In this session, we dig more deeply into the evolution of the complex behaviors and patterning found in vertebrates. We begin with an whiteboard video which investigates why sexual reproduction provides an evolutionary advantage over asexual reproduction, in most cases. In the second video, we wonder why you jump when someone startles you. Melina Hale explains how her research into the startle response in fish may help us to understand the evolution of neural circuits in vertebrates. And in the last video, Neil Shubin explains how studying the expression of hox genes in fish and mice may help to explain the evolution of limb development from fish, to early tetrapods like Tiktaalik, to modern mice and humans.
All Course Materials for this Session (Educators only)
- Duration: 05:17
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.
[00:00:56.18]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,
[00:03:25.11]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,
[00:07:24.25]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
[00:10:48.01]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,
[00:14:43.05]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,
[00:16:25.20]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,
[00:20:49.15]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.
- Youreka Science: Types of Reproduction: Sexual Versus Asexual Reproduction
- Melina Hale iBioSeminar: The Evolution of Neural Circuits and Behaviors
- Neil Shubin iBioSeminar: Hox Genes: The Evolution of Limbs from Fins
Melina Hale is a professor of Organismal Biology and Anatomy and Neurobiology and Computational Neuroscience at the University of Chicago. Using predominantly zebra fish, Hale’s lab studies neural circuits that control limb and axis movement and how that movement changes over time. Movement changes can be seen both in the short time frame of development… Continue Reading
Dr. Neil Shubin is a Professor in the Department of Organismal Biology and Anatomy and the Committee on Evolutionary Biology at the University of Chicago. Shubin’s research focuses on understanding the evolutionary origins of new anatomical features such as limbs. Shubin is well known for his discovery of Tiktaalik roseae,the 375 million year old fossil… Continue Reading
Youreka Science was created by Florie Mar, PhD, while she was a cancer researcher at UCSF. While teaching 5th graders about the structure of a cell, Mar realized the importance of incorporating scientific findings into classroom in an easy-to-understand way. From that she started creating whiteboard drawings that explained recent papers in the scientific literature… Continue Reading