, if we have a stimulus in the circuit,
00:10:03.07 coming from the right,
00:10:04.28 then here's the circuit...
00:10:06.17 the stimulus is coming from the right...
00:10:08.03 the Mauthner cell on the right,
00:10:10.04 which is this big, dark blue cell,
00:10:12.12 is going to fire.
00:10:13.29 When it fires, it's actually generating
00:10:16.14 what's called an action potential,
00:10:18.01 a signal that's transmitted
00:10:19.28 all the way down its process,
00:10:21.12 down its axon.
00:10:22.25 In this case, it goes
00:10:25.06 from being in the hindbrain,
00:10:26.18 which is that top group of cells on this image,
00:10:28.16 down into the spinal cord.
00:10:30.11 In the spinal cord,
00:10:32.21 it'll signal other interneurons and motor neurons
00:10:35.16 to signal muscle
00:10:37.11 to cause the body bending
00:10:39.03 that I showed you in those videos.
00:10:40.17 So, if I'm the fish
00:10:42.14 and this is, you know, this is me,
00:10:44.17 a stimulus coming from this side of my head
00:10:46.19 would cause the Mauthner cell to fire,
00:10:49.19 it would send a signal down my spinal cord,
00:10:52.03 and I would bend in that direction,
00:10:54.16 forming that C-shaped bend
00:10:56.12 that you saw in those videos.
00:11:00.28 Now, one of the really interesting things
00:11:04.03 about the Mauthner cell
00:11:05.19 is that there's this special part of the circuit
00:11:08.03 that's really not diagrammed in detail here.
00:11:10.07 It's called the axon cap,
00:11:12.09 and the axon cap has all sorts of interesting things
00:11:14.20 going on in it.
00:11:16.00 It has cells coming in
00:11:17.22 that excite the Mauthner cell
00:11:19.03 and get it to fire more readily.
00:11:20.18 It also has neuron processes coming in
00:11:24.15 that inhibit the Mauthner cell
00:11:26.07 and prevent it from firing.
00:11:27.19 So, there's a lot going on in this region.
00:11:30.02 Because the Mauthner cell
00:11:31.23 is so big and obvious,
00:11:33.26 and so conserved across a lot of taxa,
00:11:36.16 we can use the cell as an anchor
00:11:39.15 to understand the circuit components
00:11:41.27 that are in the axon cap structure.
00:11:46.18 So, let me show you a Mauthner cell firing
00:11:49.19 and then we'll talk a little bit more about it.
00:11:52.28 So, this is a Mauthner cell
00:11:55.02 that I've injected with
00:11:56.24 what's called a calcium-sensitive dye.
00:11:59.00 So, when the Mauthner cell fires,
00:12:01.22 calcium floods into the cell,
00:12:03.27 and the dye actually
00:12:05.27 reacts with the calcium
00:12:07.13 and becomes brighter when the cell is active.
00:12:10.09 Now, we've color-coded brightness
00:12:12.06 with this heat map,
00:12:13.21 so that when this cell fires
00:12:15.20 you'll see more reds and oranges.
00:12:19.01 So, here's the Mauthner cell
00:12:20.17 and you can imagine I'm tapping the fish on the head
00:12:23.07 from this side,
00:12:24.17 and you can see what happens.
00:12:27.16 So, that's a Mauthner cell activation;
00:12:30.16 that's the activity
00:12:32.18 that's initiating that big bend
00:12:34.18 that we showed you.
00:12:37.07 Let me show you one more time...
00:12:39.04 touch the fish
00:12:41.05 and it fires really, really quickly.
00:12:42.29 Something I didn't point out before
00:12:44.25 is that these various videos I've shown you,
00:12:46.14 and even this image,
00:12:48.00 are shown slowed down quite substantially.
00:12:50.14 A typical Mauthner cell response...
00:12:53.25 the behavior would occur
00:12:55.29 within tens of milliseconds,
00:12:58.02 so in less than the blink of an eye.
00:13:02.00 So, studying the Mauthner cell overall,
00:13:04.23 and this is not my lab specifically,
00:13:06.18 but in this broad group of researchers
00:13:08.24 that have looked at the Mauthner cell
00:13:10.18 over 100+ years,
00:13:12.09 we've learned a lot about neuroscience.
00:13:14.20 This simple system has been really, really valuable.
00:13:17.22 We've looked and been able to understand
00:13:19.24 more about the subcellular structure
00:13:21.18 of vertebrate neurons,
00:13:23.10 how neurons communicate to each other
00:13:25.13 through different types of synapses.
00:13:28.05 We've learned about this transmission
00:13:30.01 of electrical signals between neurons as well.
00:13:33.26 In addition, the Mauthner circuit
00:13:35.28 has been used to study learning
00:13:38.06 and to look at the relationship
00:13:40.00 between neural activity and behavior.
00:13:43.00 So, it's given us access to circuits
00:13:45.16 in a way that we just can't get
00:13:47.17 in a human brain, for example.
00:13:50.29 Alright, so let's talk a little bit
00:13:53.24 about evolution of this circuit now.
00:13:56.02 So, I introduced you to the Mauthner cell
00:13:58.12 and told you that's found
00:14:00.03 in lots of different animals,
00:14:01.24 and I also mentioned this structure
00:14:03.12 called the axon cap,
00:14:04.24 which has excitatory inputs and inhibitory inputs,
00:14:07.05 and gives us a little window
00:14:09.04 into how neural circuits might work.
00:14:11.12 So, this is a phylogeny
00:14:13.12 that I showed on the previous video
00:14:15.08 that I did on an introduction to evolution.
00:14:18.20 It's again of just vertebrate animals,
00:14:21.01 and only a very, very few of them
00:14:23.10 where we see on the left
00:14:25.09 the jawless fishes, like lampreys.
00:14:27.21 We move up the tree through the chondrichthyans
00:14:30.02 into fishes,
00:14:32.06 and then tetrapods as well.
00:14:34.29 Now, for the Mauthner cell,
00:14:36.22 we see its presence
00:14:39.02 in many of these different groups.
00:14:41.00 Lampreys have the Mauthner cell.
00:14:42.24 Chondrichthyans, in a few species we see Mauthner cells.
00:14:46.04 Many, many fishes,
00:14:48.00 the vast majority of the 30,000 species of fishes
00:14:51.02 that are known have Mauthner cells.
00:14:53.11 And even in amphibians,
00:14:55.15 in both tadpoles and in some frogs,
00:14:57.25 as well as in salamanders,
00:14:59.10 we also see Mauthner cells.
00:15:02.04 When we move up into the tree, though,
00:15:04.20 we have quite a mystery to solve.
00:15:06.29 We do not know what happens
00:15:09.10 to these giant, really important neurons
00:15:12.05 once we move up into the tetrapod animals
00:15:15.24 that have become terrestrial
00:15:17.16 and have changed their startle behaviors.
00:15:19.12 It's one of these big questions
00:15:21.27 that I'm so curious about and my lab's trying to investigate.
00:15:27.10 So, let's look at that cap
00:15:29.05 and see where we've gotten
00:15:30.20 in terms of understanding how the Mauthner cells
00:15:32.07 and their circuits have evolved.
00:15:34.27 So, remember where those yellow stars were
00:15:37.12 on that very simplified diagram
00:15:39.04 of the Mauthner circuit.
00:15:40.13 This is what we're looking at here on the left,
00:15:42.19 that cap structure,
00:15:43.29 and as I mentioned there are excitatory neurons
00:15:46.23 that are connecting to the Mauthner cells.
00:15:48.15 Those are shown as these cells that are...
00:15:50.29 this process that's spiraling around the base
00:15:53.19 of the cell on the far left.
00:15:55.17 Then we see this sort of egg-shaped capsule
00:15:57.26 around that process of the cell,
00:16:01.09 and those little black nubbins coming in
00:16:04.17 are the inhibitory interneurons,
00:16:06.05 that are shutting down the Mauthner cells.
00:16:07.27 So, we have excitatory and inhibitory components.
00:16:11.01 On the right, you can see what that actually looks like
00:16:14.06 in a section of a brain of one of these fishes.
00:16:17.02 So, this is what we call the composite axon cap.
00:16:19.09 It has all these different components to it.
00:16:21.20 This is the cap that you would see
00:16:24.09 in fish that...
00:16:25.26 if you go to an aquarium
00:16:27.11 and are looking at a lot of fish swimming around in a tank,
00:16:29.20 or at a pet store,
00:16:31.04 most of them have Mauthner cells that look like this.
00:16:35.10 Now, I showed you this image of the startle
00:16:38.16 in the top panel, here,
00:16:40.12 this is shown in a trout,
00:16:42.15 and the composite cap and Mauthner cell
00:16:45.08 generates this type of startle.
00:16:47.14 But startles in fish are actually
00:16:48.28 a bit more diverse than that,
00:16:50.25 and in particular if we look down in the lampreys,
00:16:52.28 which diverged off the of the main lineages of vertebrates
00:16:55.22 at the base of the tree,
00:16:57.01 we see a very different type of startle.
00:16:59.08 It's a retraction response,
00:17:00.28 shown down below here,
00:17:02.22 where the animal, actually, when stimulated,
00:17:05.24 kind of contracts or accordions its body.
00:17:08.17 In the lampreys, the Mauthner cells
00:17:11.05 are there,
00:17:12.20 but they don't have this axon cap structure,
00:17:14.16 so without these inputs
00:17:16.12 they generate a very, very
00:17:18.14 different behavioral output.
00:17:23.22 So, here are the two primary basic
00:17:27.16 types of caps that have been known for awhile:
00:17:30.12 this composite cap of typical teleosts fishes,
00:17:32.29 typical bony fishes,
00:17:34.11 and this no cap condition
00:17:36.11 that we see in these retracting animals like lampreys.
00:17:41.06 In my lab,
00:17:43.11 one of my students, Hilary Bierman,
00:17:45.01 and a colleague, Steve Zottoli,
00:17:47.06 worked with me to try to get
00:17:49.11 a better sense of how this cap structure
00:17:51.25 varies across species,
00:17:53.17 and whether there is actually variation
00:17:55.29 between those two basic morphs.
00:17:58.05 When we look at lots and lots of different species,
00:18:00.25 we actually found that there are
00:18:03.11 four types of caps.
00:18:04.21 In addition to those first two that I talked about,
00:18:06.22 we have what's called a simple cap,
00:18:08.14 that's shown on the top,
00:18:10.03 where we have those winding excitatory fibers
00:18:12.19 at the base of the Mauthner cell,
00:18:14.19 and then we have what we're calling
00:18:17.05 a simple dense cap,
00:18:18.13 where we have those same winding fibers,
00:18:20.21 but they're much, much more populous,
00:18:22.17 much denser in the axon cap region.
00:18:25.26 Note both of these cases,
00:18:27.20 and these images in the diagrams shown here,
00:18:30.09 we don't have that nice glial...
00:18:35.04 that nice cap around the axon
00:18:37.13 where we have those inhibitory interneurons coming in,
00:18:40.07 so they differ quite a bit from the composite cap.
00:18:43.26 So, let's map the Mauthner cells
00:18:46.10 and the Mauthner cell axon cap
00:18:47.27 onto the phylogenetic tree of vertebrates,
00:18:51.07 and this is a bit more of a complicated tree,
00:18:53.11 a detailed tree,
00:18:54.29 than what I've shown you previously.
00:18:56.19 At the base, shown in yellow,
00:18:58.23 are two clades that actually even aren't vertebrates.
00:19:01.08 We see the amphioxus there
00:19:03.15 and hagfishes.
00:19:05.05 Amphioxus and hagfish
00:19:06.24 actually don't even have Mauthner cells.
00:19:08.09 We think the Mauthner cells
00:19:10.05 arose before the lampreys,
00:19:12.03 which are shown in white,
00:19:14.03 branched from the rest of the vertebrates.
00:19:16.00 Now, as I said previously,
00:19:18.06 the lampreys do this retraction response
00:19:19.17 and they don't have axon caps at all.
00:19:23.07 As we move up the tree,
00:19:24.11 we see several lineages
00:19:26.11 that are shown in purple
00:19:27.29 that have a simple cap structure
00:19:30.07 that I showed you.
00:19:32.06 Those includes things like sharks
00:19:34.17 and, as we move up, amphibians and lungfish,
00:19:37.08 as well as some of the basal bony fishes
00:19:40.08 called bichers and ropefishes.
00:19:43.14 Moving up in the tree from there,
00:19:45.07 we see the evolution of the simple dense cap,
00:19:47.28 and that's in these species
00:19:50.23 that we don't actually have a lot of extant today,
00:19:53.13 they are some species, but not a ton,
00:19:55.23 of sturgeons and gar,
00:19:57.29 and there's really only one extant species
00:20:00.03 of bowfin
00:20:02.01 that was a very diverse group
00:20:03.21 many, many hundreds of millions of years ago.
00:20:07.03 They all have a simple dense cap.
00:20:09.23 Interestingly, where we see a transition
00:20:13.06 into this composite complex cap structure,
00:20:17.12 is right at the base of the teleosts.
00:20:19.22 Now, the teleosts are the modern bony fishes.
00:20:22.13 It's this amazingly diverse
00:20:25.08 and speciose group.
00:20:26.26 There are many, many species.
00:20:28.17 It's the dominant group of fishes
00:20:31.16 in the world today, the extant fishes.
00:20:34.03 Nearly all of them
00:20:36.04 have this composite cap.
00:20:37.20 It seems to be characteristic of that group of organisms.
00:20:41.21 Now, evolution isn't quite that simple,
00:20:43.17 and we do see some differences
00:20:46.01 from this nice pattern
00:20:48.13 that we've looked at up the tree.
00:20:50.08 In particular, if you go back over into the purple area,
00:20:53.11 where the sharks are,
00:20:55.02 a branch in that tree called the ratfish,
00:20:56.19 or the chimeras,
00:20:58.08 have a simple cap.
00:20:59.21 And in the teleosts,
00:21:01.11 you can see that one white branch
00:21:03.06 and that's the eels.
00:21:05.13 And so in a few places through the tree,
00:21:07.19 we will see variation.
00:21:09.23 Now, if we look at how
00:21:14.08 the cap structure and this variation
00:21:16.02 relate to behavior,
00:21:17.21 if we map behavior onto this tree,
00:21:19.14 we can pull out a couple key points.
00:21:21.28 The first is that when we have
00:21:24.24 the absence of this axon cap,
00:21:26.10 you just have the Mauthner cells
00:21:28.00 with none of this excitatory and inhibitory inputs
00:21:30.07 coming to them,
00:21:31.19 we tend to see a retraction response,
00:21:33.20 which is that pulling back of the head.
00:21:38.01 We see that in lampreys
00:21:39.15 and also in the eels,
00:21:41.01 which lack the axon cap structures.
00:21:47.01 All of these other animals
00:21:48.25 perform more of a C-start type of response,
00:21:51.23 although it varies in its strength
00:21:53.29 and how high performance,
00:21:56.11 how fast it is.
00:21:58.00 But one thing that's noticeable
00:22:00.17 is that when we get to this composite cap
00:22:01.28 at the base of teleosts,
00:22:03.13 we see a really high performance response,
00:22:06.20 like there's some innovation
00:22:08.17 of how that cap works
00:22:10.10 that allows the performance
00:22:12.01 to be very, very high, a very strong response.
00:22:15.11 This middle area of the tree,
00:22:17.05 we're still figuring out.
00:22:18.27 Fish with the simple and simple dense caps
00:22:21.08 have more diverse startles
00:22:23.11 - the same basic pattern,
00:22:24.20 but there's a diversity in performance
00:22:26.22 and how it's generated by muscle
00:22:28.13 that we're still trying to understand.
00:22:30.17 And the motor patterns are quite complex.
00:22:34.26 Now, if we get up into the amphibians,
00:22:36.25 as you could imagine,
00:22:38.19 I mentioned earlier that
00:22:40.13 not only do the tadpoles
00:22:42.22 have Mauthner cells, but also frogs.
00:22:45.05 You probably can, you know,
00:22:46.25 predict what a frog's startle response would be,
00:22:49.17 it will be a bilateral hop.
00:22:51.17 One of the things we're trying to figure out now
00:22:54.01 is how that relates to the Mauthner cells
00:22:55.27 and this circuit.
00:22:57.17 Well, what about the startles
00:22:59.01 of humans and other mammals?
00:23:00.08 This is an example
00:23:02.01 of an armadillo startle,
00:23:04.04 where the animal jumps up on its legs
00:23:06.13 into the air when it's startled.
00:23:08.11 Are these types of behaviors, and our startles,
00:23:10.27 also generated by Mauthner cells
00:23:13.03 and this Mauthner cell circuit?
00:23:14.27 We actually don't know.
00:23:16.23 There are similarities in the startle behavior,
00:23:18.27 as you might imagine.
00:23:20.17 They're really fast, really high performance,
00:23:22.23 but they're different
00:23:24.05 and we haven't been able
00:23:25.25 to associate the neural circuit of the startle in mammals
00:23:28.20 with that of the startle of fishes.
00:23:31.23 It's one of the things we really want to know in my lab,
00:23:34.24 and we're beginning to take steps
00:23:38.07 to figure out.
00:23:41.05 So, let's conclude.
00:23:43.16 First, I want to make the point that
00:23:46.25 there are a lot of ways of understanding
00:23:48.16 how the brain works,
00:23:49.27 lots of labs doing beautiful imaging work
00:23:51.25 studying healthy human brains,
00:23:54.05 and also studying injured human brains,
00:23:56.04 where we can associate damage
00:23:58.11 to a particular region
00:24:00.03 with changes in behavior or function.
00:24:02.20 In addition, we can use more tractable brains,
00:24:04.29 or more tractable nervous systems, in other species,
00:24:07.21 like C. elegans and the sea hare,
00:24:11.15 to understand how circuits are put together.
00:24:14.14 Looking at those simple circuits,
00:24:16.17 we can use them to understand
00:24:19.05 basic principles
00:24:20.25 that also may apply in tetrapods
00:24:22.27 and in other vertebrates.
00:24:25.10 Now, in addition to using models,
00:24:27.25 comparing across models
00:24:29.25 or other species
00:24:31.05 gives us a different sort of power
00:24:32.21 of looking at how things are similar
00:24:34.20 or how evolution may have
00:24:36.15 come up with different solutions
00:24:38.19 to generating certain functions in the body
00:24:40.22 and in the brain.
00:24:42.02 So, we use comparisons and evolutionary approaches
00:24:44.20 to relate structure and function more broadly.
00:24:48.02 Now, on the Mauthner cell system in particular...
00:24:51.18 I hope I've shown you that the Mauthner cell
00:24:53.19 is an example of a simple neural circuit
00:24:56.21 that we can use
00:24:58.25 to understand not only basic principles of the nervous system,
00:25:01.19 but also nervous system evolution.
00:25:04.17 In particular, because we can follow this cell
00:25:07.09 and use it to anchor this circuit,
00:25:09.21 through brains from a wide variety of animals,
00:25:12.05 we're able to then track the circuit
00:25:14.29 over a large swath of evolutionary time,
00:25:17.19 from the divergence of lampreys on up,
00:25:20.02 and that diverge occurred
00:25:21.26 about 500 million years ago,
00:25:23.10 so we're talking about a really long time frame here.
00:25:28.29 So, as I end, I just want to acknowledge
00:25:30.19 some of the people who were important to this work.
00:25:32.27 In particular, some of my students.
00:25:35.25 These are all students who were in the lab
00:25:38.01 over the past decade or so.
00:25:40.11 Hilary Bierman, who was a graduate student,
00:25:42.13 and Rachel Fremont and Julie Schriefer,
00:25:44.01 who were both undergraduates
00:25:45.19 at the University of Chicago.
00:25:47.05 In addition, my collaborators
00:25:48.20 at the University and at other institutions,
00:25:50.29 include Steve Zottoli,
00:25:52.20 Mark Westneat, John Long,
00:25:54.00 and Vicky Prince,
00:25:55.11 and they've all been important to the ideas
00:25:57.00 as well as the data collection and analysis of this work.
00:26:00.25 In particular, though, I'd also like to thank Glenn Northcutt.
00:26:03.07 The images of axon caps that I showed you...
00:26:06.11 some of them were made in my lab,
00:26:07.27 others were actually photos of collections
00:26:10.19 that he's taken over a number of years,
00:26:13.01 and they've been so important for our work,
00:26:15.07 so I wanted to thank Glenn,
00:26:16.15 and also to make the point that collections,
00:26:19.26 biological collections,
00:26:21.23 are just really important,
00:26:23.15 not only for what we've learned up 'til now,
00:26:25.07 but for the future of discovery in science.
00:26:28.17 And of course I want to thank our funders,
00:26:31.04 the National Science Foundation,
00:26:33.23 and grants that we've had for work in the lab,
00:26:35.13 have been really important
00:26:37.19 to our research and our ideas.
00:26:39.10 So, thank you.

[00:00:07.15]My name is Neil Shubin from the University of Chicago,
[00:00:09.25]and we're going to talk today
[00:00:11.07]about organogenesis in deep time.
[00:00:13.27] In particular, we're going to look at this:
[00:00:15.15]we're going to try to compare a fish to a human.
[00:00:17.10]How do you compare a fish fin
[00:00:19.25] to a human limb?
[00:00:21.02]How did the limb come about from fins?
[00:00:23.00] And what are the different ways we pull together
[00:00:25.26] different types of data?
[00:00:27.20]Let's think of it this way,
[00:00:29.04]this is a nice starting point.
[00:00:31.11] Think about comparing, like I say,
[00:00:33.05]a human arm to a fin of a fish,
[00:00:34.27] shown on the left here.
[00:00:36.14]They look very different.
[00:00:38.01] Right, if you look at the bones, shown in black,
[00:00:39.25]there doesn't really seem to be a whole lot of correspondence.
[00:00:43.02]Fish have lots of bones,
[00:00:44.11]we have the one bone, two bone, little bone, finger pattern
[00:00:47.10] seen in chickens and whales and everything with limbs.
[00:00:50.22]Also, fish have fine rays, which we don't have.
[00:00:53.12]Big differences,
[00:00:56.18]yet we can bridge these gaps when we look at fossils.
[00:00:59.12] If we were to fill this diagram with some of the fossils,
[00:01:01.29]what you see here are
[00:01:04.19]you start to see lots of finned creatures,
[00:01:06.27]creatures with fin webbing, fin rays,
[00:01:09.25]but also having the one bone, two bone, little bone pattern as well.
[00:01:15.07]So what this means is, if we want to bridge the gap
[00:01:17.10] between fins and limbs,
[00:01:19.02]what we need to do is
[00:01:21.12]to have expeditions targeted to key parts of the tree of life.
[00:01:25.10]That is, we can target certain time periods to find fossils,
[00:01:28.18]and some of those fossils
[00:01:30.10]will start to bridge the gap between fins and limbs.
[00:01:34.25] But that's not the only thing that's important here.
[00:01:37.01] When we start to have these fossils,
[00:01:39.00] we can start to compare
[00:01:41.02]living creatures in different ways.
[00:01:42.09] That is, we can start to compare
[00:01:43.22] a human arm
[00:01:45.14]to the fin of a fish
[00:01:47.06]by seeing correspondences
[00:01:49.27]that would have been absent to us without the fossil evidence.
[00:01:52.01]What that enables us to do is to design experiments,
[00:01:54.24] that is, we can design new experiments
[00:01:56.18] based on our paleontological understandings
[00:01:59.00]on the developmental genetics of all kinds of different kinds of fish,
[00:02:02.12] non-model organisms.
[00:02:04.24]And in fact it works all ways.
[00:02:06.04]Once we have these experiments
[00:02:08.04] based on non-model organisms,
[00:02:09.20]we can begin to target new parts of the tree of life
[00:02:11.22]where we may be missing fossil data.
[00:02:14.01]So, the central idea here is
[00:02:16.25]that fossils enable us to bridge gaps in the record,
[00:02:21.05]the anatomical record of the tree of life,
[00:02:23.23]that enables us to design experiments in developmental genetics
[00:02:27.05] on living creatures,
[00:02:28.06]and the more we understand about developmental genetics of living creatures,
[00:02:30.15] the more we understand about what gaps exist in the fossil record
[00:02:33.17]and where we need to lead the next expeditions.
[00:02:36.01] So, really, the fossil and genetic data
[00:02:38.11]work hand in hand.
[00:02:39.21]So, let's work through an example here.
[00:02:42.21]Well, our work obviously begins with the origin of tetrapods,
[00:02:45.03]the transition, say,
[00:02:46.15]of something like a fish on top
[00:02:48.03]to a limbed animal on the bottom,
[00:02:50.16]and we can design expeditions that bridge this gap,
[00:02:54.05]and what we do is we look for places in the world
[00:02:56.27] that have rocks of the right age,
[00:02:58.16]rocks of the right type,
[00:03:00.03]and rocks that are exposed to the surface
[00:03:02.22] for us to find fossils.
[00:03:03.20]Using that tool kit, we can begin to bridge this gap.
[00:03:07.00]It turns out to understand the origin of tetrapods
[00:03:08.29]we need to focus on environments like this,
[00:03:11.06] near-shore marine environments like ancient seaways,
[00:03:13.25]but likewise ancient highlands,
[00:03:16.10]so delta systems turn out to be really perfect for us,
[00:03:20.14]because when we have a system like this
[00:03:22.12]we can sample ancient seas,
[00:03:23.29]ancient estuaries,
[00:03:25.11]ancient rivers and streams,
[00:03:26.20]you know, the whole enchilada, as they say.
[00:03:29.14] So, really, it became clear
[00:03:31.29]very early in our study
[00:03:34.13]that the best places that had these kind of delta systems
[00:03:36.18]of the right age, in the late Devonian period,
[00:03:39.14]were centered in three general places in North America
[00:03:42.07] -- this is a slide actually taken from an undergraduate college geology textbook,
[00:03:46.13] which helped us launch a number of expeditions --
[00:03:48.27]but it became very clear to us that
[00:03:50.27] two of these areas that were seen in this diagram
[00:03:52.10]were known by scientists before.
[00:03:54.18]We had previously worked
[00:03:56.21]on the so-called Catskill rocks of eastern Pennsylvania.
[00:03:59.10]Other colleagues had worked in East Greenland,
[00:04:02.18]these are very well studied rocks.
[00:04:04.16]If in this diagram you can see what led us to the Arctic in the first place
[00:04:08.07] is rocks of the right age, rocks of the right type,
[00:04:09.20] rocks exposed across the surface,
[00:04:11.20]in an area of the Arctic
[00:04:14.14] that was completed explored by vertebrate paleontologists.
[00:04:16.28]So we had ideal geology,
[00:04:18.18]but really very few of our colleagues had worked on these rocks.
[00:04:21.05] So off we went.
[00:04:23.16]Anytime you talk about a fossil expedition
[00:04:25.06]you're talking about teamwork,
[00:04:26.22]and I just want to give credit where credit's due.
[00:04:28.01]My graduate mentor, Ferris Jenkins,
[00:04:30.00]he and I have these big smiles on our faces
[00:04:32.00] -- the reason why is something that's in this plaster jacket, here.
[00:04:35.18]My good friend and colleague Ted Daeschler,
[00:04:38.03]shown in the upper left,
[00:04:39.07]he's been a partner in these expeditions for decades.
[00:04:41.24] Likewise, all the field crews
[00:04:43.23]that we've had over several decades
[00:04:45.04]as well as the lab team as well.
[00:04:46.10]This is a team effort, discovering fossils.
[00:04:48.18]We don't go out there alone,
[00:04:50.02]we go out there in teams of very talented people.
[00:04:52.20]So, we started these expeditions in 1999,
[00:04:56.28]based on this kind of map,
[00:04:58.21] and what you see on this map
[00:05:00.12]are the islands of the Canadian Arctic,
[00:05:03.05]and surrounded in red are where the Devonian-age rocks are exposed.
[00:05:07.06] And the first set of them that we did,
[00:05:08.18]we had to get to by these helicopters and planes
[00:05:11.09] because it's pretty far away.
[00:05:12.28] This sort of dictates the kind of science that we can do.
[00:05:16.10]Using this, spending several hours
[00:05:19.01] on a helicopter or a plane,
[00:05:20.07]we got to the western part of the Canadian Arctic,
[00:05:22.24]shown on the arrow here.
[00:05:25.01]This area was, you know, ideal for exposures.
[00:05:27.04] What you see is a vista,
[00:05:29.10]a plain of Devonian-age rocks
[00:05:31.20]all across this landscape, here,
[00:05:33.27]but this was the wrong fossil environment
[00:05:36.20]to hold the kinds of creatures we were interested in.
[00:05:39.11] This was an ancient marine system,
[00:05:41.08] this was a system that had ancient deep-water sediments,
[00:05:44.27]so we weren't finding the kind of critters we were on the hunt for,
[00:05:48.15] which is, say, a flat-headed fish with fins.
[00:05:50.20]So we had to retool a little bit,
[00:05:52.14]so we used the geological understandings here.
[00:05:54.24]This is an ancient delta system...
[00:05:56.20]we were in the ancient seaway...
[00:05:58.27]we needed to move upstream.
[00:06:00.16]To move upstream in the ancient geological rocks
[00:06:03.20] meant moving east.
[00:06:05.00]So, we went east, and then the next year,
[00:06:07.05]you can see here in 2000, where the arrow is,
[00:06:09.19]we went to southern Ellesmere Island.
[00:06:11.00]This is what it looked like; it's a really marvelous place.
[00:06:13.16]Moraine with, you know, with red rocks.
[00:06:15.18]This contained ancient rivers and streams
[00:06:18.17] that held a number of lobe-finned fish critters.
[00:06:22.09]We homed in on a particular valley
[00:06:23.27] that had a layer of fossil fish
[00:06:27.09]that were preserved one on top of the other.
[00:06:29.20] These fish were very well preserved
[00:06:32.06] and it really wasn't until 2004
[00:06:34.09] that one of my colleagues removed a rock from this layer...
[00:06:37.16] Steve Gates, who is a professor at Brown University here on the left,
[00:06:41.06]removed a rock here and he saw a V.
[00:06:43.24] And he called us over and he said, "What's this bone here?"
[00:06:45.28] You can barely see it in this picture,
[00:06:47.29]but it was beautiful,
[00:06:49.25]because what it is is it's a snout of a fish,
[00:06:52.11]and not just any fish,
[00:06:54.07]it's a snout of a flat-headed fish.
[00:06:55.18]And one of the big transitions
[00:06:57.08]is going from a conical head to a flat-headed animal.
[00:06:59.05] Here I had a flat-headed fish looking right at me.
[00:07:02.03]So we bring these things move,
[00:07:03.18] it turns out we found four of them this first year,
[00:07:05.22] they come home and the preparers begin to work on them,
[00:07:08.22]removing the rock grain by grain,
[00:07:11.06]and you could see what's emerging here
[00:07:13.02] is a flat head with eyes on top.
[00:07:15.14]Several months later, you could see this thing exposing even more.
[00:07:18.08]You could see the head revealing itself.
[00:07:20.15]You can even see the shoulder girdle, here,
[00:07:23.06]revealing itself,
[00:07:24.25]and maybe this creature even has a neck.
[00:07:27.02] Remember what this quest is all about:
[00:07:28.29]sort of bridging this gap
[00:07:30.20]between lobe-finned fish and a limbed animal.
[00:07:33.22]Maybe finding a flat-headed fish with fins...
[00:07:35.23] this is what the expedition led to,
[00:07:37.13]a flat-headed fish with fins.
[00:07:39.02]Like a fish, it has scales on its back
[00:07:41.16] and fins with fin webbing.
[00:07:43.09]Like a tetrapod, it has a flat head
[00:07:45.09]with eyes on top, a neck,
[00:07:47.15]and, when we cracked open the fin, w
[00:07:49.15]e found bones that correspond to upper arm, forearm,
[00:07:52.02]even parts of a wrist.
[00:07:54.10] Here's a CT scan of the fin
[00:07:56.24]and you can see what it has is a humerus,
[00:07:58.09]and then two bones here,
[00:07:59.22]a radius and an ulna,
[00:08:01.06]and shown in blue are the fin rays,
[00:08:03.06]so it's a real mix of characteristics.
[00:08:05.10]Shown on the left, this is the work of Justin Lemberg,
[00:08:09.01]a graduate student in my laboratory,
[00:08:10.10]shows the joints of this animal.
[00:08:12.10]In a you see the shoulder of this animal,
[00:08:15.25] the socket of the shoulder on the left
[00:08:18.00]and the ball of the humerus on the right.
[00:08:20.23] This is a fish with an elbow, you can see the elbow in b,
[00:08:23.27]and there are even two parts of a wrist,
[00:08:25.16] a proximal carpus and a distal carpus.
[00:08:28.12] This is a fish with components
[00:08:30.12]of our own anatomy inside.
[00:08:32.16]And we can use CT scanning,
[00:08:34.09]as you can see in the image here...
[00:08:37.07]we can begin to dissect the skull using CT scanning
[00:08:40.14]and begin to see the individuals bones
[00:08:42.23]and how they suture together.
[00:08:44.29] It turns out that when we use living animals...
[00:08:47.10]this is an alligator gar shown on the bottom right, here,
[00:08:49.19]and the alligator gar will bite animals in the water,
[00:08:52.10]but as it does so the bones of the skull
[00:08:55.02]show cranial kinesis.
[00:08:56.20]They move in particular ways relative to one another,
[00:09:00.00]and when we analyze Tiktaalik's skull, here,
[00:09:02.19]which is this fossil creature I'm showing you,
[00:09:04.25]we can begin to see that the joints of this animal's skull
[00:09:07.22] can actually move.
[00:09:09.10] It has cranial kinesis,
[00:09:10.25]much like a living alligator gar.
[00:09:12.28] So, what I'm saying is when we find these fossils,
[00:09:14.28]the discovery is really only the beginning,
[00:09:17.11]because then we can start to work on their anatomy,
[00:09:20.23] compare them to other creatures,
[00:09:22.07]and begin to assess their biomechanics
[00:09:24.01] -- how they ate, how they walked,
[00:09:25.26]and how they lived in these aquatic environments,
[00:09:31.03] in Devonian streams.
[00:09:32.11]So, we have this creature, Tiktaalik roseae,
[00:09:33.24]it's an animal that has lungs and gills,
[00:09:35.25] it has fins that have components of limbs inside,
[00:09:38.06]it has a neck...
[00:09:40.24]it really has a mix of characteristics.
[00:09:42.07] And when we map this in the phylogenetic tree,
[00:09:45.06]what we see is it holds a relatively special place.
[00:09:48.16]That is, you can see the fish on the bottom
[00:09:50.18]and the limbed animals on top.
[00:09:52.12]Tiktaalik sits right here in the middle.
[00:09:54.20]It shows us the sequence
[00:09:56.25]of the acquisition of tetrapod characteristics,
[00:10:00.05] whether it's necks, fingers, wrists, toes,
[00:10:03.09]and so forth.
[00:10:05.09] Well, how is this relevant to developmental biology?
[00:10:07.17] Well, remember what we're saying
[00:10:10.10]is when we have fossils like Tiktaalik
[00:10:12.02] we can compare the arms of, say,
[00:10:14.04]people and chickens and mice
[00:10:16.10]to the fins of fish in novel ways.
[00:10:18.28]What creatures like Tiktaalik are showing us
[00:10:22.16]is that fish, back in the Devonian, had wrists.
[00:10:25.24] They had components of the distal,
[00:10:29.18]the terminal ends of the appendage,
[00:10:30.23] such as seen in our own limbs.
[00:10:33.06]So what that means is,
[00:10:35.06]if the fossil should be read at face value,
[00:10:36.28]is that sometime in the distant past,
[00:10:38.28]and maybe even in living fish,
[00:10:40.13] there should be the machinery
[00:10:42.16]by which limbs and toes and fingers and wrists and ankles
[00:10:46.07]are developed.
[00:10:48.01]So, let's get back to this comparison here.
[00:10:49.24] If you look at a zebrafish, say,
[00:10:52.24]the fish on the left,
[00:10:54.23]and a human here on the right,
[00:10:56.29] you know, the bones of the fins don't look very similar.
[00:10:59.14]Where the similarities start to emerge
[00:11:02.03]is when we compare them to the fossils,
[00:11:03.16]like I just showed you,
[00:11:04.29]but also when we compare their development.
[00:11:07.02]See, what we have here is a chicken limb in its development,
[00:11:09.23]taken from a textbook,
[00:11:10.29]and you can see the limb bud shown on the left.
[00:11:13.08] It develops this little bud, sticks out of the body,
[00:11:15.12]and as it develops the cartilage skeleton begins to form.
[00:11:20.06]Now, what's driving the development of that cartilage skeleton
[00:11:23.17]are a set of interactions
[00:11:26.00]among signaling centers, like this region here,
[00:11:28.18] the AER, and another one seen at the bottom here,
[00:11:31.26] the ZPA,
[00:11:33.04] but as well as other factors, genes and proteins,
[00:11:34.22]that are turned on and off,
[00:11:36.00]driving the patterns of development
[00:11:37.21]and pattern formation
[00:11:39.14]that are so characteristic of limbs.
[00:11:41.06]Really, the comparison we want to make
[00:11:42.27] is not just between the structures of fins and limbs,
[00:11:45.24]but the developmental mechanisms
[00:11:47.26]by which the skeletal patterns of fins and limbs emerge.
[00:11:51.00]How similar are they and how different are they?
[00:11:53.09]So, to do that,
[00:11:55.19]we focus on a variety of different signaling systems,
[00:11:57.02]as well as transcription factors.
[00:11:58.23] One of the transcription factors that's been incredible important to us
[00:12:02.20] are the Hox genes.
[00:12:03.27]The Hox genes have been shown to be important
[00:12:05.25] in a variety of processes of development
[00:12:07.24]from hindbrains to the axial skeleton to the limbs.
[00:12:10.28] To give you an example of why they're considered so important,
[00:12:13.12]here's a wild type mouse limb.
[00:12:15.22] If you knock out some of the Hox genes
[00:12:18.01] what are known as the 13 paralog groups, Hoxd13 and Hoxa13,
[00:12:22.20]you can develop a mouse limb
[00:12:26.06]that has no fingers, toes, or wrists or ankle bones.
[00:12:29.24]And these are segment-specific modifications of the appendage.
[00:12:33.16]And if you knock out elements
[00:12:36.11] of the 11 cognate groups,
[00:12:37.19]you're missing the middle segment of the appendage.
[00:12:40.13] So, these are genes that are really involved
[00:12:42.12] with the specification of different components of our appendages.
[00:12:46.04]They take a very, very special role
[00:12:48.24]in our understanding of the origin of digits from fish fins.
[00:12:52.22]The question is, how have these patterns of expression
[00:12:56.03]and the patterns of activity
[00:12:57.28]of these genes evolved?
[00:12:59.13]Are they present in fish?
[00:13:00.20]Are they doing similar things in fish?
[00:13:02.00]What's involved in their regulation and their activity?
[00:13:03.20]How is this assembled
[00:13:06.06]going from fish to limbed animals?
[00:13:07.23]Well, there have been a number of studies
[00:13:09.25]of the expression of these genes
[00:13:11.22] in diverse limbed animals,
[00:13:13.01]and interestingly they follow two phases of expression.
[00:13:16.05]Look at the limbed skeleton on the left.
[00:13:19.03]That has three components.
[00:13:20.10]It has a top component consisting of one bone,
[00:13:22.18]it has a central segment composed of two bones,
[00:13:26.19]and then it has a distal segment composed of multiple bones.
[00:13:29.21] It turns out there are two phases in the expression of Hox genes
[00:13:32.14] that are involved with the specification of these components of the appendage.
[00:13:36.05]The earliest phase, shown on top here,
[00:13:38.10]shows the different genes of the Hox system
[00:13:41.19]expressed within one another,
[00:13:44.05]so these are like nested sets of expression
[00:13:46.24]of one set of genes
[00:13:49.13] in the domain of expression of another --
[00:13:51.05]think of Russian dolls.
[00:13:52.29]This phase of expression acts early in limb development
[00:13:55.15]and is involved in the specification
[00:13:57.20] of the first two segments of the appendage.
[00:14:00.28] Coming on later
[00:14:03.05]is a late phase pattern of these same genes.
[00:14:05.24]This involves expression
[00:14:08.08]across the entire distal domain
[00:14:10.04] of what will become the digits and wrist of the limb,
[00:14:13.17]and activity of the late phase
[00:14:16.17]is what's driving specification of the distal component,
[00:14:19.18]that component which includes the wrist
[00:14:22.21]and finger bones of tetrapods.
[00:14:24.18] It's an open question:
[00:14:26.02]To what extent is the origin of the tetrapod limb
[00:14:28.03]based on the origin of a novel,
[00:14:30.08] late phase pattern of Hox expression?
[00:14:32.13] How did this come about?
[00:14:33.27] Is this something we see in fish?
[00:14:35.13] Is this something that comes about with tetrapods?
[00:14:37.06] How is it assembled over evolutionary time?
[00:14:39.23] If you take creatures like Tiktaalik at their word,
[00:14:41.24]the fossils,
[00:14:43.05]it would suggest that perhaps late phase expression
[00:14:44.28]already existed in fish fins,
[00:14:46.26]and maybe it's doing something else.
[00:14:48.15] Let's look at that.
[00:14:50.12]So, the question is really,
[00:14:52.24]when did late phase Hox expression come about?
[00:14:54.08] Did it come about... is it unique to tetrapods
[00:14:57.15]or is it something that we see, primitively, in fish.
[00:15:00.18] So, one of my graduate student, Marcus Davis,
[00:15:02.27] started this quest to understand
[00:15:05.05]patterns of activity of Hox genes in limbs and fins,
[00:15:07.18]and he started by looking at paddlefish,
[00:15:09.27]and you wonder, why paddlefish?
[00:15:11.03]Well, here's a paddlefish.
[00:15:12.17] Paddlefish, it turns out... you can get a lot of embryos of these things
[00:15:15.01]and they have big, fleshy fins.
[00:15:17.23]As you can see in blue, here,
[00:15:19.19]this is the cartilage of the fin.
[00:15:21.12] It's big, fleshy cartilage,
[00:15:22.25]so these are really relatively easy to analyze.
[00:15:26.04]Furthermore, these critters
[00:15:29.06] have a phylogenetic position that's very relevant.
[00:15:31.22] They're very basal ray-finned fish,
[00:15:33.25]so they're sort of close to the branch point
[00:15:36.02]of creatures like Tiktaalik,
[00:15:37.09]so it gives us a window into that.
[00:15:38.28]So, in looking at Hox expression,
[00:15:41.12]Marcus found looking at early expression,
[00:15:42.12]he found they have an early phase pattern of Hox expression.
[00:15:44.19] If you look later on,
[00:15:46.05]they have a late phase pattern of Hox expression.
[00:15:47.29] So, it really does appear they have
[00:15:49.22]two phases of Hox expression,
[00:15:51.01]and that late phase Hox expression
[00:15:52.26]correlates to just like a distal strip of cells
[00:15:55.03]that you see in the distal terminus of the appendage.
[00:15:59.13] The real question here is
[00:16:01.07]if we look at these two phases of Hox expression...
[00:16:03.15]if you look at a mouse,
[00:16:06.12]early phase Hox expression is one
[side] of the chromosome,
[00:16:08.23] on the telomeric phase of the chromosome,
[00:16:11.04] and late phase expression,
[00:16:12.23]that expression that's driven in the wrists and digits and so forth,
[00:16:17.27] is on the centromeric side of the chromosome.
[00:16:19.27]So there's a real structural organization
[00:16:21.21] to the enhancers and regulatory apparatus,
[00:16:24.05]the architecture,
[00:16:25.20]that drives these patterns of late and early phase activity.
[00:16:28.21] And this is well known from mouse
[00:16:30.21]from the work of Denis Duboule's laboratory.
[00:16:32.09] So what we thought we would ask is,
[00:16:33.23]how is this pattern of regulation generated?
[00:16:37.11] Is it present in fish and what is it doing?
[00:16:39.17]And the problem is we don't know much about fish,
[00:16:42.02]so we really had to assemble those data.
[00:16:46.01]But here's the problem:
[00:16:47.24]if you look at late phase expression,
[00:16:50.27] the potential in some of these enhancers
[00:16:52.22]that are present in fish,
[00:16:55.14]you actually have some of the late phase enhancers,
[00:16:57.12]such as this one here, CsB,
[00:16:59.15]and it turns out if you make a reporter of the fish elements,
[00:17:02.10] say from fugu,
[00:17:04.08]and put it in a mouse reporter,
[00:17:05.20]you don't get any activity in the limb.
[00:17:07.16] So the earliest analyses seem to suggest
[00:17:11.28]that late phase enhancers, regulatory apparatus,
[00:17:14.24]are present in fish genomes,
[00:17:18.23]but they're not active in limbs,
[00:17:20.26] that they're not capable of driving late phase expression.
[00:17:22.22]This kind of analysis suggested
[00:17:25.02] that late phase expression is unique to digits,
[00:17:28.24] unique to tetrapods,
[00:17:30.08]and that regulatory apparatus is there,
[00:17:32.18]but not functional in the same way.
[00:17:35.15]Well, it turns out if you look at this,
[00:17:37.00] it seems to be maybe we're not
[00:17:39.24]relying on the right animals for comparison.
[00:17:41.09]So, let's take this area here.
[00:17:44.05] Here is a chromosome,
[00:17:45.17]you can see the Hox cluster on the left,
[00:17:47.01]and on the right, shown in green,
[00:17:48.08] are early phase enhancers.
[00:17:50.12]If you look at a VISTA plot of this enhancer,
[00:17:55.13]comparing human through fish,
[00:17:58.01]through zebrafish and pufferfish,
[00:18:00.27]and you compare the similarities of these regions,
[00:18:02.17]what you'll see is...
[00:18:04.04]you have curves on the top, the human and the chicken,
[00:18:05.29]which suggest they are very similar,
[00:18:07.25] that they have this early phase enhancer.
[00:18:10.03]But if you look at the zebrafish and the pufferfish,
[00:18:11.29]no lines whatsoever --
[00:18:13.17]there's no conservation at all.
[00:18:15.08]So, this kind of conservation analysis
[00:18:17.29]would suggest that these enhancers aren't even present
[00:18:20.15] in fish fins.
[00:18:21.19] Well, it turns out we might not be comparing the right animals,
[00:18:24.14]and the reason for this is that
[00:18:26.26]fish have a whole genome duplication.
[00:18:28.15]And there are three people from my lab
[00:18:29.29]who have been working on this particular problem:
[00:18:31.17]Andrew Gehrke is a graduate student,
[00:18:33.20]our colleague and collaborator José Luis Gómez-Skarmeta,
[00:18:36.05]and Tetsuya Nakamura, a postdoc in my laboratory.
[00:18:40.09]And they've been interested in this whole genome duplication
[00:18:42.28] as perhaps a reason
[00:18:45.19]for maybe why we're not seeing these enhancers
[00:18:47.08] in certain kinds of fish.
[00:18:49.08]Look at it this way:
[00:18:50.26]if we look at the Hox clusters,
[00:18:52.11] in humans there are four Hox clusters,
[00:18:54.01]you can see it in the middle here...
[00:18:55.15] if you look at basal creatures such as Amphioxus or jawless fish,
[00:18:57.24]what you'll see is there is a set up duplications
[00:19:00.26] that go from the single Hox cluster shown in Amphioxus
[00:19:04.21] to the four clusters that are shown in humans.
[00:19:08.01] But if we look at living fish,
[00:19:09.13] the ones that have been the basis for the comparisons
[00:19:11.01]we've already talked about,
[00:19:12.21]you can see they're taken this duplication one step further.
[00:19:15.13] Zebrafish have eight of these clusters,
[00:19:18.05] in fact even salmon have sixteen of them.
[00:19:20.05]So how do you know what to compare?
[00:19:21.19]Maybe functions have been shuffled between them.
[00:19:23.24] So the idea of Andrew in the laboratory is, maybe,
[00:19:27.07]what if we took a fish that didn't have this whole genome duplication,
[00:19:31.16]say a gar,
[00:19:33.13]and use that as the basis of comparison?
[00:19:34.18] Maybe having the right fish system
[00:19:36.26]would allow us to pick up these enhancers
[00:19:38.28]which we're not seeing in other fish.
[00:19:42.17] The good news for us is working with John Postlethwait
[00:19:44.26]and Ingo Braasch from Oregon,
[00:19:46.14] the genome of a spotted gar is now available,
[00:19:49.10]and this was very fortunate for us,
[00:19:50.21]we were able to apply this genome.
[00:19:52.14]So, when we take the gar and put it in this comparison...
[00:19:55.00]you recall, what we showed before is...
[00:19:56.09]here is the human and the chicken
[00:19:58.24]shown on the baseline comparison to a mouse.
[00:20:00.18]You can see there's lots of similarity.
[00:20:02.05]Remember, the take-home message before was zebrafish and pufferfish
[00:20:04.27]don't show any similarity.
[00:20:06.08]When we take the gar as a unit of comparison,
[00:20:09.02]the story changes dramatically.
[00:20:10.18]Here, you have the human and the chicken,
[00:20:12.12]but look, the gar now shows this enhancer peak conserved,
[00:20:15.07]and even having the gar as an intermediate taxa
[00:20:17.07]enabled us to pull out small peaks
[00:20:19.17] for both the pufferfish and the zebrafish,
[00:20:21.27] which were invisible to us before.
[00:20:24.21]Now the question is,
[00:20:26.14] is this chromatin accessible
[00:20:28.13] at the right stage of development?
[00:20:29.15] Are these functioning like real enhancers?
[00:20:31.10]For that we used a new technique
[00:20:33.19]known as ATAC-seq,
[00:20:35.24]which shows us the accessibility of the chromatin
[00:20:37.28] at the right stage.
[00:20:39.01] We can ask the question, by looking at this,
[00:20:40.23]is this enhancer, CNS65, accessible?
[00:20:43.13] And you can see here those large peaks
[00:20:45.18]you see in whole-body,
[00:20:47.21]24-hours post-fertilization,
[00:20:49.15]they show that that chromatin is accessible,
[00:20:52.04] functioning likely as an enhancer.
[00:20:54.08]Now, when we take that enhancer region
[00:20:57.08]and we put it in a fish with the reporter,
[00:20:59.15]here's how it drives expression.
[00:21:01.05] It drives expression throughout the fin
[00:21:03.19] in 31 hours post-fertilization
[00:21:06.21]and then knocks out at 60 hours post-fertilization.
[00:21:10.05]When we take the fish element
[00:21:11.21]and put it in a mouse
[00:21:13.29] we get the same pattern.
[00:21:15.07] Early in mouse limb development
[00:21:17.03]it's driving expression throughout the forelimb,
[00:21:18.28] and in late development
[00:21:21.08] it begins to knock out in the area
[00:21:23.18] that will form the distal part of the appendage.
[00:21:25.18] So, the fish element in mouse
[00:21:27.28] is functioning just as it should for an early phase enhancer.
[00:21:30.26]So, this is a case where having the right model organism
[00:21:33.08] allowed us to find an enhancer
[00:21:36.21]present in fish that has a conserved function with mouse.
[00:21:39.01]Now, the real question we're interested in is
[00:21:41.02] not just these early phase telomeric enhancers;
[00:21:43.12]what we're interested in
[00:21:45.09] is those centromeric enhancers all the way on the right,
[00:21:46.28] because these are the ones that are driving digit expression.
[00:21:49.27]So here we're asking the question,
[00:21:51.13]do fish have the genetic apparatus
[00:21:54.10]that drives Hox activity
[00:21:56.10]which drives the formation of digits?
[00:21:59.22]And here's the whole Hox cluster...
[00:22:01.08] just to make a long story short,
[00:22:02.25]there's the Hox cluster itself,
[00:22:04.16] these are the early phase enhancers, here,
[00:22:07.00]on the left in yellow are the late phase enhancers.
[00:22:09.20]And what I just showed you is
[00:22:11.24]early phase enhancers are present in fish,
[00:22:13.17] this is what I just showed you,
[00:22:14.28]and you can see they report both in mouse and in gar,
[00:22:17.16] and in very similar ways,
[00:22:19.05]and they function as early phase enhancers...
[00:22:20.27]you'll notice how the expression is knocked down
[00:22:23.17]in the distal fin.
[00:22:25.08] Now when we look at the late phase enhancers,
[00:22:27.26] indeed they are present in fish fins
[00:22:30.06]when you add the gar to the comparison.
[00:22:33.02]They report in very similar ways in mouse and in gar,
[00:22:36.14]you'll see the expression activity driven
[00:22:38.17]by the gar element in a mouse,
[00:22:39.22] it's very similar to that
[00:22:41.20]driven by a mouse element in a mouse.
[00:22:43.17]And indeed, when we look at their expression in the fin,
[00:22:46.02]what they do is they drive expression
[00:22:47.22]across the entire fin in early development,
[00:22:49.15]but only in the distal fin in later development,
[00:22:51.29]and the same is true for other late phase enhancers
[00:22:54.24] seen in mouse.
[00:22:57.01] They drive activity both endogenously in mouse,
[00:22:59.26]but the gar element also drives activity within the mouse,
[00:23:03.04] and you can see all the way on the left, here,
[00:23:05.12]just like a late phase enhancer should,
[00:23:06.26] it drives activity throughout the fin in early development
[00:23:09.12]and just in a distal strip of tissue in late development.
[00:23:13.20]What's interesting here is when we take the gar
[00:23:15.29]and put it in mouse
[00:23:16.27] and the endogenous mouse,
[00:23:19.06]they are very similar.
[00:23:20.13]Yet the zebrafish, an animal with that duplicated genome,
[00:23:22.22]barely even reports in the mouse genome.
[00:23:25.06]So, this is a case to show,
[00:23:27.25]when you have the right genetic model,
[00:23:30.16] the right genomic model,
[00:23:32.07]you can see hidden similarities
[00:23:34.10] that would be hidden to you otherwise.
[00:23:36.04]The zebrafish doesn't report easily in mouse,
[00:23:37.29]probably because it has that duplicated genome,
[00:23:40.06]whereas the gar, which has the unduplicated genome,
[00:23:42.13] reports very much like a mouse,
[00:23:44.11] it behaves very much like a mouse.
[00:23:46.21]So, just to give you a sense, looking at other Hox genes,
[00:23:48.10]I just want to show this for one simple point...
[00:23:50.10]when you look at the endogenous activity of these enhancers
[00:23:54.27]in a fin,
[00:23:56.13]what you'll notice is they drive expression
[00:23:58.10]in a distal strip of tissue,
[00:24:00.08]just like fish,
[00:24:01.20]late phase expression of Hox genes.
[00:24:03.03]These same elements in mouse
[00:24:04.28]drive distal expression across the mouse paddle,
[00:24:08.28]which becomes the digits and the wrist.
[00:24:11.03]So, the idea here is a developmental
[00:24:13.12]and genomic equivalency
[00:24:15.17]between that distal strip of tissue you see on the right of the fish fin
[00:24:17.27]and the entire distal paddle of a mouse limb.
[00:24:21.10] So, this leads us to the evolutionary comparison,
[00:24:24.04]supported by Tiktaalik, fossils like Tiktaalik,
[00:24:26.25]and supported by the developmental biology,
[00:24:29.09] is that fish indeed do have wrists,
[00:24:31.27]and if we take the developmental data at face value,
[00:24:34.04] it seems like the distal region of a fish fin,
[00:24:36.02]which consists of those little blobs shown in yellow, on the left,
[00:24:39.27]correspond to the wrists of humans.
[00:24:44.24] So, the take-home message here is
[00:24:47.08]we can leverage multiple lines of data
[00:24:49.12]to understand evolutionary history.
[00:24:51.04] Look, I'm a paleontologist,
[00:24:52.23] I don't find enhancers buried in rocks,
[00:24:54.15] but what I have is the means
[00:24:56.29] to compare the enhancers of living creatures
[00:24:59.20] that are separated by huge phylogenetic distances.
[00:25:02.17] The way we do it is first start with fossils
[00:25:04.29] to bridge the gaps
[00:25:06.18]and then devise experiments
[00:25:08.06]which help really bridge those gap
[00:25:10.05]s in a mechanistic way.
[00:25:11.13]So it's really an exciting time for science
[00:25:13.08]because we can begin to analyze evolutionary transformations
[00:25:16.17]using both genetic and molecular data,
[00:25:18.27]as well as classic paleontological data.
[00:25:20.27]To do an analysis like this takes amazingly talented people,
[00:25:23.24] including the artist who drew this lovely diagram,
[00:25:26.22] as well as my members of my laboratory,
[00:25:29.20]my good colleagues,
[00:25:31.03]who have co-led the Tiktaalik expeditions with me,
[00:25:33.21]the Inuit community and Canadian government,
[00:25:35.21]which has supported our work for several decades,
[00:25:39.04]my molecular colleagues
[00:25:40.27]and the colleagues who have provided access
[00:25:42.17]to the gar genome,
[00:25:44.14] and of course the funders of our work.
[00:25:46.07]Thank you very much.