Chromosome mis-segregation in inviable Xenopus hybrids

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00:00:12.01 Imagine that you’re taking a walk with me outside.
00:00:15.24 It’s a beautiful sunny day,
00:00:18.11 and we’re climbing up a small hill,
00:00:20.28 and we’ve reached the peak.
00:00:23.07 Take a deep breath in,
00:00:25.08 maybe catch your breath a little bit,
00:00:27.12 and look around.
00:00:29.12 What do you see?
00:00:31.20 There’s a beautiful view,
00:00:33.23 and there’s lots of greenery around you.
00:00:36.24 Maybe you also notice some really vibrantly colored flowers
00:00:41.01 or the different types of trees that are around.
00:00:44.11 Maybe there are birds flying above you,
00:00:47.06 chattering to each other or looking for their next meal.
00:00:51.26 You might also even notice the insects
00:00:54.26 underneath your feet.
00:00:56.26 It’s a really beautiful scene
00:00:59.11 and there’s lots of species around you.
00:01:02.14 But have you ever stopped to think about
00:01:05.11 what makes us unique from other species?
00:01:07.27 Or more broadly speaking, how did we all get here?
00:01:13.08 My name is Maiko Kitaoka,
00:01:15.21 and I’m a PhD candidate at UC Berkeley.
00:01:19.03 This is a photo of me in front of the Golden Gate Bridge,
00:01:22.17 and I hope you can also appreciate
00:01:24.14 that there are plants around me
00:01:26.06 as well as some hidden marine animals behind me in the water.
00:01:30.12 Again, there’s a lot to see here,
00:01:33.10 and I hope that by the end of my talk,
00:01:35.06 I’ll be able to convince you that
00:01:37.11 all of us got here through a combination
00:01:39.07 of cell division and evolution.
00:01:43.01 Now, these are two very different scales in biology,
00:01:47.03 but they’re both crucial to helping us
00:01:49.13 get to where we are today.
00:01:52.00 So, let’s start with cell division.
00:01:54.18 This is an essential process
00:01:56.12 that all of our cells go through,
00:01:58.16 and actually, every organism starts with cell division.
00:02:03.08 For humans, we all start as single fertilized egg cells,
00:02:07.18 but actually the human body is made up of trillions of cells.
00:02:11.09 So, in order to create that sheer number,
00:02:14.10 we have to go through many, many cell divisions
00:02:16.16 in the first few hours of life.
00:02:19.02 And in this video, you’re looking at a single fertilized frog egg
00:02:23.02 that goes through many, many rapid and successive divisions
00:02:26.09 in the very first hours of life.
00:02:30.09 So, you can imagine that
00:02:33.10 if this process didn’t happen at all,
00:02:35.13 or if it went horribly wrong,
00:02:38.20 you would not be able to build a frog at the end of the day.
00:02:41.25 And that is true for humans, for insects,
00:02:44.29 and for any other organism.
00:02:47.29 But remember, we’re also thinking about
00:02:50.12 how we got here on the grander time scale of evolution.
00:02:54.15 And I’m specifically interested in a process called hybridization,
00:02:58.14 where two species mix to create new species
00:03:01.23 and new organisms.
00:03:04.10 You’ve actually probably already run into some hybrids,
00:03:08.09 perhaps at the fruit section of your grocery store,
00:03:11.02 where a lot of summer fruits are actually hybrids,
00:03:14.06 like this example of plutots and apriums,
00:03:16.21 which are made from plums and apricots.
00:03:20.10 But actually, a lot of the Tree of Life
00:03:24.12 uses hybridization in its evolutionary history.
00:03:28.28 Even humans have evidence of interbreeding
00:03:32.18 with early human hominids in our evolution.
00:03:35.18 So, it’s really not just a tool that farmers use
00:03:38.16 to make more delicious summer fruits.
00:03:42.05 But there’s a catch to this.
00:03:44.03 In the Tree of Life, and when you go outside
00:03:46.13 and see the different species around you,
00:03:49.00 you’re really only able to see the species
00:03:51.01 that have survived, or the hybridizations that worked out,
00:03:55.17 in the evolutionary time scale.
00:03:58.13 But not all hybrids work.
00:04:01.10 Many end up being sterile and cannot continue reproducing.
00:04:05.12 But also many pairings don’t work out.
00:04:09.22 Now, this is probably intuitive to you
00:04:12.16 if you think about the example of the princess and the frog,
00:04:15.06 where in the absence of magic
00:04:17.28 it makes sense that this princess and this frog
00:04:20.04 are probably not going to make a hybrid
00:04:22.26 that works out.
00:04:25.13 But this is where my research comes in.
00:04:28.22 I use closely related frog species to ask the question of,
00:04:32.25 what failures arise in cell division due to hybridization?
00:04:38.09 Now, to answer this question,
00:04:40.02 I first need to introduce you to
00:04:42.29 my cast of froggy characters.
00:04:45.22 I use three different species of african clawed frogs
00:04:48.15 from the genus Xenopus.
00:04:50.23 And here I’m showing Xenopus laevis, the big frog on the left.
00:04:55.13 In the middle we have the smaller frog, Xenopus tropicalis.
00:04:58.20 And on the right, we have Xenopus borealis,
00:05:01.01 the medium-sized frog.
00:05:03.24 These frogs are really easy to keep and maintain in the lab,
00:05:07.12 but importantly, for my work,
00:05:10.12 we’re able to isolate their eggs and sperm
00:05:14.03 in order to figure out which hybrids work
00:05:15.21 and which ones don’t
00:05:18.02 by mixing and matching them.
00:05:19.24 As you can see in this matrix,
00:05:21.11 if we do these types of experiments,
00:05:23.05 we’re actually able to create many different hybrids
00:05:25.12 from these three species.
00:05:28.02 But there are two that don’t work out,
00:05:30.19 and these are the ones that I’m interested in.
00:05:33.19 These are hybrids that are created when you take
00:05:36.09 the Xenopus tropicalis egg
00:05:38.06 and fertilize it with either the Xenopus laevis
00:05:40.06 or Xenopus borealis sperm.
00:05:42.19 And throughout my talk,
00:05:44.08 I’ll be referring to these as Hybrid 1 and Hybrid 2.
00:05:48.18 So, let’s see what happens when you make these hybrids in the lab.
00:05:52.15 I’m going to show you a video
00:05:54.19 where we have the Xenopus tropicalis embryo on the left
00:05:57.16 and the two hybrids next to it.
00:06:00.06 As the video starts, you’ll notice that these hybrids
00:06:04.06 are able to go through cell division seemingly very normally…
00:06:07.10 but then they run into a problem,
00:06:09.18 and they die in these really dramatic
00:06:12.04 and pretty horrible ways.
00:06:14.16 Clearly, these are not going to make frogs at the end of the day.
00:06:19.02 We’ve shown that these dramatic deaths
00:06:21.11 are due specifically to the loss of paternal DNA.
00:06:25.16 In these three images,
00:06:27.15 I’ve stained embryos with a DNA dye
00:06:29.15 so that you can see the DNA.
00:06:32.09 On the left, we have the Xenopus tropicalis embryo,
00:06:35.07 where the two populations of DNA are clearly and cleanly
00:06:38.27 getting separated into two distinct cells.
00:06:42.16 But in the hybrids, there are bits and pieces of DNA
00:06:46.07 that are lost in the center of that dividing plane
00:06:49.01 or tossed around the cell pretty haphazardly.
00:06:54.04 So, when I first saw this data,
00:06:56.08 I got really interested in figuring out,
00:06:58.07 what happens to the paternal DNA
00:07:00.22 during this process of cell division in these hybrids?
00:07:05.22 It’s worth noting that DNA goes through
00:07:08.24 a lot of changes in cell division.
00:07:11.00 I’m showing you a movie here,
00:07:12.22 where the DNA is labeled in yellow,
00:07:15.15 and it’s going through many, many changes
00:07:18.00 and actually also has to coordinate with microtubules.
00:07:22.01 These are cables in the cell
00:07:24.13 that will eventually attach to the DNA
00:07:26.10 and physically pull them apart at division.
00:07:30.08 So, DNA has to go through
00:07:33.13 two really big changes during this whole cycle,
00:07:36.26 the first of which is replication,
00:07:39.17 where the entire genetic materia
00:07:43.06 l is completely and accurately duplicated
00:07:45.17 so that eventually, when you get to segregation,
00:07:48.29 these DNA pools can be pulled apart
00:07:52.01 as cleanly and accurately as possible.
00:07:54.29 So, if we stop a little bit and look at this in more detail,
00:07:59.11 you realize that the DNA changes shape a lot in this process.
00:08:04.14 In particular, during the segregation process,
00:08:07.03 they have to become what are called chromosomes.
00:08:10.16 Here in this image,
00:08:12.28 normally you can see DNA as one big, circular lump in a cell.
00:08:16.15 But after going through replication,
00:08:18.24 they start to condense and become these X-shaped rod structures.
00:08:22.23 These are the chromosomes,
00:08:24.11 and these are the structures that then attach to those microtubule cables
00:08:28.16 and get pulled apart during segregation.
00:08:33.10 Importantly, this cycle happens over and over and over again,
00:08:37.20 so after the cell has divided,
00:08:40.04 the DNA comes back together into one circular lump
00:08:43.02 and repeats the process all over again.
00:08:46.15 Now, in an embryo in the very first hours of life,
00:08:51.09 DNA has to go through this process
00:08:53.26 over and over and over again,
00:08:56.07 very rapidly, and in an error-free manner.
00:08:59.28 So, it’s a big challenge for the embryo and for the DNA.
00:09:04.08 I was really interested to see if evolution
00:09:06.20 had co-opted any of these processes
00:09:08.25 to create mismatches between different species
00:09:12.12 so that, potentially, maternal and paternal species
00:09:17.04 now are no longer compatible with each other.
00:09:19.21 And in particular, I was really interested
00:09:22.00 in a special region of every chromosome
00:09:24.16 called the centromere.
00:09:26.13 This region is unique because it is the place
00:09:29.28 that builds the machinery so that microtubules
00:09:33.04 can attach to the chromosomes and eventually
00:09:36.04 pull them into the dividing cells.
00:09:38.19 And paradoxically,
00:09:42.05 this is one of the most rapidly evolving regions of the genome,
00:09:44.27 despite the fact that it is so essential
00:09:47.17 for this process of cell division.
00:09:50.18 So, I hypothesized that,
00:09:53.01 perhaps, in a hybrid
00:09:56.24 we’re running into a situation
00:09:59.02 where the maternal chromosome and the maternal centromere
00:10:01.06 is well matched with the surrounding maternal centromeric proteins,
00:10:04.23 which then means that we can
00:10:07.13 maintain the centromere properly
00:10:10.00 and allow it to attach to those microtubule cables.
00:10:13.08 But perhaps on a paternal chromosome,
00:10:15.29 there’s been enough evolutionary time
00:10:18.22 that the centromere is now very different,
00:10:20.27 and that means that the proteins
00:10:23.29 from the maternal environment
00:10:26.03 are no longer compatible to maintain that paternal centromere.
00:10:31.10 Now, in order to answer this question,
00:10:33.02 I needed a system that would help me
00:10:35.29 parse out the individual contributions from the paternal DNA
00:10:40.09 as well as the maternal environment that these chromosomes are in.
00:10:43.27 And actually, a lot of hybrids
00:10:46.19 make that really complicated
00:10:48.15 because we also have to deal with the maternal half
00:10:51.12 of the hybrid genome.
00:10:54.03 But this is where my Xenopus frogs
00:10:56.09 are the perfect system to answer this question,
00:10:59.04 because we can make what’s called Xenopus frog egg extract,
00:11:03.07 where we can collect the thousands and thousands of eggs
00:11:06.08 that female frogs lay,
00:11:08.09 spin them very, very quickly
00:11:10.07 to separate out the cellular layers,
00:11:12.10 and then isolate just the cytoplasm from these eggs.
00:11:16.07 This is essentially a cellular soup.
00:11:18.12 It has all of the proteins and other biomolecules
00:11:21.15 necessary to maintain cellular activity.
00:11:24.16 And importantly, it’s just from the maternal species,
00:11:28.25 without any other DNA or membranes
00:11:31.25 or other contaminating components.
00:11:35.09 And so, using this system,
00:11:37.12 I’m able to add in different species’ paternal DNA
00:11:40.08 to see what happens when they enter maternal cytoplasm.
00:11:45.03 And because the cellular soup is still really active,
00:11:48.05 we can look at DNA replication,
00:11:50.06 as well as mitotic chromosomes,
00:11:52.21 to see what’s happening
00:11:54.27 in a much more simplified and controllable environment
00:11:58.05 that mimics the embryo in vitro.
00:12:01.19 So, I was really excited to use this system
00:12:03.20 and look for the centromere or any other markers
00:12:06.20 on mitotic chromosomes.
00:12:10.10 And here, I’m showing you
00:12:12.16 a single Xenopus tropicalis chromosome
00:12:14.25 that has a very normal centromere,
00:12:17.10 and you can see on this chromosome
00:12:19.15 that there is one particular spot labeled
00:12:21.14 with this green centromeric protein.
00:12:24.23 But i was really curious to see that in the hybrids
00:12:29.23 some chromosomes actually had no centromeric protein marks
00:12:33.16 at all.
00:12:35.05 And so, I wanted to count this up
00:12:37.15 and see how prevalent this was in the population of chromosomes.
00:12:40.20 Here in this graph,
00:12:43.04 I’m looking at two different time points of chromosomes,
00:12:45.24 where t=0 corresponds to a time point before DNA replication
00:12:51.08 and 1 h corresponds to a time point after DNA replication.
00:12:55.14 And on the y-axis,
00:12:56.29 I’m looking at the number of chromosomes
00:12:58.26 that have centromeres in the total population.
00:13:01.20 And when I did this quantification,
00:13:06.28 I was really interested to see that the hybrids
00:13:09.00 start to lose some centromeres only after going through DNA replication.
00:13:14.13 So, this gave me a really interesting phenotype to study,
00:13:18.01 and I was really interested in trying to figure out
00:13:20.27 how I could rescue these centromeres in the hybrids.
00:13:24.03 Is there something that I could do to save the centromeres
00:13:27.05 and make sure that they are maintained
00:13:29.22 in hybrids?
00:13:31.27 So, first off, let’s start with Hybrid 1.
00:13:34.24 I wanted to see if the rapid evolution of the centromere
00:13:40.11 and the centromeric proteins
00:13:42.03 was contributing to this loss of centromeres in hybrids,
00:13:45.00 so I tried to add in to my system
00:13:48.20 just a maternal version of a specific centromere protein
00:13:51.12 called CENP-A.
00:13:53.11 And as you can see from this graph,
00:13:55.09 that actually didn’t give me any rescue of any centromeres.
00:13:59.04 But when I added in the paternal version
00:14:02.22 of the centromeric protein CENP-A,
00:14:05.20 I started to see more centromeres in the Hybrid 1 chromosomes,
00:14:09.12 and this was even more evident
00:14:12.00 when I also added in a paternal-specific
00:14:15.00 centromere helper protein called HJURP,
00:14:16.28 so that essentially,
00:14:19.26 now all of these chromosomes still maintained their centromeres
00:14:22.10 after DNA replication.
00:14:25.09 And this is really exciting
00:14:27.09 because you can essentially see evolution at work.
00:14:30.06 Clearly, there’s something about these paternal chromosomes
00:14:33.13 and these paternal centromeres that are a little bit different,
00:14:36.17 that only the paternally matched proteins
00:14:39.13 are able to help maintain that locus,
00:14:41.26 and the maternal proteins in the hybrid
00:14:44.01 were not doing the job.
00:14:47.26 Now, you may also recall
00:14:50.13 that I actually have two hybrids that I’m interested in.
00:14:53.09 And at this point, you might be wondering
00:14:55.07 what happened to Hybrid 2.
00:14:57.09 And I was really excited,
00:14:59.26 after seeing my results from Hybrid 1,
00:15:01.25 that I went and tried all of the analogous experiments
00:15:04.21 for Hybrid 2,
00:15:06.18 to look for the same type of centromere incompatibility.
00:15:10.06 But actually, all of my data was negative.
00:15:13.07 I was really disappointed —
00:15:16.00 and really confused, because there’s a lot that happens in cell division,
00:15:20.22 and I had no hints of where to go next.
00:15:23.23 So, this is essentially what my brain looked and felt like,
00:15:27.22 and I didn’t know what to do next.
00:15:32.00 Until one day, I was looking at a data set from Hybrid 2,
00:15:36.21 and again I was looking for centromere rescues,
00:15:38.29 so I was really disappointed
00:15:41.07 that I wasn’t getting any positive data.
00:15:44.21 And then I noticed something interesting about the chromosomes.
00:15:48.01 Normally, chromosomes look like
00:15:50.21 these rod-shaped spaghetti structures.
00:15:53.01 But some chromosomes in my Hybrid 2 data set
00:15:56.05 had these really weird ultra-thin stretched regions.
00:16:00.21 And at first I brushed this off
00:16:03.18 because maybe it was just an anomaly —
00:16:05.26 perhaps I did something that day to change the prep a little bit.
00:16:09.12 But then I started noticing them more and more and more
00:16:12.21 in this data set,
00:16:15.03 so I counted them up,
00:16:17.13 and I was really surprised because,
00:16:19.21 when I looked at the number of stretched chromosomes
00:16:21.16 across my different time points
00:16:23.29 both before and after DNA replication,
00:16:27.09 the stretched chromosomes
00:16:29.25 were really prevalent in Hybrid 2 alone,
00:16:32.22 and only after DNA replication.
00:16:35.16 This was really exciting for a number of reasons.
00:16:39.17 It was one of the first hints I had
00:16:42.13 of something going horribly wrong in the hybrid,
00:16:44.27 but also other people have seen these types
00:16:48.20 of chromosome shapes and morphologies before.
00:16:51.11 They’re found in other organisms,
00:16:53.27 and it’s also seen when chromosomes
00:16:57.24 have specific points in their DNA
00:17:00.14 that are more fragile and thus more prone to instability.
00:17:04.28 This actually changed my whole paradigm
00:17:07.11 about how I thought about this part of my project.
00:17:11.04 Up until this point,
00:17:12.29 I had been focusing almost exclusively
00:17:15.02 on the segregation half of this DNA cycle,
00:17:17.29 and I was completely ignoring the replication half of it —
00:17:21.18 how the DNA prepares itself and duplicates itself
00:17:26.04 prior to segregation.
00:17:29.22 And it’s well appreciated that DNA replication
00:17:32.20 can be really stressful,
00:17:34.24 and this could actually lead to DNA breakage
00:17:36.25 or other aberrations.
00:17:39.09 Specifically, sometimes the replication machinery
00:17:42.15 hits a stressful point in the chromosome
00:17:45.00 and it stops.
00:17:46.26 When this happens,
00:17:48.26 there’s a protein called p97
00:17:51.00 that comes in and helps remove that replication machinery
00:17:53.24 off of the chromosome.
00:17:56.08 This can lead to small nicks and breaks in the DNA
00:17:59.17 that are quickly repaired.
00:18:01.18 But I was really interested to see
00:18:04.07 if my Hybrid 2 chromosomes
00:18:07.00 were undergoing more replication stress than normal,
00:18:10.22 so I wanted to see if I could inhibit the action of p97
00:18:14.05 so that the replication machinery
00:18:16.14 will stay on the chromosome even if it’s stalled,
00:18:20.01 but then there wouldn’t be
00:18:23.11 any resulting chromosome breakage or stretching, perhaps.
00:18:26.06 And when I did this experiment,
00:18:28.11 I was so excited,
00:18:30.08 because when I treated this Hybrid 2 with this drug,
00:18:34.21 I was able to rescue and maintain
00:18:37.19 many, many more centromeres
00:18:40.00 and reduce the number of stretched chromosomes
00:18:42.06 that I saw in Hybrid 2.
00:18:44.27 So, in my project looking at two different hybrids,
00:18:48.26 I’ve actually discovered two different failures in cell division.
00:18:54.10 In my first hybrid, we have centromeric incompatibility,
00:18:57.16 where the paternal chromosome
00:19:01.11 has evolved and changed over time enough
00:19:03.10 that now the maternal proteins are no longer well matched
00:19:06.10 to the paternal centromere
00:19:08.14 to keep that region of the chromosome well maintained.
00:19:13.25 And my second hybrid
00:19:17.14 suffers from increased amounts of replication stress,
00:19:20.05 so that, eventually, many of the chromosomes
00:19:22.18 end up stretched as a result.
00:19:26.15 And this is really interesting to me
00:19:29.05 because these are two different hybrids
00:19:31.11 with two completely different mechanisms
00:19:33.27 that failed in cell division
00:19:37.05 because of hybridization.
00:19:39.11 And I know, and many other people know,
00:19:41.12 that there are more mechanisms
00:19:43.27 and more potential avenues of failure
00:19:46.04 in cell division alone,
00:19:47.28 so it’s very cool to see how evolution
00:19:50.17 has co-opted this single essential process of cell division
00:19:54.17 to create barriers across species.
00:19:58.01 And it really points to this fact
00:20:00.10 that not only did we get here
00:20:02.17 due to early cell divisions that built up our cells,
00:20:06.06 but we also got here through this combination with evolution.
00:20:11.09 Importantly, I want to point out that
00:20:15.12 just because hybrids fail
00:20:17.11 doesn’t necessarily mean that it’s a bad thing.
00:20:19.07 The failures in hybridization
00:20:21.06 also help to keep species unique and separate from each other,
00:20:24.24 contributing to the world’s biodiversity.
00:20:27.27 And particularly in a time
00:20:30.06 when we’re often losing more biodiversity,
00:20:33.10 studying these types of failures in hybridization
00:20:36.15 helps us to understand how we might
00:20:40.07 protect, enhance, or even recreate some biodiversity in our world today.
00:20:46.03 Thank you.

This Talk

Speaker: Maiko Kitaoka

Audience:

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Recorded: June 2021

Talk Overview

When we think of what makes two species distinct, we often think of their ability (or inability) to produce viable offspring. But is there anything we can learn from inviable offspring? In this Share Your Research talk, Maiko Kitaoka discusses her work on closely related Xenopus frogs. She reveals how examining hybridization between these species at the cellular level can provide insight into only the mechanisms that restrict cell division and development, but also broader patterns of how new species are able to evolve.

Speaker Bio

Maiko Kitaoka

Maiko Kitaoka is a PhD candidate in the Heald Lab in the Department of Molecular and Cell Biology at the University of California, Berkeley. Broadly interested in what the cell cycle and cell division reveal about developmental and evolutionary biology, she uses three different Xenopus frog species to study spindle morphometrics and the mechanisms driving… Continue Reading

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Kevin McLean (iBiology): Producer
Brittany Anderton (iBiology): Producer
Eric Kornblum (iBiology): Videographer
Jiefei Yuan (iBiology): Editor
Chris George (iBiology): Graphics
Maggie Hubbard (iBiology): Graphics

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