The Role of Homeotic Genes in Development

Part 1: Patterning the Anterior-Posterior Axis: The Role of Homeotic (Hox) Genes

00:00:07.20 Hi, my name is Nipam Patel.
00:00:09.12 I'm a faculty member at the University of California, Berkeley, in the departments of
00:00:13.00 Molecular Cell Biology and Integrative Biology.
00:00:15.27 And in my lectures today, what I hope to tell you about is patterning of the animal
00:00:21.19 along the anterior-posterior axis, from the front to the back, and a set of genes
00:00:26.09 called the homeotic or Hox genes, which are intimately involved in this process.
00:00:30.04 But as a way of introduction, like many scientists, I'm fascinated by the diversity that you see.
00:00:36.04 And this slide, here, illustrates that.
00:00:38.15 It's a very animal-centric slide, but it gives you an idea of the kind of
00:00:42.13 incredible diversity of morphology and of life histories that you find in animals on Earth today.
00:00:48.11 And so we can ask a lot of questions about how this diversity arose in terms of
00:00:53.04 thinking about the evolution of this... of this process.
00:00:55.20 So, we can look at this at many different levels.
00:00:58.10 So, for example, we can think about... in the case over here,
00:01:02.04 this is a single species of butterflies that shows many different variations in its color pattern.
00:01:07.21 And so this is an example of a microevolutionary process -- variation within a population.
00:01:12.11 And we can also study it at the extreme opposite, which is variation, say, between phyla
00:01:17.06 of animals that look very, very different from one another.
00:01:20.01 And so scientists have been engaged for a long time in understanding evolution
00:01:23.22 at many different levels of organization.
00:01:25.25 So... but it in all of this, though, there's also the understanding that all of these animals
00:01:32.06 start off as a single-cell embryo.
00:01:35.00 And many times when you look at that single cell, you can't really tell what kind of
00:01:40.02 species that's going to be.
00:01:41.02 But then it goes ahead and develops into these different adult forms that are
00:01:45.09 quite remarkably different from one another.
00:01:47.12 And so when evolution occurs to give you different organisms, one of the ways we can study that
00:01:52.17 is thinking about how there's changes in the process of embryonic development
00:01:57.04 that lead to those differences in the final organization of the organism.
00:02:00.23 And in fact Darwin, when he wrote On the Origin of Species, he devoted a whole section
00:02:05.15 to embryology, because he understood that changes in this pattern of embryogenesis would underlie
00:02:11.10 many of the changes in morphology that you see in evolution.
00:02:15.02 And so here's just one example of embryonic development.
00:02:18.13 These are frog embryos.
00:02:19.19 They're just at the two-cell stage.
00:02:21.26 And over the next couple days, they're gonna develop, and turn into tadpoles.
00:02:24.27 And here, you're gonna watch that process.
00:02:27.04 So, the cells are dividing very rapidly.
00:02:29.18 And then you get a process called gastrulation, where tissue goes inside.
00:02:33.13 And in a moment, you'll see the nervous system beginning to form in these embryos.
00:02:37.28 And then you're going to have internal organs developing.
00:02:41.03 And then these guys are gonna hatch out as little tadpoles.
00:02:43.20 So, a huge amount is happening there, in those few days of development.
00:02:49.10 And so, we can sort of think about, what are some of the things that are happening?
00:02:52.20 So, one thing, of course, is that cells are simply dividing.
00:02:55.06 You're going from a single cell to many thousands, millions of cells.
00:02:58.20 Okay?
00:02:59.20 But it's not... those cells are no longer identical to one another.
00:03:02.12 So, one of the key things that's going on in embryonic development is cell differentiation.
00:03:06.20 So, the cells are becoming different from one another.
00:03:09.11 But at the same time, there's also a process of morphogenesis.
00:03:12.04 So, the right cells have to be in the right place at the right time.
00:03:16.08 And it's that process that really gives you the overall form of an organism.
00:03:20.14 Okay.
00:03:21.14 So, developmental biologists have been studying the phenomena of development, of embryogenesis,
00:03:25.27 in many different ways.
00:03:27.02 So, you can try to just watch the embryo, and you understand a lot just from watching.
00:03:32.00 The other thing you can do, of course, is do experimental manipulations to perturb cells,
00:03:35.27 maybe to kill a cell, do something like that.
00:03:38.06 But what people have been very successful at in the last few decades is really taking
00:03:44.00 a genetic analysis.
00:03:45.24 So, the idea is that we want to understand the development of an organism at a very detailed
00:03:52.17 molecular and genetic level.
00:03:54.04 And then we understand how development, then, might be able to generate new morphologies.
00:04:00.05 And so as I mentioned, one of the most successful approaches to trying to understand development
00:04:04.20 is to take a genetic approach, which means to actually do things to manipulate the genes
00:04:09.24 in the embryo.
00:04:10.24 So, it could be just random genetic mutagenesis of the animal.
00:04:15.02 Or it could be very directed against individual genes.
00:04:19.13 But to understand development at that sort of level, we need model species.
00:04:23.15 We need species which are really amenable to genetic analysis.
00:04:27.03 And that's led to the adoption of a relatively small number of species, in which
00:04:31.22 we've focused a lot of energy in understanding development at an incredibly detailed level.
00:04:36.11 And those include things like the nematode (C. elegans) and the zebrafish (Danio rerio).
00:04:41.08 But what I'm gonna focus on today, for the start of my talk, is another one of the workhorses
00:04:45.20 for genetic analysis of development, which is Drosophila melanogaster.
00:04:49.00 So... otherwise known as the fruit fly.
00:04:51.11 So, why choose Drosophila?
00:04:53.19 What are the benefits of it?
00:04:54.28 So, one of the striking things about Drosophila is that it's a relatively complicated organism.
00:04:59.24 It's small and easy to keep, but it has a rapid life cycle.
00:05:03.11 So, the adult flies mate.
00:05:05.15 They lay eggs.
00:05:06.21 Those eggs hatch out, actually, as larvae.
00:05:09.20 And then those larvae undergo metamorphosis as pupae, and emerge adults, again,
00:05:13.27 which then can mate, and start the whole cycle over again.
00:05:16.13 So, this whole process in Drosophila takes only about two weeks.
00:05:20.17 And so you can do fairly rapid experiments.
00:05:23.14 And especially for doing genetics, it's a very tractable system.
00:05:26.26 Okay.
00:05:27.26 So, one of the things that's undergone intensive study at the genetic level is patterning
00:05:33.15 along the anterior-posterior axis of the animal.
00:05:35.25 So, you're looking here, first, at an adult fly.
00:05:38.12 Okay.
00:05:39.12 And it's pretty obvious that it has a head and a tail end, right?
00:05:42.07 So, the head is over there with the eyes.
00:05:44.14 And then you've got a thorax -- three segments of the thorax, with legs on those segments.
00:05:49.00 The second segment of the thorax has wings on it.
00:05:51.24 The third segment has a little structure called the haltere, which I'll show you more of later.
00:05:56.04 And then you've got segments along the abdomen.
00:05:58.24 Okay?
00:05:59.24 But that's the adult form.
00:06:01.08 And down below you're seeing a larva.
00:06:03.15 So, this is what's hatched out of the egg.
00:06:06.03 And you can see, I've numbered the segments here, at the top.
00:06:09.01 So, you've got a series of segments of the... of the head, and then you have three thoracic segments,
00:06:14.06 and then, at this stage of development, you can clearly see about nine of the abdominal segments.
00:06:19.20 Now, fortunately for us, in Drosophila the segments are clearly defined by these things
00:06:23.07 called denticle belts.
00:06:24.09 Now, I've false-colored them here.
00:06:26.06 They're normally just white.
00:06:28.01 But they're sort of stiff hairs on the bottom of the animal.
00:06:30.19 And they demarcate the separation between segments that you're seeing here.
00:06:34.14 But if you stare at these long enough, you actually realize that there are slightly different patterns
00:06:37.26 to these denticles, and you can recognize the individual segments separately from one another.
00:06:43.17 So anyway, this is all to say that the organism is very, very clearly organized into segments,
00:06:48.14 and that that body... those segments also show regional specification,
00:06:52.03 so that different segments are different from one another.
00:06:54.20 And so this was the starting point for a very intensive set of genetic analyses,
00:06:59.20 which has led to a very detailed understanding of the genes that actually set up segments,
00:07:04.14 and then give identity to those segments.
00:07:06.14 So, this is known as a... sort of a cascade of genetic interactions that lead to segmentation
00:07:11.20 of the embryo.
00:07:12.23 And finally, a set of genes, which I'm going to focus on today, called the Hox or homeotic genes,
00:07:16.26 which are the ones that actually give regional identity.
00:07:19.18 So, I'm not gonna go through this process of segmentation.
00:07:22.08 Like I said, I'm gonna focus on the Hox genes.
00:07:24.07 But I just wanted to mention that this sort of... this was one of the high points of
00:07:28.23 this sort of genetic approach in Drosophila, early on.
00:07:32.15 And it led, in 1995, to the awarding of the Nobel Prize in Medicine and Physiology
00:07:38.13 to Christiane Nusslein-Volhard, Eric Wieschaus, and Ed Lewis.
00:07:41.15 So, Nusslein-Volhard and Wieschaus were primarily working on the process of segmentation,
00:07:47.09 and Ed Lewis is the person who did much of the pioneering work looking at genes
00:07:52.13 that control regionalization of the body plan, the so-called homeotic or Hox genes,
00:07:56.13 which are gonna be the main focus of my talk today.
00:07:58.27 So, the hallmark of Hox genes, or homeotic genes, is their phenotype.
00:08:03.20 Okay?
00:08:04.20 And maybe one of the most famous of those phenotypes is shown here.
00:08:07.21 This is the phenotype that results from a mutation in a gene called ultrabithorax,
00:08:12.02 often referred to as Ubx.
00:08:14.04 So again, over here is a wild type fly, okay?
00:08:17.07 And what I want to highlight is that it has, again, three thoracic segments.
00:08:21.18 Each segment of the thorax has a pair of legs.
00:08:24.27 But then there's a pair of wings on the second thoracic segment.
00:08:28.12 And the third thoracic segment has a structure called the haltere.
00:08:31.22 So if you look here, this inset shows this haltere.
00:08:34.02 It's this very, very small structure that's right there.
00:08:37.26 And it looks kind of like a tiny wing, but it has a very different function.
00:08:41.06 It functions as a gyroscope, and it helps to orient the animal as it's flying.
00:08:45.23 So again, the second thoracic segment has the pair of wings,
00:08:48.20 and the third thoracic segment has the pair of halteres.
00:08:51.16 Here, in a Ubx mutant, what you're seeing is that the third thoracic segment,
00:08:57.08 the segment that normally has the halteres, is transformed into the second thoracic segment.
00:09:02.11 This results in a really remarkable change in the morphology of the animal.
00:09:06.02 It has two pairs of wings, or four wings, now, instead of the normal single pair of two wings.
00:09:12.02 And so this is, again, showing the hallmark of these homeotic mutants.
00:09:15.13 The number of segments is still the same, but what's happened is the identity
00:09:20.04 of the segments have been transformed.
00:09:21.22 In this case, the third thoracic segment has been transformed into the second thoracic segment,
00:09:25.27 causing a repeat of those wings on this animal.
00:09:29.14 And so, again, this is a mutation in a gene called ultrabithorax.
00:09:32.19 Now, they're called homeotic or Hox genes.
00:09:36.03 And part of the reason they're called that actually was from much older literature.
00:09:40.17 So, a guy by the name of William Bateson... in 1894, he published a book called
00:09:45.26 Materials for the Study of Variation.
00:09:48.04 So, Bateson was really fascinated by animals that have serially repeating segments, right?
00:09:53.08 And that includes things like flies, other insects, arthropods, and even us.
00:09:57.09 As humans, we have repeating vertebrae down our axis.
00:10:00.17 And one of the things that Bateson noticed was that he would often see transformations
00:10:06.05 of one region into another.
00:10:07.20 So, this again shows a fly that Bateson had observed.
00:10:11.25 And in a normal... this is the normal antenna on this side, okay?
00:10:15.22 But in this particular fly, this is not an antenna, a normal antenna anymore.
00:10:19.23 It's been transformed into a leg.
00:10:21.19 So, you can see the sort of claw parts at the end of it.
00:10:24.18 And so Bateson was fascinated by these kinds of transformations.
00:10:28.11 And he termed them homeotic or homeosis.
00:10:30.27 So, it comes from the Greek root for the word the "same".
00:10:34.04 So, one part of the body is transformed into the likeness of another.
00:10:38.14 So in this case, the antenna is transformed into a leg.
00:10:41.28 And Bateson really felt that that was telling us something about the guiding principle...
00:10:46.10 principles of development, of how the organism was organized.
00:10:49.17 And so Bateson collected literally thousands of examples of these kinds of homeotic mutants,
00:10:55.22 and other kinds of variation that he would see in organisms that he thought were
00:11:00.10 very useful for thinking about how organisms were put together.
00:11:02.20 The problem, of course, from a genetic viewpoint was that these were sort of one-off animals,
00:11:07.23 right?
00:11:08.23 These were sort of perturbations that probably happened... they may not have had, really,
00:11:12.04 a particular genetic basis to them.
00:11:14.05 And so they couldn't breed true and be reproduced.
00:11:17.03 But in fact, another example of a homeotic mutation in Drosophila looks
00:11:21.17 remarkably like this fly that Bateson has drawn.
00:11:24.02 And it's from a mutation called antennapedia, another Hox gene.
00:11:28.06 So again, in a wild type fly, here... this is a scanning electron micrograph of the head of a fly.
00:11:33.18 And what I want to do is point out those antennae that are there in the... in the...
00:11:37.06 in the head of the fly, right here.
00:11:39.12 Okay.
00:11:40.12 And in an antennapedia mutant, the antennae are replaced by legs.
00:11:44.08 So, that's really a dramatic transformation of the body plan.
00:11:47.09 So, where there were antennae, now there are legs on this fly, right?
00:11:51.00 And this is due to a mutation in this gene called antennapedia, another one of the Hox genes.
00:11:56.19 So, through the work of many hundreds or thousands of people, now, we have
00:12:01.21 a really detailed understanding of these Hox genes in Drosophila to begin with.
00:12:06.15 And in flies, there are eight of these genes, and they're... and they're listed, here,
00:12:10.15 down at the bottom.
00:12:11.15 So, going from anterior to posterior, they're labial, proboscipedia, deformed, sex combs reduced,
00:12:16.07 antennapedia, Ubx, abdominal-A, and abdominal-B. And you can see how they're
00:12:23.16 organized in their expression pattern along the axis of the animal.
00:12:26.09 So, you're looking, here, at a side view of a fly embryo.
00:12:30.00 This side is the ventral side, and this side is the dorsal side of the embryo.
00:12:34.03 And then the colors indicate the different domains of expression of these various Hox genes.
00:12:40.13 So, you can see they're expressed in these block-like patterns
00:12:43.08 -- and sometimes there's overlap in those patterns --
00:12:45.22 headed from the anterior end, the head, to the posterior end.
00:12:50.06 One of the remarkable things is that these genes are also organized on the chromosome
00:12:54.06 in the same order that they're expressed along the axis of the animal.
00:12:57.26 And this is actually still an area of intense investigation, trying to understand
00:13:02.20 exactly why those genes are really organized that way.
00:13:05.08 But for the purposes of our talk, the important thing is that they are expressed
00:13:09.23 spatially along the anterior-posterior of the axis like this.
00:13:12.10 Okay.
00:13:13.10 But remember, their hallmark is that their mutant phenotype... is that they give homeotic transformations --
00:13:18.05 they transform one region of the body into another.
00:13:21.04 So, they don't change the number of segments or repeats of the animal.
00:13:24.15 But they cause their identity to be shifted.
00:13:27.03 So, here's just an illustration of how these genes are expressed.
00:13:30.22 In this case, it will be a particular example, Ubx.
00:13:33.00 So, now I'm showing you a ventral view of the fly embryo.
00:13:36.06 The head is up here; the tail is down here.
00:13:38.18 The grooves you see here are the segments.
00:13:40.22 So, this embryo is about halfway through embryogenesis,
00:13:43.18 which for flies is around 12 hours or so of development.
00:13:47.08 And again, you can very clearly see the segments.
00:13:48.22 And what we're gonna do... we'll put this movie into motion.
00:13:51.25 And you can see we're just, like, imaging through half of the embryo.
00:13:54.13 And if we peel away that surface, and now we're looking at a gene called engrailed,
00:13:58.20 which marks the posterior boundary of each segment in green.
00:14:01.20 And in red, now, we have one of these Hox genes, Ubx, which, again, is expressed
00:14:06.01 in a particular domain of the embryo, shown here by that... by that red coloring.
00:14:10.21 Here's another example of looking at the localization of some of these Hox genes.
00:14:15.05 So in this case, sex combs reduced, antennapedia, ultrabithorax, and abdominal-B.
00:14:20.08 And again, you can see that these genes are expressed along the anterior-posterior axis of the animal.
00:14:24.23 Anterior is to this end; posterior is here.
00:14:27.11 And the darkened area towards the middle is the nervous system of the animal.
00:14:30.24 But again, the point is that these genes have very spatially restricted expression patterns.
00:14:35.18 Okay.
00:14:36.18 So, it turns out that all of these genes are actually related to one another,
00:14:40.26 and that they have a common sequence in them called a homeodomain, or the... sorry, the DNA sequence
00:14:46.19 is called a homeobox.
00:14:47.21 And it includes a structure called the homeodomain, which is illustrated here.
00:14:52.04 And this domain allows the protein to bind specifically to sequences... to DNA sequences.
00:14:58.01 So, these are what are called transcription factors.
00:15:00.07 So, they turn out to be regulatory molecules that turn on and off hundreds if not thousands
00:15:05.24 of other genes.
00:15:06.24 So, these genes are expressed in very specific places along the axis.
00:15:10.22 Each one is slightly different, and each one controls different sets of downstream targets,
00:15:15.02 which gives, in the end, the final morphology of the particular segments that those genes
00:15:19.02 are expressed in.
00:15:20.06 Okay.
00:15:21.06 So, I told you...
00:15:22.06 I showed you some examples of Hox mutants that actually make it to adulthood,
00:15:26.12 and you can see transformations of the animal.
00:15:28.20 But in fact, for any of these Hox genes, if you completely delete the gene or you
00:15:32.23 make a mutation that we would call a null mutation, a completely non-functional copy of the gene,
00:15:37.24 what happens is that these embryos surely die at the end of embryogenesis.
00:15:41.26 But when you look at them, you can again see these spectacular homeotic transformations
00:15:46.15 of the body plan.
00:15:48.02 And I'm gonna mostly talk about the most posterior of the three genes, so, ultrabithorax, abdominal-A,
00:15:53.15 and abdominal-B.
00:15:55.09 And this is traditionally called the bithorax complex of Drosophila.
00:15:59.15 And what I'm illustrating here is the phenotype that you see.
00:16:02.22 So first, if you look at this line that says wild type, what I've done is I've given you
00:16:07.02 the names of the different segments of the animals, starting in the posterior part of the head
00:16:10.18 -- mandible, maxilla, labial -- and then you've got the three thoracic segments
00:16:15.02 -- T1, 2, and 3 -- and then here I'm illustrating eight of the abdominal segments.
00:16:19.22 So first, let's start with Ubx.
00:16:20.26 So, what happens when you completely remove the Ubx gene?
00:16:24.00 So again, the animal dies as an embryo.
00:16:26.22 But when you look at it, at the end of embryogenesis, it forms those denticle belts.
00:16:30.26 You can look at them, and you can tell what transformations have occurred.
00:16:33.24 And what happens is that T3 and A1 now are transformed into T2.
00:16:38.09 So now, you have an animal with three T2 segments, and no T3 and A1.
00:16:42.24 Likewise, in an abdominal-A mutant, A2 through...
00:16:45.00 A2 through A4 are eliminated... or, they're transformed, sorry, into A1.
00:16:50.18 Right?
00:16:51.18 So, no segments are deleted; there's just a transformation of the segments.
00:16:55.17 And finally, in an abdominal-B mutant, it's even more posterior in its phenotype,
00:16:59.09 and now what happens is that A5 on back are transformed into A4.
00:17:03.17 So, this is the hallmark of the homeotic mutants, that they... you don't change the number of segments,
00:17:07.28 but you're transforming their identity.
00:17:10.09 Okay.
00:17:11.09 So, this is how those three genes are expressed.
00:17:13.19 So, Ubx shown there in green, abdominal-A shown in this purple color, and abdominal-B in red.
00:17:20.28 And what you can see is they have this sort of nested pattern of expression.
00:17:24.17 And I'll show you those domains in a little more detail in a second.
00:17:27.06 But this is, like, again, the typical kind of patterns that you see for Hox genes
00:17:31.11 during embryogenesis.
00:17:32.11 And again, in this animal, the head is towards the top and the tail is towards the bottom.
00:17:36.19 So, now what we can do is we can take those expression patterns I just showed you
00:17:40.22 and relate them to the segments and the phenotypes that you see.
00:17:44.08 So now, we have the domains of expression of Ubx, abdominal-A, and abdominal-B
00:17:48.24 relative to the different segments.
00:17:50.07 And now... we can think, now, that there's actually a really simple set of rules, now,
00:17:54.20 that we can apply to understanding the phenotypes.
00:17:57.05 So, let's start with Ubx.
00:17:58.16 So, Ubx begins expression in T3.
00:18:01.21 And so the transformation begins in T3 and A1, and those segments are transformed
00:18:06.03 into the segment in front, which is T2.
00:18:07.22 But that transformation ends when you start at the... when you get to the anterior boundary
00:18:12.11 of the next gene, abdominal-A.
00:18:14.21 Likewise then, when you delete abdominal-A, the transformation
00:18:17.13 starts at the leading edge of abdominal-A, which is here in A2, so that segment
00:18:22.05 gets transformed to the segment in front, A1.
00:18:24.22 That transformation to A1 extends until you get to the anterior boundary of the next Hox gene,
00:18:30.01 which is abdominal-B. And then, likewise, the abdominal-B transformation starts
00:18:35.02 at that boundary, there, at A5, and goes on back.
00:18:37.21 And those segments, again, now take on the identity of the segment in front, which is A4.
00:18:42.19 So, this leads to a relatively simple rule that we apply to Hox genes, which is that
00:18:47.27 when you delete expression of a Hox gene then those segments that lose expression
00:18:53.08 take on the identity of the segment in front of them.
00:18:55.13 And now, I'm telling you this...
00:18:56.13 I'm illustrating it for this set of genes -- Ubx, abdominal-A, abdominal-B,
00:18:59.23 the so-called bithorax complex
00:19:01.26 Things get more complicated in the head, but this is the set of genes we're going to
00:19:05.08 focus in quite a bit on, so just remember that set of rules for this set of genes.
00:19:09.08 So, how is it, then, that you end up, though, with adult phenotypes?
00:19:14.00 So, I told you that when you delete these genes, you get these transformations.
00:19:17.06 The embryo makes it to the end of embryogenesis, but then dies with these transformations
00:19:21.09 that you can see.
00:19:22.09 So, the way that you can think about how you get adult transformations is, first,
00:19:26.16 you have to understand where the adult tissues come from.
00:19:28.28 So, early in embryogenesis, you know, the external morphology of the embryo is set up,
00:19:34.09 but sets of cells are set aside that are going to be the adult structures.
00:19:38.15 So, these form structures called imaginal discs.
00:19:41.06 And so, these are inpocketing of cells, that in the larvae end up inside, and end up
00:19:46.07 growing quite large inside those larvae.
00:19:48.17 And those imaginal discs give rise to the different adult structures.
00:19:51.24 So, there's an imaginal disc for the wings, an imaginal disc for the halteres,
00:19:55.19 imaginal discs for the legs, and so on.
00:19:58.01 And so those structures are set aside in development.
00:20:01.02 But they're set aside in the same associated segments.
00:20:04.02 So, the haltere disc, for example, is set aside in the third thoracic of the embryo.
00:20:09.04 The wing discs are set aside from cells of the second thoracic segment.
00:20:13.22 And remember, then, again, where these Hox genes are expressed.
00:20:16.14 So, for example, Ubx is expressed from T3 on back.
00:20:19.21 Okay?
00:20:20.21 So, that means it's also expressed in those imaginal discs in those regions, right?
00:20:24.17 And in this case, the important one is gonna be in T3.
00:20:28.12 And so what you have is that in the second thoracic segment of the larvae,
00:20:33.12 when it's growing, you have an imaginal disc... sorry... that's going to make the wing.
00:20:37.15 And then you have an imaginal disc that's going to make the haltere.
00:20:40.16 And the wing disc is located in the second thoracic segment, and the haltere disc
00:20:44.24 is located in the third thoracic segment.
00:20:46.23 So, in keeping with that, Ubx is expressed in the third thoracic segment,
00:20:50.27 so what you see is that the haltere disc is expressing Ubx, shown in green here,
00:20:55.17 but the wing disc proper is not expressing Ubx.
00:20:58.26 And so it's that difference it actually sets the difference between T2 and T3.
00:21:03.04 So, what happens in that Ubx mutant, that gives rise to this four-winged fly,
00:21:08.25 is that the gene is perfectly normally expressed in the embryo, but that expression is lost later.
00:21:14.15 So, the haltere disc, which should be expressing Ubx, loses Ubx expression.
00:21:19.09 It then transforms into the equivalent disc in the second thoracic segment,
00:21:22.24 which is the wing disc.
00:21:24.10 And when the animal finishes metamorphosis, then you end up with this four-winged fly,
00:21:28.04 instead of the normal two-winged fly.
00:21:30.16 And so, this kind of transformation that goes... and you see as a phenotype in the adults,
00:21:35.17 in this case it's loss of Ubx expression from T3 late in development.
00:21:39.08 So, that's why it makes it through embryogenesis but then it has this hallmark adult transformation.
00:21:44.26 So, how does that happen?
00:21:46.22 So again, if you just think back, again, to what you know about the general structure of genes...
00:21:51.24 so, we often think of a gene as its coding region, but just as importantly
00:21:55.16 are these flanking regions called regulatory regions.
00:21:58.24 And so, if we were to eliminate the coding region, that would give us a null allele,
00:22:03.02 a complete loss of function.
00:22:04.20 But instead, these kinds of mutations that make it to adulthood are generally
00:22:08.26 mutations in the regulatory DNA.
00:22:10.02 So, they're affecting the expression.
00:22:11.19 And they're often in some part of the regulatory DNA which is only affecting late expression
00:22:16.21 of the gene.
00:22:17.21 And that gives us these kinds of phenotypes.
00:22:19.18 And in fact, these regulatory regions can be quite complicated, so you can have
00:22:23.05 different regulatory regions for different parts of an expression pattern.
00:22:26.17 In this case, I've just made up an example in a mouse, where there's some gene expressed
00:22:30.07 in the brain and the spinal cord, but there's different enhancers
00:22:33.20 -- what we call the regulatory regions -- for that.
00:22:35.22 So, all that is to say that, in fact, you can get mutations that give very, very specific phenotypes
00:22:40.10 because they're only knocking out a portion of the function of a gene.
00:22:45.22 Okay.
00:22:46.22 So, back to our... our point about these genes then being deployed along the anterior-posterior axis
00:22:52.25 of the animal and being transcription factors.
00:22:55.21 So then, these control the overall patterning along the whole axis.
00:22:59.25 And I told you in a little detail about the more posterior three, but then there's
00:23:04.08 five more genes, which are controlling more anterior regions of the animal.
00:23:07.15 So, one of the huge discoveries in the field of developmental biology a couple decades ago
00:23:11.24 was the finding that this was not a unique phenomenon in Drosophila.
00:23:16.01 And in fact, you could find this same complex of genes, essentially, in all animals.
00:23:20.05 And I'm just showing you an example here in mice, where you have a similar complex.
00:23:24.20 It's expressed in a similar way, being that it's in particular parts of the anterior-posterior axis
00:23:29.24 of the animal.
00:23:32.15 And I'll show you in a minute that these also are responsible for regionalization of
00:23:37.03 the body plan of a mouse, and also, presumably, a human, where you have the same complex.
00:23:41.19 The one thing that this diagram doesn't show you is an additional complexity,
00:23:46.09 which is that in mice and humans and other vertebrates there's actually four copies of the complex.
00:23:49.17 So, there's a little bit more complexity there.
00:23:52.02 But overall, there's that same similar organization.
00:23:55.04 And if you start to mutate these genes in something like a mouse,
00:23:58.16 you get the same sort of phenotypes that you see in flies.
00:24:01.07 So, in this case, I'm showing here a wild type mouse, where you have
00:24:05.13 the specialized vertebrae of the thorax with the ribs, and then you've got lumbar vertebrae,
00:24:09.10 which are the back, and sacral, which are even more posterior.
00:24:12.16 And in this particular mutant mouse, we knocked out a couple of different Hox genes,
00:24:17.04 and what happens is that now the lumbar and sacral vertebrae are transformed to be more thoracic-like.
00:24:23.06 So, they have ribs on them.
00:24:24.14 So, remember back to the whole idea that, in fact, what happens is when you delete a Hox gene,
00:24:29.01 then segments take on a more anterior identity.
00:24:32.10 And that's exactly what you're seeing in this mouse mutant.
00:24:36.04 So, from the perspective of developmental biology, this was of course a spectacular discovery,
00:24:42.19 that this set of genes that had been discovered in flies really gave rise
00:24:47.20 to a very universal understanding of a conserved mechanism for setting up regionalization
00:24:53.20 along the anterior-posterior axis of the animal.
00:24:55.22 And in fact, the presence of these Hox genes is often... by some... used by some as a signature
00:25:01.04 of being an animal, that you use these genes to set up your regionalization of the body.
00:25:05.14 Or more particularly, the set of animals called bilaterians
00:25:08.19 -- the ones that have a left and a right, and an anterior-posterior axis.
00:25:12.00 So, that's great from understanding the development.
00:25:15.06 Why were evolutionary biologists so excited by this kind of discovery?
00:25:19.05 And for that, let's go back to this slide, again, that shows the kind of transformation
00:25:24.08 that you get.
00:25:25.08 So, we end up with this four-winged fly, right?
00:25:28.19 But that might remind you of something, right?
00:25:30.25 So, that looks a little bit like other insects that are four-winged insects.
00:25:34.17 So again, here's our homeotic mutants of a... of a fly which now has four wings.
00:25:38.22 But if you think about it, and to some people, that looked a little bit like a dragonfly
00:25:42.07 or another four-winged insect.
00:25:43.27 And so people thought, well, maybe this is telling us something about the evolution of insects,
00:25:47.14 insects, about how you change the pattern of the body plan in insects.
00:25:51.21 Okay.
00:25:52.21 So, we can take that a little bit further.
00:25:54.18 So, as evolutionary... if thinking from an evolutionary viewpoint, one of the things
00:25:58.20 we'd like to know, then, is, what was the ancestor of a fly and a dragonfly?
00:26:03.19 Did it have one pair of wings, like the extant flies?
00:26:06.18 Or did it have two pairs of wings, like this dragonfly?
00:26:09.22 Okay?
00:26:10.22 So, how would we answer that question?
00:26:12.08 So, one way to think about that is to think about the phylogeny of insects,
00:26:16.22 so their evolutionary history and the branching pattern that you see.
00:26:20.17 And that's what's shown here.
00:26:21.24 So, different orders of insects are shown here.
00:26:25.05 And if you think about it, right, you can ask the question, well, what's the most parsimonious explanation
00:26:30.05 for a common ancestor way back here?
00:26:32.21 Did it have two pairs of wings or did it have a single pair of wings?
00:26:36.18 And one way we can answer that is if we look at this distribution, one of the things
00:26:40.08 that you notice is that, in fact, the only insect with just a single pair of wings, right,
00:26:45.07 the two wings, is Drosophila, is Diptera.
00:26:47.08 And in fact, Diptera means "two wings".
00:26:50.20 And so, if you look at other insects, like butterflies, or wasps, or grasshoppers,
00:26:55.03 or damselflies and dragonflies, what you see is all of those insects have two pairs of wings,
00:26:59.09 or four wings.
00:27:00.27 And then, ancestrally, though, something like silverfish, the realization is that
00:27:06.13 initially you didn't have wings.
00:27:07.17 You evolved wings.
00:27:08.17 But when you evolved wings, you had at least two pairs of wings.
00:27:11.26 And then the very derived state is what you see in Drosophila, with only a single pair of wings.
00:27:16.14 So, that means the common ancestor to flies and these other winged insects would
00:27:20.15 have had four wings, or two pairs of wings.
00:27:23.05 So, that gives us a very specific scenario for thinking about... well, could Hox genes
00:27:27.25 explain this change in wing number?
00:27:30.12 Okay?
00:27:31.12 So, now we're going from understanding a gene that's involved in development of Drosophila,
00:27:36.14 and thinking about how it might be involved in evolution of insects.
00:27:40.08 So, I'll give you sort of a specific scenario here, that you had a common ancestor,
00:27:44.28 which is some, you know, made up drawing that we have down here, but the key point is that
00:27:49.06 it has two pairs of wings.
00:27:51.04 Okay?
00:27:52.04 And then you have extant insects that still have two pairs of wings, and in this example,
00:27:56.26 I'm showing you a butterfly which clearly has two pairs of wings.
00:28:00.03 And then here's our fly, which has a single pair of wings.
00:28:03.01 And on T3, instead of wings, it has a haltere.
00:28:05.22 And we know that Ubx is expressed in T3, and that haltere on back, okay?
00:28:10.27 And so now we can come up with a couple of hypotheses about how we might explain
00:28:15.02 the evolution of insects, or the wing number in insects, from thinking about a Hox gene like Ubx.
00:28:21.19 So, one hypothesis -- and this is actually one of the things that Ed Lewis first thought about --
00:28:25.06 was, well, maybe this gene Ubx is actually unique to flies, to the...
00:28:31.24 its set of insects, Diptera, that only have this single pair of wings.
00:28:35.05 And that the Ubx gene arose somewhere in this lineage leading to flies.
00:28:39.07 And it wasn't present in that common ancestor.
00:28:41.16 And it's not present, then, presumably, in extant two-winged insects or... sorry...
00:28:47.28 insects with two pairs of wings, like a butterfly.
00:28:49.21 So, we know that this isn't true, because we now know that Ubx is a very ancient gene,
00:28:55.14 and you find copies of it in all insects, all arthropods, things like that.
00:28:58.28 So, we can rule this hypothesis out right away.
00:29:01.25 A very attractive hypothesis, though, was that, in fact, that four-winged mutants of flies
00:29:07.11 was actually telling you something about the kinds of regulatory changes that
00:29:12.01 might have occurred.
00:29:13.01 So, in this scenario... again, in flies we know the data that Ubx is expressed from T3
00:29:18.14 on back.
00:29:19.16 But in this hypothesis, what we would say is that in the common ancestor it was actually
00:29:23.13 expressed just from A1 back.
00:29:25.04 Okay?
00:29:26.04 So, Ubx is no longer expressed in T3, so then T3 has a pair of wings on it like T2.
00:29:31.26 And that we would see the same pattern in extant insects with two pairs of wings,
00:29:36.21 like a butterfly.
00:29:37.21 So, the difference between a butterfly and a fly would be that in flies Ubx
00:29:42.23 is expressed from T3 on back, but in something like a butterfly it's expressed from A1 on back, okay?
00:29:48.07 And that's presumably, then, also telling us something about this ancestor.
00:29:51.19 There's a third hypothesis, which is that, in fact, Ubx hasn't been involved in
00:29:56.00 this process at all.
00:29:57.00 So, Ubx has the same expression pattern in butterflies as it does in flies.
00:30:01.09 And it's always had that pattern.
00:30:03.00 So, it's hypothesis 2 that we can actually test pretty quickly.
00:30:06.20 So, we can ask, okay, so is the pattern of expression between Ubx different in flies
00:30:13.19 from insects with two pairs of wings, like a butterfly or a grasshopper or a beetle
00:30:18.13 or something like that?
00:30:19.17 And from that, we can make some sort of inference about the common ancestor.
00:30:23.10 Unfortunately, these common ancestors died out a long time ago, so we can't look at them,
00:30:26.28 okay, but we can look at extant animals.
00:30:29.19 So first of all, let's just look at their embryos.
00:30:31.12 So, here's a fly embryo.
00:30:33.25 And Ubx, again, is expressed from T3 on back.
00:30:35.17 But lo and behold, if we look at a beetle or a grasshopper
00:30:38.26 -- and these are examples of insects with four wings, two pairs of wings --
00:30:43.23 we actually see the exact same pattern.
00:30:45.16 It's expressed from T3 on back.
00:30:47.15 An even more convincing illustration comes from work that was done in butterflies.
00:30:51.13 So, butterflies, again, are a very clear example of an insect with two pairs of wings,
00:30:56.05 a forewing on T2 and a hindwing on T3.
00:30:59.06 Okay?
00:31:00.06 So, now we can ask, well, what do you see?
00:31:02.06 So, these are the imaginal discs for those two segments.
00:31:05.03 So, this is a forewing disc in T2, and a hindwing disc in T3.
00:31:09.08 And when we look at Ubx expression, what we see is that, in fact,
00:31:12.13 the hindwing expresses Ubx and the forewing doesn't.
00:31:15.15 So in fact, the pattern is just like in Drosophila.
00:31:18.12 It's expressed in the imaginal disc of T3 and not an imaginal disc of T2.
00:31:22.28 Okay, but then... but why... but butterflies have two pairs of wings.
00:31:25.26 But of course, if you realize and think about it, those two wings are not identical to each other.
00:31:32.03 So, in this particular butterfly, the pattern and shape of the forewing is very different
00:31:35.11 than the pattern and shape of the hindwing.
00:31:37.26 And so, in fact, the idea would be that Ubx still plays a role.
00:31:41.14 We call both of these wings, but they have different patterns, and different shapes,
00:31:46.03 and so on, those two wings.
00:31:48.00 And in fact, you can get homeotic mutants in butterflies as well.
00:31:52.00 So, this is showing an example, here.
00:31:53.17 So, a normal butterfly.
00:31:55.04 And in this butterfly, this region of the hindwing has been transformed into forewing.
00:32:00.09 So, you can see this little patch where the pattern, and in fact the scale morphology,
00:32:04.05 is the same as the forewing.
00:32:05.20 And this is actually an area of the wing in which Ubx is not being expressed properly.
00:32:09.23 So, it should be expressed uniformly in this hindwing, and this is a little area
00:32:13.17 of the wing where Ubx is not being expressed, and you see this homeotic transformation.
00:32:17.04 So, what it means is that hypothesis 3 is actually the right conclusion,
00:32:22.03 that in fact Ubx has always been expressed from T3 on back.
00:32:26.10 Okay.
00:32:27.10 But then, why is it that flies don't have what we would call a wing on T3.
00:32:31.10 They have this haltere structure.
00:32:32.28 So, the answer is is that... what's happened is that Ubx has always been
00:32:37.26 distinguishing T3 from T2.
00:32:39.21 But how it does that has changed, because the downstream targets have changed.
00:32:43.21 So, this pattern has been conserved from the ancestor, with whatever that may have
00:32:48.03 looked like in the... in the T3 wing.
00:32:50.05 You still always have it in the T3 wing.
00:32:52.13 But in flies, it's now somehow repressing the normal pattern of a wing,
00:32:56.15 the growth of the wing, and so on.
00:32:58.15 And turning on or off genes that end up giving you this haltere morphology.
00:33:01.26 Wherein butterflies, you're maintaining those downstream targets.
00:33:04.16 So, you still have a wing, though, then, now the color and the shape of that wing is different
00:33:08.17 than T2.
00:33:09.22 And so, this is an example, then, where we can see, now, we've made great strides
00:33:15.02 in understanding the development of organisms by understanding what these Hox genes do
00:33:19.22 in individual species like in Drosophila and mice and so on.
00:33:23.13 But it seems that at least in the case of insects, these genes are actually
00:33:27.23 quite static in their expression pattern.
00:33:30.03 And they're not necessarily moving around to give different morphology.
00:33:35.05 And so what I'm going to do now is... is... is... in the next talk, I'm gonna
00:33:41.01 give you an example, though, where we can say that Hox genes are moving around and giving rise
00:33:46.14 to changes in evolution.
00:33:48.01 So, in this first part, really again, just to reiterate, what I've really tried to drive home
00:33:52.04 is our understanding of what Hox genes do from the detailed analysis in animals like Drosophila.
00:33:58.13 And that gives us an idea of what they might do in evolution.
00:34:00.28 I ended my talk with maybe a slightly, you know, negative note by telling you that, well,
00:34:05.13 in the case of flies, moving around the Hox genes, a really good hypotheses for what they
00:34:09.21 might do in evolution, turned out not to necessarily be true, though we still gained
00:34:14.22 some really interesting insight into how downstream changes to the Hox genes may be involved in evolution.
00:34:19.17 But as I said, what we're gonna do in the... in the next section is jump outside of insects
00:34:24.14 and give you an example where we can show you that it looks like changing around
00:34:28.23 Hox gene expression has in fact led to evolutionary changes in animals.

Part 2: The Role of Ubx in the Development of Crustacean Body Plan

00:00:07.20 Hi, my name is Nipam Patel, and I'm a faculty member at UC Berkeley in the Department of
00:00:12.00 Molecular Cell Biology and in the Department of Integrative Biology.
00:00:15.28 In the first of my talks,
00:00:17.10 I explained to you the role of Hox genes in patterning the anterior-posterior axis of animals,
00:00:24.27 and the early attempts to associate Hox gene expression with evolutionary changes in morphology.
00:00:31.28 And in that example, the very first time people had looked in insects to see whether
00:00:35.27 it might explain the transition from four wings to two wings, it looked like the explanation
00:00:39.21 was that Hox gene... changes in Hox gene expression, in fact, weren't responsible.
00:00:44.16 But what I'd like to do now is to move on, in the second part of my talk,
00:00:48.08 to now give you an example where we can show that the change in Hox gene expression
00:00:53.06 early in embryonic development does seem to play a role in the evolutionary change of morphology.
00:00:58.11 And in this case, I'm going to illustrate this by moving outside of the insects.
00:01:03.07 So, insects are one of the largest groups in the phylum Arthropoda, but here
00:01:08.16 you're seeing a phylogenetic tree of the arthropods.
00:01:11.06 And so, you've got the insects, here, and then things like chelicerates,
00:01:14.23 which are spiders and scorpions, here,
00:01:17.27 and then you've got the myriapods, which are millipedes and centipedes.
00:01:21.26 But then this other huge clade that you have are crustaceans.
00:01:25.26 So now, actually, it turns out that crustaceans are paraphyletic, so technically an insect
00:01:30.15 is just a type of terrestrial crustacean.
00:01:33.03 But... my lab has increasingly begun to focus on crustaceans in addressing a number of questions.
00:01:39.18 And I'm gonna focus on these questions of the role of Hox genes in body morphology and
00:01:43.25 evolutionary changes.
00:01:45.09 So, one of the reasons that crustaceans are an excellent group to look at...
00:01:49.10 I mean, first of all, they are reasonably closely related to Drosophila, where we have
00:01:53.17 a really detailed understanding of the molecular and genetic basis for development,
00:01:58.19 so we can actually quickly have candidate genes and some idea of pathways and things to look at
00:02:03.28 when we go into crustaceans.
00:02:05.13 But the other reason is that they actually have incredible diversity of their body plans,
00:02:10.18 much more so than insects.
00:02:12.08 So, for example, as embryos, insects are all stuck with the same number of segments.
00:02:16.17 But segment number is actually quite variable in crustaceans between different species,
00:02:20.12 and there's even a few species of crustaceans that have variable numbers of segments
00:02:24.12 while they're developing.
00:02:25.23 But again, the overall body morphology, and especially the patterns of appendages,
00:02:31.05 which is what we're really gonna focus on, are much more varied in crustaceans that there are...
00:02:35.09 than they are in insects.
00:02:37.09 And so, here's an example of a typical crustacean.
00:02:41.02 This is a stomatapod, a side view of a stomatapod.
00:02:44.15 And the thing I wanna really point out here is that, unlike insects,
00:02:48.02 they have a pair of appendages on every single segment of their body.
00:02:51.14 So, starting from the antennae and working their way all the way back.
00:02:55.00 So, they don't have appendages just in their head and thorax; they actually have appendages
00:03:00.07 on all of their abdominal segments as well.
00:03:02.24 Okay.
00:03:03.24 And the idea is that they actually have a lot of different appendage morphology,
00:03:08.05 even along the axis of a single individual.
00:03:10.18 So, here you're seeing a diagram of the appendages dissected off here, in a crayfish.
00:03:15.11 And you're seeing examples of all the different types of appendages that they have.
00:03:19.14 So, again, up at the very front they have antennae, and then they have specialized appendages
00:03:23.27 for feeding, picking up food, claws for fighting, legs for running, and then on the abdomen
00:03:31.02 they have appendages for gas exchange, and for holding eggs, and then they have an appendage
00:03:37.26 to anchor themselves into the substrate.
00:03:39.27 So, they have this incredible diversity of appendages, both along the axis of
00:03:43.26 an individual animal and, if you look between species, lots of variation in appendage morphology.
00:03:50.03 And so some people have likened this to kind of a Swiss Army knife approach to a body plan.
00:03:54.27 So, if you think of these different things like the blades and the screwdriver
00:03:58.27 and things like that as different appendages, the idea is that you have, actually, a lot of tools
00:04:03.20 at your disposal.
00:04:04.24 And you put different morphologies of appendages, which serve different functions, on different segments.
00:04:10.15 So, if you've ever, like, dealt with a lobster that didn't have rubber bands on its claws,
00:04:13.18 you kind of understand this, because the animal can actually attack you, it can eat,
00:04:18.22 and it can run away simultaneously, because has different appendages on its body for different functions
00:04:23.00 that it can use all at once.
00:04:25.05 And so, what we'd like to do, then, is understand how you get this variation.
00:04:30.15 And again, crustaceans are great for this because they have incredible variation
00:04:34.22 in appendages between species as well as along the axis.
00:04:37.22 And so I'm illustrating that here with two different examples, that are maybe kind of
00:04:42.08 the extremes of appendage specialization in crustaceans.
00:04:45.11 So, this animal is a brine shrimp, or Artemia.
00:04:48.11 And it's a filter feeder, but it has 11 thoracic segments.
00:04:51.25 But those all actually have appendages that all look very similar to one another.
00:04:56.00 And they basically just act like oars as the animal rows through the water.
00:05:00.01 And you can contrast that to something like, again, the crayfish, here, which has
00:05:05.18 many, many different kinds of appendages for different things.
00:05:08.15 And so one of the things that we wanted to do was to understand how it is that
00:05:12.18 you get these specializations, and how you change them between species.
00:05:16.07 And we focused on a particular specialization that occurs in the anterior part of the thorax.
00:05:22.01 Okay.
00:05:23.01 And this is a structure called a maxilliped.
00:05:24.27 And I'm illustrating that here, in a crustacean called the mysid shrimp.
00:05:28.25 It's a little shrimp, and this is a scanning EM view of a mysid, and it's colorized
00:05:33.19 for a couple of the different appendages.
00:05:36.04 So, what I'm showing here is the T3 appendage.
00:05:40.01 And this is a typical swimming appendage in this animal.
00:05:42.27 So again, this is a swimming animal, and T3 on back, and the thorax, look very much like this,
00:05:47.26 and they're used for swimming.
00:05:49.14 But if you look at the first thoracic appendage, T1, it has a very different morphology,
00:05:53.22 and in fact it has a completely different function.
00:05:56.07 This appendage is actually used for feeding instead.
00:05:59.06 And it, morphologically, looks much more like a jaw appendage from the head than it does
00:06:04.10 one of these swimming appendages.
00:06:05.23 So, it's been called a maxilliped, or literally a jaw foot.
00:06:09.10 So, it's a thoracic appendage, right?, but it actually looks more like a head appendage.
00:06:14.25 And functionally, it's used like a head appendage, because it's used for feeding.
00:06:19.00 Interesting enough is T2 -- which is obviously between T1 and T3 --
00:06:22.27 and it's sort of intermediate in its morphology.
00:06:25.07 So, it's used for locomotion, but you can see that it actually has a sort of intermediate morphology.
00:06:31.03 So, it turns out that these maxillipeds are actually important for classifying
00:06:35.01 different groups of crustaceans.
00:06:36.01 So, what I'm showing you here is a... is a very rough phylogeny of some of the crustaceans.
00:06:42.20 And then you're seeing their body plans, here.
00:06:44.14 And I've highlighted the maxillipeds in red.
00:06:47.08 So, a couple points to make.
00:06:48.26 One is that you can have different numbers of maxillipeds.
00:06:51.01 So, I already showed you the example of something like brine shrimp, which doesn't have any maxillipeds,
00:06:55.10 and has all locomotory legs across the thorax.
00:07:00.10 And then I showed you the example of the mysid, that has one pair of maxillipeds in T1,
00:07:06.06 and then locomotory appendages more posterior.
00:07:09.05 But it turns out that you can have, in fact, two pairs of maxillipeds
00:07:12.25 -- you see that in something like a lobster --
00:07:15.17 and you can have three pairs of maxillipeds as well.
00:07:18.01 An example of that is a cleaner shrimp.
00:07:20.00 Now, if you look at the phylogeny, what you realize is probably the ancestral state
00:07:25.05 was to not have any maxillipeds.
00:07:27.11 And that maxillipeds, potentially, have evolved more than once.
00:07:30.16 So, you see, in evolution, here... these are copepods.
00:07:33.02 And then you see another evolution of maxillipeds, first with just one, and then
00:07:39.06 potentially with increasing numbers, in this group of crustaceans.
00:07:42.27 So, you see this various... this distribution of maxillipeds within this group of organisms.
00:07:49.28 So, what we want to do is ask if we can understand what might be the molecular basis for
00:07:56.03 the transition from maxillipeds to locomotory appendages.
00:07:59.17 And then, if that molecule that we're looking at, or gene that we're looking at,
00:08:04.13 that we think might be correlated with that... whether, then, it also explains the different numbers
00:08:08.21 of maxillipeds, either zero, one, two, or three pairs.
00:08:13.04 And so, I'll start showing you, then, the data that we collected to try to look at this issue.
00:08:18.19 And we did this by looking at the crustacean ortholog of the... of the Drosophila Hox gene
00:08:25.09 ultrabithorax, Ubx.
00:08:27.08 And so what I'm showing you here is... we're starting with an example of a crustacean
00:08:31.08 that has no maxillipeds.
00:08:32.15 And again, it's the animal I showed you before, the brine shrimp.
00:08:35.19 So, all of the thoracic appendages look very similar, and they're all locomotory.
00:08:40.26 And so, if you look, shown in black here, is Ubx expression.
00:08:44.18 So, if we look at a slightly older animal, you can already see the limbs.
00:08:47.26 And you see that Ubx is expressed starting in T1, going all the way back.
00:08:53.00 And that expression is actually established even much earlier in embryos,
00:08:56.20 before you see the limbs appearing.
00:08:58.22 So, in this animal, the black staining is actually a gene called engrailed,
00:09:02.13 which is expressed in the posterior part of each segment.
00:09:04.27 And in brown is Ubx expression.
00:09:06.24 But it's still the same result, that Ubx starts being expressed at T1 along the AP axis,
00:09:12.08 and then is expressed from there more posterior.
00:09:14.15 So, it's expressed at the transition between the head and the thorax, which is the transition
00:09:19.10 between the feeding appendages and the locomotory appendages.
00:09:22.28 But now let's go to an animal that has a pair of maxillipeds.
00:09:26.18 And again, we'll use the mysid, which I introduced you to before.
00:09:29.27 So remember, in the head it has these feeding appendages.
00:09:34.03 And then from T2 and T3, these are basically locomotory appendages.
00:09:38.11 We'll get back to the T2 being a little bit intermediate in a minute.
00:09:41.25 And that the maxilliped is on T1.
00:09:44.02 So, this appendage is clearly functionally used for feeding, and its morphology
00:09:48.13 is much more like a jaw appendage of the head than it is like one of these locomotor appendages.
00:09:53.02 So, what do you see here in terms of Ubx expression?
00:09:56.04 So, if you focus on the middle panel to start with, what you see is that it expresses
00:10:01.17 from T2 on, more posterior.
00:10:04.05 So remember, Artemia expression started in T1.
00:10:07.21 So here, relative to Artemia, the anterior boundary is shifted posterior one segment.
00:10:12.26 Okay?
00:10:13.26 So, that correlates, then, with that transition of where you go from a maxilliped to a locomotory appendage.
00:10:20.19 And you can see... here's an embryo where the Ubx staining is in black, and again,
00:10:24.25 it's from T2 on back.
00:10:26.19 And engrailed is in brown, which shows you the position of the segments
00:10:30.05 before there's any morphology of the limbs.
00:10:32.10 Now, let's revisit this question of T2, because T2 is intermediate in its morphology.
00:10:37.20 It is functionally a locomotory appendage, but it's got some aspects that look
00:10:41.28 a little bit like a maxilliped in some aspects, which look a little bit more like
00:10:47.04 the rest of the locomotory limbs.
00:10:48.21 And in fact, it's mosaic in another interesting way.
00:10:51.10 It's the tip of it, here, the distal tip, that looks more like the maxilliped,
00:10:56.05 and the more proximal part of the limb looks a little bit more like this T3 locomotory limb.
00:11:01.06 So, that's interesting because if you look here, I showed you expression starts in T2
00:11:06.00 and then goes more posterior, but you can see that there's lower expression in T2
00:11:09.26 relative to T3.
00:11:10.26 So, there is this expression level difference that correlates with this intermediate morphology.
00:11:15.11 Furthermore, if you look later in development... so now it's sort of a side view,
00:11:18.15 you're seeing the limbs off... going this way, and this dotted line indicates the boundary
00:11:24.22 of this T2 limb... and what you see is that at this stage, late in development, T2 is expressing
00:11:29.09 Ubx very strongly at its base, and then not at all in more distal regions.
00:11:34.07 Which again correlates with the fact that the more distal tip of it looks more like
00:11:38.25 a maxilliped, and the more proximal base looks more like T3.
00:11:42.16 So, think about this, now, in terms of what we told you before about the genetics
00:11:47.14 of these Hox genes in flies and mice.
00:11:49.22 So, if we compare Artemia to this mysid, in Artemia Ubx started in T1,
00:11:56.13 and in the mysid it starts in T2.
00:11:58.18 So, that means we've lost expression in T1 in this species.
00:12:02.22 And if you think, again, like I said, back to the genetics in flies or in mice,
00:12:07.01 if we lose Hox gene expression in a segment
00:12:10.08 -- and in this case, the example that we had before of Ubx in flies --
00:12:14.13 then that segment takes on the morphology of the segment in front of it.
00:12:19.00 So that means that if we shift Ubx back from... out of T1, and now the boundary is in T2,
00:12:24.18 that T1 should look more like a head appendage.
00:12:27.04 And that's exactly the definition of the maxilliped.
00:12:29.26 So now, this segment... this appendage looks much more like a jaw appendage than it does
00:12:34.11 like a locomotory appendage.
00:12:35.22 So, we see this shift in expression that correlates, then, with this change in the number of maxillipeds.
00:12:42.06 So, what if we go to other crustaceans that have different numbers of maxillipeds?
00:12:46.04 So, an example of a crustacean that has two pairs of maxillipeds when it hatches
00:12:51.08 is a lobster embryo.
00:12:52.11 So, you're seeing here that you've got a big claw already, on T3.
00:12:55.08 But [T1 and T2], which are shown by these white arrow heads here, they just have maxillipeds
00:13:01.11 on them.
00:13:02.11 And so, sure enough, when you look at Ubx expression in this animal, which is shown here,
00:13:06.03 Ubx expression starts in T3 and goes from there back.
00:13:10.11 And it's absent from T1 and T2.
00:13:13.03 And those are lacking Ubx expression, and again, as the embryo grows up and turns into
00:13:18.20 a hatchling, it has maxillipeds on those segments.
00:13:21.25 And finally, here's an example of a crustacean that has three pairs of maxillipeds.
00:13:25.22 So, these are cleaner shrimp.
00:13:27.21 And so their claws, their big legs, start on T4, and T1, T2, and T3 have maxillipeds.
00:13:34.00 You can kind of see that a little bit better in this embryo, here, that these three segments
00:13:38.15 have maxillipeds.
00:13:39.21 And then you get the much larger segments.
00:13:41.21 Sure enough, if you look at expression of Ubx, it actually starts, now, in T4.
00:13:46.28 So, it's shifted three segments back relative to Artemia.
00:13:50.08 Okay.
00:13:51.08 And this is showing expression in the nervous system, which shows the same boundary.
00:13:54.18 So in the end, what we see is illustrated here.
00:13:58.08 So now, the maxillipeds are still shown in red.
00:14:01.04 But now the domain of expression of Ubx is shown in blue, at least the...
00:14:04.19 the important thing is the anterior boundary of expression.
00:14:07.17 And what you see is a correlation between the expression of Ubx and the transition
00:14:13.01 from feeding appendages, like maxillipeds, to locomotory appendages.
00:14:16.12 So, what you see is that as Ubx moves more posteriorly -- okay, between species --
00:14:22.24 that those segments that have lost expression become maxillipeds, and they have the morphology
00:14:27.19 of head appendages.
00:14:28.26 So, this really, again, like I said, matches very well with the genetics that we know
00:14:33.10 from flies and mice, that when you shift a Hox gene more posteriorly, then segments
00:14:37.27 that lose expression take on a more anterior morphology.
00:14:40.19 So, unlike the case in insects, where we didn't see the correlation between the wing number
00:14:45.24 and the boundary of Ubx, in this case we are seeing a very nice correlation between
00:14:51.05 the boundary of a Hox gene and the morphology of the appendage,
00:14:54.21 not only within a single species but between species.
00:14:58.20 And so this implicates this change in Ubx expression is possibly playing an actual functional role
00:15:04.15 in the evolutionary change of these body plans, obviously, here, changing the number
00:15:09.16 of maxillipeds.
00:15:10.24 But... it's a great correlation, but then, this is one of the problems that we run into
00:15:15.08 when we start working with non-model species, or species that are newly emerging
00:15:20.11 and we don't necessarily have all the tools that we would have in something like Drosophila,
00:15:24.00 C elegans, or zebrafish to actually address gene function.
00:15:27.19 So, my lab, a number of years ago, we really wanted to test this hypothesis that Ubx
00:15:33.00 was controlling this boundary between the feeding appendages and the locomotory appendages.
00:15:37.13 But what we needed to do was to develop a crustacean species in which we could do
00:15:42.27 manipulative genetic experiments.
00:15:45.07 And so, a number of years ago, we did exactly that.
00:15:48.09 So, we've been working on this crustacean called Parhyale hawaiensis.
00:15:51.22 So, it's kind of a mouthful.
00:15:53.05 You can just call it a beach hopper.
00:15:55.17 And they're small sort of nondescript little gray animals that live on beaches all over the world.
00:16:01.14 This particular species lives in tropical water.
00:16:03.22 If you've ever been to a beach anywhere and you see great things jumping in the sand,
00:16:08.02 they are these amphipod crustaceans that belong to this same group.
00:16:11.10 Like I said, our particular species, Parhyale, is found in tropical water around the world.
00:16:16.12 But the place we isolated it from was interesting.
00:16:19.02 I was actually a faculty member at the University of Chicago at the time.
00:16:22.20 And a student in my lab, Bill Brown, went to the Shedd aquarium.
00:16:25.09 But Bill didn't go to the place where they have display tanks.
00:16:28.16 He went to the filtration system of the aquarium.
00:16:30.25 And these animals live in the filtration system.
00:16:33.11 Nobody takes care of them, and they just eat garbage that comes through the filters.
00:16:36.11 So, they're a perfect lab animal, because they're just adapted to living in pretty awful conditions.
00:16:42.20 And so they do well in the lab for that reason.
00:16:44.24 They have a number of really useful attributes.
00:16:46.17 So, they're a marine species, but they're actually incredibly tolerant of water conditions.
00:16:51.07 They're very easy to maintain at high density.
00:16:52.28 And as I'll show you in a minute, they have a relatively good generation time.
00:16:56.23 So, we can go generation to generation in about seven weeks.
00:17:00.11 And so we've established this animal over the last two decades, now, actually as a system
00:17:06.02 for really being able to do manipulations.
00:17:08.17 And we've been joined by quite a number of colleagues, now, who've also adopted this species.
00:17:13.06 So, I'm going to introduce you a little bit to the animal, and then explain to you
00:17:16.23 how we do genetic manipulations in this species.
00:17:19.16 So, this is how they live in the lab.
00:17:21.27 So, they just live in flats of seawater that we keep in the lab.
00:17:26.15 And you'll see them here in a minute.
00:17:27.23 That's a carrot that they're actually eating.
00:17:29.18 And so, they're the little animals that are all over the carrot.
00:17:32.03 So, they're only, you know... the full-grown adults might grow about a centimeter
00:17:36.13 or a little bit more, and so you'll see them there.
00:17:39.02 And we can feed them just on carrots, and they do fine.
00:17:41.04 They get very orange, and they presumably see really well,
00:17:44.04 but otherwise they do really well just growing on carrots.
00:17:47.11 And we've characterized their embryonic development.
00:17:50.22 So, they take about ten days to come to hatching, to go from the fertilized egg all the way
00:17:56.27 through development.
00:17:58.10 And they hatch out looking like little versions of the adult.
00:18:01.00 So, unlike Drosophila, they don't have a larval stage.
00:18:04.06 They come out at the end of ten days being miniature versions of the adults.
00:18:09.04 And this also helps to illustrate the different things that we've been studying in Parhyale,
00:18:14.06 which I'm gonna only describe to you very briefly, just so you get some idea of the organism.
00:18:18.23 So, we've been interested in the early development that sets up the lineages of different cells
00:18:22.19 giving rise to different tissues, the pattern by which it makes segments, and then,
00:18:26.20 what I'm gonna mostly focus on, is the Hox genes and regionalization of the body plan.
00:18:31.14 But I thought I'd introduce you a little bit to some of these other topics very quickly.
00:18:34.26 So, we've characterized these early lineages of the blastomeres.
00:18:38.28 And so, in this animal, after three divisions there's this 8-cell stage where there are
00:18:44.01 4 micromeres, small cells, and then there's 4 macromeres, very big cells.
00:18:48.11 And what we've shown is that for the macromeres, they give rise to anterior... posterior...
00:18:54.14 anterior left and right and posterior ectoderm and visceral mesoderm.
00:18:57.17 And then for the micromeres, you have two micromeres which give rise to the somatic mesoderm,
00:19:02.16 and you have a micromere for the germline, and a micromere for endoderm.
00:19:05.26 And you're seeing that in a cartoon fashion in this lower part of the figure.
00:19:10.01 This will become important in some experiments we do later, because, basically,
00:19:14.07 the first division of the animal divides most of it into a left and a right side.
00:19:18.09 Okay.
00:19:19.09 The other thing that's really interesting is the way that this animal makes segments.
00:19:23.02 So here, you're seeing an animal that's about... a little less than a third of the way
00:19:27.23 through embryogenesis.
00:19:28.23 The blue is just staining for DNA in all of the cells.
00:19:32.03 And the red is this gene, which I've mentioned a couple of times, called engrailed.
00:19:35.21 And in all arthropods that have been examined, engrailed is expressed in the posterior part
00:19:39.14 of the segment.
00:19:40.14 But if you focus on this blow-up of part of the embryo, one of the things that you realize
00:19:44.22 is that these lines of cells are incredibly well organized.
00:19:48.19 These are the ones making the segments of the animal.
00:19:51.00 The cells are actually organized into perfect rows and columns, which is remarkable
00:19:56.02 because you don't generally see that level of organization of ectoderm in any arthropod.
00:20:01.05 We understand a little bit about how they generate this organization.
00:20:04.06 So, initially, you get a sheet of ectoderm in the very early embryo, and then,
00:20:09.06 posterior to the head, that sheet begins to almost magically organize itself into rows and columns.
00:20:14.07 And then, if you follow any one of these rows, shown here, and then you follow what happens
00:20:19.28 in the... in the course of the next few hours, that row will undergo two more divisions,
00:20:24.15 but it will do so in a completely organized anterior-posterior direction.
00:20:28.00 So, you go from one row to two rows to four rows.
00:20:31.17 And then it's the anterior-most edge of those... of that... of that set of four rows
00:20:36.11 that expresses engrailed.
00:20:38.00 And it's this very organized division that maintains that grid, and then you get
00:20:41.27 gene expression that then looks like it's in perfectly organized stripes.
00:20:46.19 And it turns out that you can watch this live in this animal, as well.
00:20:50.02 So, here's a Parhyale embryo.
00:20:51.27 And what we've done is, when it was a one-cell embryo, we injected RNA
00:20:56.03 for a fluorescent red protein that's going to the nucleus.
00:20:58.26 And we've started taping a couple days later.
00:21:01.09 So at this point, the head is oriented up, and the future posterior is down.
00:21:06.03 It's a ventral view.
00:21:07.27 And the head curves a little out of focus.
00:21:10.19 These brighter cells, right in this area, are actually the germline.
00:21:13.28 The really bright cells are in the yolk.
00:21:16.03 But what I want you to focus on are all the dimmer cells that are in this region
00:21:20.09 of the embryo, right here.
00:21:21.13 And I'm gonna put this movie into motion.
00:21:23.04 And what you're gonna see is that these cells are progressively getting themselves
00:21:27.22 organized into rows and columns, starting sort of towards the posterior part of the head
00:21:31.28 and then progressively doing that more and more as you go down the axis of the animal.
00:21:36.14 And then you'll see these waves of mitosis starting to begin in the embryo as well.
00:21:40.27 This is all much easier to see if we go ahead and artificially colorize a few rows of cells.
00:21:46.06 So, I've colorized a few of the rows, here, in this case, it's a row that's blue, red, and green.
00:21:52.04 And now when I put the movie into motion, you'll have a little bit easier time
00:21:55.02 seeing what's going on.
00:21:56.02 So you see, progressively, these rows get organized going anterior to posterior.
00:22:01.09 But then, if we follow any one row, you'll see that this wave of mitosis goes through the row.
00:22:06.17 And so the row has gone from one to two cells.
00:22:08.21 And then this row is going to divide again.
00:22:10.27 And you're eventually gonna go to a four-row stage.
00:22:13.10 So, this embryo we sort of jokingly refer to as sort of a Swiss version of an embryo.
00:22:17.23 It has a very organized way of setting itself up, much more so than most arthropods.
00:22:23.14 So... and the cells become so organized that they do something very unusual.
00:22:27.18 They actually become cubes.
00:22:29.18 Because they're organized into perfect rows and columns, they actually have right angles
00:22:32.25 on them.
00:22:33.25 And so this is something we're very interested in, how this actually plays out in terms of
00:22:37.10 setting up the segments.
00:22:39.01 Likewise, the mesoderm has a very organized way of setting itself up.
00:22:43.11 So in this embryo, actually, the head is here, and the future posterior is here.
00:22:47.24 And what we've done is we've labeled those two micromeres that make the mesoderm
00:22:51.26 -- one making the left and the right -- with a fluorescent tracer, and now we're starting to image them
00:22:56.08 a couple days later.
00:22:57.14 And what happens is some of those mesoderm cells move into the head to make the head mesoderm.
00:23:02.06 But what I want you to pay attention to are these cells here, which are the stem cells
00:23:06.02 that are going to make the rest of the mesoderm.
00:23:08.19 So, these are called mesenteloblasts, and what you're gonna see is they're gonna divide
00:23:12.11 asymmetrically.
00:23:13.11 They're gonna keep moving posterior in the embryo.
00:23:16.08 And then they're gonna leave behind rows of daughters, which make the segmental mesoderm.
00:23:19.28 So, this is going into action, here.
00:23:22.07 So you see, now, that those stem cells keep dividing, moving posterior.
00:23:25.19 They leave behind these small daughters, which are then the mesoderm of that segment.
00:23:29.02 Now unfortunately, you see suddenly the movie is ruined, because the embryo rolls.
00:23:33.01 This is one of the dangers of working in Northern California, is that actually that was an earthquake,
00:23:38.10 that it hit the lab, and it caused the embryo to roll.
00:23:41.05 But nevertheless, hopefully you see that both the mesoderm and the ectoderm have
00:23:44.20 this very highly organized way of setting themselves up.
00:23:48.07 So... but now, I want to move to the main topic, which is really trying
00:23:52.11 to understand this regionalization.
00:23:54.13 So as I said, one of the nice things is that when the embryo hatches out it actually looks like
00:23:59.00 a miniature version of the adult, and it has all of those appendages on it already.
00:24:03.20 So, the question we want to answer, then, is the relationship between Ubx,
00:24:09.07 and this difference between feeding and locomotory appendages.
00:24:12.14 So, Parhyale, this amphipod we're working on, has one pair of maxillipeds, and that's on T1.
00:24:18.23 Okay?
00:24:19.23 And so you can see that this appendage, here... and it's called a maxilliped because it has
00:24:23.20 these jaw parts at the base of it, like the other head appendages.
00:24:27.21 And this appendage is actually used for feeding, and it's not used for locomotion.
00:24:32.09 And then, going from there posterior, you have segments that don't look like head appendages
00:24:36.26 and are not used for feeding, and are primarily locomotory, though I'll go into more detail
00:24:41.06 about these in the third part of the talk.
00:24:43.18 But for now, the distinction is between the maxilliped and the locomotory limbs,
00:24:49.12 and the fact that that correlates with the boundary of Ubx expression.
00:24:52.12 So, the segments expressing Ubx, here, are shown in blue.
00:24:55.13 And the segment not expressing Ubx are shown here.
00:24:58.22 And so, just as with every other example I showed you with the crustaceans,
00:25:03.01 there's a correlation, then, between that boundary of Ubx expression and the transition
00:25:07.03 from feeding-type appendages to locomotory-type appendages.
00:25:10.11 So, the whole point was... of working on this animal was not to just get another animal
00:25:15.09 to get correlation, but to now be able to do functional experiments to test the hypothesis
00:25:20.28 that Ubx was really controlling that aspect of the body plan.
00:25:24.12 So, you can imagine that there are two types of experiments, as developmental biologists,
00:25:28.27 we might want to do.
00:25:30.14 The first one would be to express Ubx inappropriately.
00:25:34.22 So, if we express Ubx in these segments that don't normally express Ubx, then the prediction
00:25:40.09 would be that we'd be able to transform these appendages away from feeding-type appendages
00:25:46.07 and towards locomotory-type appendages.
00:25:48.25 Likewise, the other experiment we want to do is to remove Ubx expression from this part
00:25:53.28 of the embryo.
00:25:55.05 And the idea would be that, then, we should turn these locomotory appendages
00:25:58.23 into feeding-type appendages.
00:26:00.21 And that would test our hypothesis that Ubx really plays a role in this distinction.
00:26:04.19 So, how would we go about doing these experiments?
00:26:07.01 So, it turns out that in Parhyale we can make transgenic Parhyale, and so we can get genes
00:26:13.00 to be misexpressed by doing that.
00:26:15.03 We take advantage of a transposable element called Minos, which integrates very nicely
00:26:19.17 into the Parhyale genome.
00:26:21.16 And what we're gonna do is we're gonna use an inducible promoter.
00:26:24.15 In this case, it's called a heat shock promoter.
00:26:26.26 We just gently warm the embryo up, and then whatever gene is attached to this promoter turns on.
00:26:31.20 And in the first movie I'm gonna show you, what we've done is we've put under the control
00:26:35.16 of the heat shock promoter... we've put in a fluorescent red molecule.
00:26:39.06 And then you're gonna see what happens.
00:26:40.23 So, there's an embryo here, we heat shock it, and what you're going to see
00:26:44.24 is within a few hours, now, that fluorescent red protein shows up in all the nuclei, okay?
00:26:49.24 This doesn't bother the embryo, because it's just a fluorescent red protein.
00:26:52.19 It doesn't affect the development in any way.
00:26:55.12 But now, instead, what we're going to do is we're going to hook up that heat shock promoter
00:26:59.24 and we're going to use it to drive expression of Parhyale Ubx.
00:27:05.10 But now it's going to be driven inappropriately, all through the embryo.
00:27:08.22 So, it shouldn't do anything to the more posterior parts of the embryo, where Ubx
00:27:13.00 is normally expressed.
00:27:14.08 But from T1 more anterior, Ubx isn't normally expressed, but now it's going to be,
00:27:18.20 because of this promoter.
00:27:19.26 And we can ask what the effect of that is.
00:27:22.08 The other thing we can do in Parhyale
00:27:23.26 -- as I explained to you a little bit, that the first division essentially divides the animal left and right --
00:27:28.12 is we can inject our transposable element only on one side.
00:27:31.10 And then we have a control side of the embryo as well.
00:27:33.28 So, here's what happens.
00:27:34.28 So, this is... first, to show you a wild type animal.
00:27:37.08 So, you've got antennae that are shown here.
00:27:39.28 And then this is the T1 or the maxilliped on one side, here.
00:27:43.12 And then, again, here are the locomotory appendages there, more posterior.
00:27:49.11 In this animal, what we've done is that this side of the animal is normal.
00:27:53.05 So, you've got a normal T1 right there, okay?
00:27:56.19 But this other side of the embryo is now inappropriately expressing Ubx.
00:28:00.17 And so what happens is that the T1 on the other side is not a maxilliped,
00:28:04.27 but you can see is a full-blown walking leg.
00:28:07.18 And that's true for even more anterior appendages, now that they're transformed
00:28:11.15 into the appendages that are used for walking.
00:28:14.28 So, you lose the feeding appendage morphology in those segments, and you gain this morphology
00:28:20.24 of a... of a locomotory leg.
00:28:23.09 So, that shows the gain-of-function phenotype works.
00:28:26.05 What about if we do a lack of... or a knockdown of gene expression?
00:28:30.27 So the idea, then, is we should get more maxillipeds.
00:28:33.20 So again, we can do this.
00:28:35.08 And in Parhyale, when we were doing these experiments many years ago,
00:28:38.18 the way we knocked down expression was by RNAi.
00:28:41.08 So, we inject double-stranded RNA, and that interferes with either the stability
00:28:47.06 and/or the translation of the target RNA.
00:28:48.28 So, what you're seeing here are wild type embryos stained for Ubx protein.
00:28:52.26 If we inject in the double-stranded RNA for Ubx, you can see that we get a knockdown.
00:28:57.15 So, rarely does RNAi produce a complete lack of expression, but we get a noticeable reduction
00:29:03.13 in expression.
00:29:04.13 So now, what happens?
00:29:05.24 So again, here's the wild type T1 that's a maxilliped.
00:29:09.02 So, it's much shorter than a walking leg, and it's got these feeding jaw pieces
00:29:13.09 to the base of it.
00:29:14.11 And then here's a typical T2 appendage, which is clearly not a feeding appendage,
00:29:18.28 and is this sort of general class of locomotory appendages.
00:29:21.15 Okay.
00:29:22.15 So, what happens?
00:29:23.15 So, if we knockdown Ubx and we look at T2 in these knockdown animals...
00:29:28.18 and there's a couple examples shown here, and you get, you know,
00:29:32.19 various ranges of penetrance of the phenotype.
00:29:34.26 But in this more extreme one, I think it's quite obvious that this, now,
00:29:38.08 doesn't look like a normal T2.
00:29:40.08 And instead, what it does is it looks much more like T1.
00:29:42.14 Okay?
00:29:43.14 It's shortened, and it has these jaw parts at the base.
00:29:45.24 So, it's been transformed from a locomotory-type appendage into a maxilliped.
00:29:50.11 So, that confirms, then, in both directions.
00:29:53.15 By gaining expression, we can convert maxillipeds into walking legs.
00:29:57.07 And by losing expression, we can convert walking legs into maxillipeds.
00:30:01.04 So, that really, then, confirms our hypothesis that Ubx, at least within Parhyale,
00:30:07.18 clearly controls the transition from feeding appendages to locomotory appendages.
00:30:12.15 And that further supports the idea, then, that when we compare between species
00:30:16.09 and we see different boundaries of Ubx, that really then functionally indicates that the change in Ubx
00:30:21.01 is really controlling that change in morphology.
00:30:24.21 So, that was our first opportunity to really experimentally test the role of Ubx
00:30:30.22 and to bolster this hypothesis, both about function, in development in a particular species,
00:30:35.24 and function across evolution, in changing that morphology.
00:30:40.01 The thing we don't know is, really, what's responsible for this shift in expression.
00:30:45.08 And this comes down to sort of two different ideas or general categories that
00:30:50.06 we might think of as to what's responsible for this shift.
00:30:53.02 So, the first would be what we call cis regulatory changes.
00:30:56.21 And that means that the difference in Ubx expression between species
00:30:59.24 -- let's say between Artemia and a mysid --
00:31:02.10 is due to alterations in the enhancer regions of Ubx.
00:31:06.01 So, those are those flanking regions that control when and where a gene is expressed.
00:31:10.12 So, the difference in expression is actually due to genetic changes, molecular changes
00:31:15.19 in the sequence that are flanking Ubx in these regulatory elements.
00:31:20.02 But the other option is that the evolutionary changes between species are not actually
00:31:25.19 in the Ubx gene or in those regulatory elements of Ubx, but instead, where they are is in
00:31:30.24 any number of a set of genes that act upstream of Ubx to control where and when Ubx is expressed.
00:31:37.24 So, these could be repressors or activators that are expressed prior to Ubx expression,
00:31:43.02 and basically control where Ubx is expressed.
00:31:45.20 This is an important distinction, though.
00:31:47.16 Because this would mean that... either way, Ubx expression is actually playing a role
00:31:53.09 in this... in this evolutionary change and the numbers of maxillipeds.
00:31:57.26 But in the cis model, it would mean that the actual molecular changes are in
00:32:02.17 those regulatory elements of Ubx.
00:32:04.11 And in this trans model, it would mean that the actual molecular changes are in genes not...
00:32:09.10 are in... not in Ubx, but instead are in other genes that act to actually
00:32:16.28 regulate Ubx expression.
00:32:18.04 And it'll take a lot more experimental analysis to try to answer this question.
00:32:23.14 There are a variety of approaches that could be taken.
00:32:25.13 So, for example, we could try to look at the cis regulatory regions around Ubx
00:32:29.20 between different species, and see if that explains it.
00:32:32.05 And the other thing we could do is really dissect what the trans regulators of Ubx are,
00:32:36.13 and see if they're expressed similarly between crustacean species, or whether they also
00:32:41.23 show changes in expression, which would implicate that the changes are upstream of Ubx expression.
00:32:47.09 So, we've gone through, then, and shown you, then, what's responsible, we think,
00:32:53.12 for evolutionary changes in that transition from these feeding-type appendages that are, you know...
00:32:58.08 are always in the head, but then there could be variable... variable numbers of them in the anterior part
00:33:02.02 of the thorax.
00:33:03.11 And that seems to be... changes in Ubx expression.
00:33:08.11 But I've got a whole lot of other segments to try to explain in crustaceans, right?
00:33:13.19 With lots of really, really interesting morphologies and differences between different appendages.
00:33:18.10 So, in the next part of the talk, what I'm gonna try to do is now take a broader view,
00:33:23.00 and try to tell you about our more... much more recent work trying to uncover
00:33:28.07 how the Hox genes actually set up all these other types of appendages.
00:33:31.10 And give you some additional examples where, again, changes in Hox gene expression
00:33:36.02 seem to actually explain some of the variation that you have between species of crustaceans.
00:33:40.24 So, let me... umm... and... and the idea will be that we'll be able to understand some of
00:33:47.15 this spectacular variation that we have in the body plan of crustaceans.
00:33:51.28 And I'll end this part by acknowledging the people that have done the work.
00:33:55.24 So, there's the group of people that have specifically worked on the Ubx gene.
00:34:00.19 But I really also want to acknowledge the incredible people in the lab over the years
00:34:05.03 that have worked on Parhyale, different aspects, who have developed it into a model system.
00:34:09.25 Thank you.

Part 3: Homeotic (Hox) Genes and Evolution of Crustacean Body Plan

00:00:07.21 Hi, my name is Nipam Patel,
00:00:09.09 and I'm a faculty at UC Berkeley in the departments of
00:00:12.05 Molecular Cell Biology and Integrative Biology.
00:00:14.20 So, in Part I of my talk, I introduced you to patterning along the anterior-posterior axis
00:00:19.26 of Drosophila, and the important role that Hox genes play in that process.
00:00:24.25 And that these genes were evolutionarily well conserved.
00:00:27.19 And in Part II, I explained how shifts in those Hox gene expressions, particularly in
00:00:33.25 the gene Ubx, seem to be responsible for evolutionary changes in the morphology of crustaceans.
00:00:40.11 What I'm gonna do in Part III is expand out and explore the function of the remaining Hox genes.
00:00:47.08 So, what I talked about, again, the first time was the particular Hox gene Ubx and
00:00:52.11 the potential role it had in both the development of the crustacean Parhyale, and then evolutionary changes
00:00:59.21 in the relative number of feeding versus locomotory appendages in multiple crustacean species.
00:01:06.01 So, as I said, what I'm gonna do is expand out to the other Hox genes, because, of course,
00:01:11.19 explaining the difference between maxillipeds and walking or swimming legs is great,
00:01:17.17 but if you really look at crustaceans, we've got a lot of different appendages to explain.
00:01:21.26 So, this is a micro CT scan of an adult Parhyale.
00:01:25.24 And you can see that, in fact, you have this wonderful array of appendages.
00:01:30.06 And if we even just restrict ourselves to the thorax and the abdomen,
00:01:35.26 we have quite a number of morphologies to explain.
00:01:38.25 So, for example, again, the first thoracic segment has that maxilliped that we've talked about.
00:01:44.18 But then, T2 and T3 actually are specialized in that they have claws on them.
00:01:49.17 So, they're called gnathopods, and they use them for picking up food.
00:01:52.26 And then T4 and T5 are the forward walking legs.
00:01:56.23 So, these are the legs the animal uses when it wants to propel itself simply forward.
00:02:01.23 But then 6, 7, and 8 have these enormous jumping legs.
00:02:06.12 So, one of the things about amphipods is that they come up onto the beach.
00:02:10.20 And then when they want to escape, they can launch themselves through the air.
00:02:14.06 And it's using those three giant reverse-oriented legs that they can jump.
00:02:19.07 And then on the abdomen, they have two distinctly different types of appendages.
00:02:22.24 So, the first three abdominal appendages are feathery appendages called pleopods,
00:02:27.28 and they're used for swimming in the water.
00:02:30.14 Whereas the last three abdominal appendages are called uropods, and they're used for
00:02:35.05 anchoring the animal into the substrate when it doesn't wanna move.
00:02:38.20 So, we've actually got a whole array of other appendage types that we need to explain,
00:02:44.08 at least in the thorax and abdomen.
00:02:46.03 And then even more distinct appendages in the head.
00:02:48.16 But I'm gonna restrict myself, for the most part, to the thorax and the abdomen today.
00:02:52.09 So, in order to do... look at that, again, we want to explore their development.
00:02:57.23 And one of the advantages to Parhyale is that all of those appendage morphologies
00:03:02.27 are actually there at the end of embryogenesis, at 10 days.
00:03:05.03 So, as I explained, unlike Drosophila, Parhyale has direct development, and the hatchling
00:03:10.04 that comes out at the end of embryogenesis actually looks like a small version of the adult.
00:03:14.27 And you see that here in this light sheet movie.
00:03:17.13 So, this is a hatchling.
00:03:19.07 And you can see, already, all of those appendages.
00:03:21.14 And they have morphologies very similar to what they're going to have in the adult.
00:03:25.17 So, what we did to try to look at this question is, now, to expand out from just looking at Ubx
00:03:31.23 and actually go ahead and isolate the entire array of Hox genes that you find in Parhyale.
00:03:37.15 So, they have the typical array of genes that you would expect for an arthropod.
00:03:41.06 So, there's nine of them, here, that we've studied.
00:03:45.10 And that's the entire set that they have.
00:03:47.18 And shown here are in situ hybridization, in red, for these genes at about halfway through
00:03:53.02 embryogenesis.
00:03:54.02 And there's no... nothing particularly shocking about the pattern.
00:03:57.13 It's the typical kind of Hox pattern for any arthropod, that they are expressed in
00:04:00.25 these block-like domains going anterior to posterior along the axis of the animal.
00:04:05.11 And I won't show you the data, but it appears that they're organized in a complex,
00:04:09.11 which is, again, very typical of most arthropods.
00:04:13.00 So, we can line up those expression patterns, now, to the morphology of all of these appendages.
00:04:19.07 So, again, here is the schematic of the Parhyale body plan.
00:04:22.18 And down here, in a schematized way, are those domains of expression for all of those Hox genes.
00:04:29.03 And what you can see, again, is there are some particularly striking correlations between
00:04:33.13 the expression domains and the different type of limb morphologies.
00:04:36.13 So, again, for example, Ubx begins its expression at the transition between the maxillipeds,
00:04:43.24 the single maxilliped, and then the other types of thoracic limbs.
00:04:47.28 But again, for the other Hox genes, you see interesting correspondences between
00:04:52.08 those expression domains and the... and the morphology.
00:04:55.18 But as I explained in Part II of the talk, one of the advantages to working on Parhyale
00:05:00.14 was really that we would be able to go beyond these kinds of correlations,
00:05:04.07 and we'd be able to functionally test the role of these genes to see what they really do.
00:05:10.06 We can make hypotheses based on their expression domains and what we know about the kinds of
00:05:14.17 functions that they have in Drosophila.
00:05:16.27 But the real test is to do those experiments in this organism.
00:05:21.16 I explained previously, too, when we worked with Ubx, how we had done knockouts or knockdowns.
00:05:27.08 We lowered expression by doing RNAi.
00:05:30.14 But more recently, in the past few years, we've been able to take advantage of
00:05:34.16 CRISPR/Cas9 technology to actually delete the genes and make null mutations in the genes.
00:05:39.20 So, we actually take a combined approach.
00:05:41.13 We use RNAi to knock down genes, because sometimes that's actually quite informative, too,
00:05:46.02 as well as CRISPR/Cas9 to totally knock out the genes.
00:05:48.26 So, just a very brief introduction, then, to CRISPR/Cas9.
00:05:52.24 So, this is a guided gene editing system that is... works beautifully in many, many organisms.
00:06:00.11 And the idea is that you have a guide RNA that brings this enzyme, Cas9, to the target locus.
00:06:07.13 The Cas9 creates a double-stranded break.
00:06:09.12 In our case, we're really just going ahead and taking advantage of that double-stranded break
00:06:13.17 to create small indels, so we usually put our target... make our target sequence
00:06:19.05 just past the start methionine.
00:06:21.00 We get an indel that knocks the protein out of frame, and we get a complete lack of function.
00:06:27.04 And our controls are to make multiple guide RNAs and make sure we get the same phenotype
00:06:31.27 when we target different locations.
00:06:33.20 So, we've taken advantage of this approach to really rapidly go through and test function
00:06:39.10 in Parhyale.
00:06:41.05 Here's a proof of principle experiment, just showing how well it works.
00:06:44.22 So, in this case, you're looking at a Parhyale embryo about halfway through development.
00:06:48.23 The head's up here, more posterior regions here.
00:06:51.06 The red staining is for the antennapedia protein.
00:06:54.05 Okay.
00:06:55.05 But you notice in this animal that there's antennapedia staining, and it's actually
00:06:58.26 in the normal pattern on this side of the animal, but on the other side of the animal that staining
00:07:03.00 is completely lacking.
00:07:04.18 And that's because we added the Cas9 plus the guide RNA
00:07:08.23 to one of the two cells at the two-cell stage.
00:07:12.05 And that cell that we knocked out both alleles of antennapedia in, that's this side of the embryo.
00:07:17.11 And you can see that we've eliminated protein expression.
00:07:19.14 And this side is normal.
00:07:20.22 So, that's one of the nice things that we can also do in Parhyale is we can make half-mutant animals
00:07:24.22 by injecting at the two-cell stage, or we can make fully mutant animals
00:07:28.08 by injecting at the one-cell stage.
00:07:30.00 I think the other reason that the... this system works particularly well in Parhyale
00:07:34.26 is the first three divisions take quite a number of hours.
00:07:37.09 So, it takes eight hours to go through those first three divisions, so there's plenty of time
00:07:41.01 to hit your targets when you're injecting the guide RNA and Cas9 in one-cell embryos.
00:07:45.28 So, anyway, what have we found?
00:07:47.26 So, we published, a little bit more than a year ago, now, many of our findings.
00:07:53.06 And it's just sort of schematized here.
00:07:55.04 So, in this diagram, again, we've drawn... the wild type limbs are shown to the outside
00:08:01.07 of both sides of the... of the image.
00:08:03.17 And then towards the middle are the expression domains for the Hox genes that we've knocked out
00:08:08.13 so far.
00:08:09.20 And then what you're seeing here, in the inside legs on both sides, are the altered morphologies
00:08:16.11 and the homeotic transformations we get.
00:08:18.08 So, I don't want to try to go through all of these.
00:08:21.03 We've done seven of the genes, now.
00:08:23.10 But what I want to focus on are the more posterior three genes: Ubx, abdominal-A, and abdominal-B.
00:08:28.24 So, this is equivalent to the bithorax complex that I went through in some detail before
00:08:34.13 in Drosophila.
00:08:35.13 And I want to focus on what they actually do in Parhyale, and hopefully convince you
00:08:39.28 that we can understand what they do, but they actually work somewhat differently than
00:08:44.17 they do in flies.
00:08:45.17 And in fact, if we just use the information we had in flies, we would have made
00:08:50.02 inaccurate predictions about what they would do in Parhyale.
00:08:54.12 And furthermore, understanding how they work in Parhyale from these experiments,
00:09:00.11 where we've actually directly manipulated them in Parhyale, gives us, actually,
00:09:04.01 a much better idea of the kinds of things that these genes can do during crustacean evolution.
00:09:09.00 Okay.
00:09:10.00 So, like I said, I'll focus on these three most posterior genes: Ubx abdominal-A and abdominal-B.
00:09:15.12 Okay.
00:09:15.27 So, remember back, again, to Drosophila, just to remind you that these are expressed
00:09:20.26 starting in the posterior part of the thorax, and then on through the rest of the abdomen.
00:09:26.17 And what's important here, of course, are their phenotypes.
00:09:29.06 So, again, the diagram that you've seen before, showing the domains of expression in the segments,
00:09:34.00 and then the homeotic transformations that occur.
00:09:36.19 And so again, the simple rule that we had when we were thinking about these genes
00:09:40.27 is that the segments that lose expression take on the identity of the segment just in front,
00:09:45.16 and then that transformation moves posteriorly until it gets to the boundary of the next segment.
00:09:51.19 So, that's... for example, in Ubx, then, T3 and A1 are transformed to T2.
00:09:55.17 In abdominal-A, then, these segments, A2 through A4, are transformed into A1.
00:10:01.14 And so on, okay?
00:10:02.26 This also illustrates another property of these genes, which is something called
00:10:06.19 posterior prevalence, which is that the more posterior gene basically dominates.
00:10:10.20 So, in these more posterior segments, here, you're actually expressing Ubx, abdominal-A,
00:10:14.21 and abdominal-B.
00:10:16.03 But it's really abdominal-B that's setting the fate of these segments.
00:10:19.15 Another way to illustrate this is that in flies we could actually inappropriately express
00:10:24.10 abdominal-B across the whole animal.
00:10:25.21 And that would override, essentially, the function of all the other Hox genes.
00:10:29.25 And the animal is completely transformed into a very posterior abdominal fate, okay?
00:10:34.20 And so that's a property of posterior prevalence.
00:10:36.13 And it will become clearer why I'm going through this when I try to describe to you
00:10:40.26 what we see in Parhyale.
00:10:42.00 So, again, this is another way of schematizing the expression domains of all of the Hox genes
00:10:47.24 relative to the body morphology and the different types of limbs.
00:10:51.22 But I'm just gonna focus on those more posterior three genes, and we'll start with...
00:10:55.18 actually, with abdominal-A.
00:10:57.16 So, abdominal-A, as shown here, is expressed in those three reverse legs.
00:11:02.21 Okay?
00:11:03.21 So, T6, 7, and 8.
00:11:05.18 And then it's expressed in the three pleopods, in A1, A2, A3.
00:11:09.17 Okay.
00:11:10.17 So, now what we can do is we can knock it out and think about what we're going to see.
00:11:14.13 So, one prediction that you would have from flies, right?, is that the genes...
00:11:18.23 the segments that lose expression would take on the identity of the segment in front.
00:11:22.26 So, we would think that... in this abdominal-A domain, that these legs... these...
00:11:27.04 for example, the reverse jumping legs, would take on the identity of the forward walking legs.
00:11:32.22 Okay.
00:11:33.22 So, here's what happened.
00:11:34.28 Here... and let's look and see if that's the case.
00:11:36.24 So, here is, again, a wild type Parhyale, a scanning EM.
00:11:40.06 And we've colorized, in false color, those three big reverse jumping legs.
00:11:45.04 Okay.
00:11:46.04 So, what happens when we knock out abdominal-A. So, sure enough, we turn those reverse legs
00:11:51.15 into forward legs.
00:11:52.24 So now, this animal doesn't have those big reverse jumping legs.
00:11:56.03 And instead of having just two pairs of the forward walking legs, it now has
00:12:00.12 an additional three pairs of them.
00:12:02.14 Okay?
00:12:03.14 So, that's a striking homeotic transformation.
00:12:05.06 And it's very much what we would predict from Drosophila.
00:12:07.26 Okay.
00:12:08.26 But, what happens to the more posterior part of the animal, in the abdomen,
00:12:12.28 where abdominal-A is also expressed?
00:12:15.06 And here we got a bit of a surprise.
00:12:17.13 So, again, the A1 through A3 have these feathery pleopods on them, okay? that you see here
00:12:23.20 in the wild type animal.
00:12:24.20 But when we knocked out abdominal-A, what happened was those were actually transformed
00:12:29.27 into uropods.
00:12:31.00 And the uropods, remember are actually characteristic of much more posterior abdominal segments,
00:12:37.21 the last three abdominal segments.
00:12:39.06 So, we got, actually, a reverse transformation for this part of the animal.
00:12:43.25 So, for the more anterior domain in the thorax, the transformation was more anterior,
00:12:48.16 the kind of expected result.
00:12:50.05 But here in the abdomen, where abdominal-A is expressed, the transformation went in
00:12:53.26 the reverse direction.
00:12:54.26 So, a very different result than when you knock out abdominal-A in Drosophila.
00:12:58.15 So, we can, again, summarize here.
00:13:00.27 That's the wild-type animal.
00:13:02.05 This is the transformed animal.
00:13:03.21 As we pointed out when we saw this animal before, we've lost the reverse legs,
00:13:07.26 and we've got more of these forward legs.
00:13:09.18 But you can again, actually, appreciate, here, that you've lost the pleopods, and actually,
00:13:14.00 you've more uropods.
00:13:15.00 So, the transformation has actually gone in both directions.
00:13:18.00 Okay.
00:13:19.00 And again, here's another way of looking at it that I thought might be useful.
00:13:22.17 So, here, we've colored in the claw-bearing legs in the dark blue,
00:13:26.09 and then the forward legs in purple -- and this is a wild type animal, here --
00:13:30.21 and then the reverse legs in red,
00:13:31.23 and then we've got our pleopods in these green segments,
00:13:34.27 and our uropods are in these yellow segments.
00:13:37.17 So, what's happened when we've knocked out abdominal-A is a transformation in both directions.
00:13:42.09 We've lost the red fates -- so that's the reverse legs.
00:13:45.17 We've lost the green fate, which is the swimming legs.
00:13:48.15 But we've replaced in... transformed in both directions, so now we have more of the forward legs,
00:13:53.02 and we have more of the uropods.
00:13:55.21 Okay.
00:13:56.22 So, what we find, then, or what the hypothesis would be, is it, again... the observation, sorry...
00:14:02.17 is that the transformations go in both directions.
00:14:05.04 And so it looks like, instead, abdominal-A works in a combinatorial way, with Ubx and with abdominal-B.
00:14:12.27 So, when you express Ubx plus abdominal-A, your fate is to be a reverse leg.
00:14:18.00 And when you express abdominal-A plus abdominal-B, your fate is to be a pleopod, okay?
00:14:25.19 And that when you lose, then, abdominal-A, then the reverse legs turn into forward legs.
00:14:31.25 And the... sorry, the reverse legs turn into forward legs.
00:14:35.02 And then the pleopods turn into uropods, as shown there.
00:14:38.13 Okay.
00:14:39.13 So, now I'll move on to the next gene.
00:14:41.00 Okay.
00:14:42.00 Which is abdominal-B.
00:14:43.05 So, abdominal-B is expressed in these last six segments.
00:14:46.08 So, it's expressed in the three pleopod-bearing segments, in the A1 through A3,
00:14:51.23 and then in the uropod-bearing segments, in the last three abdominal segments.
00:14:55.13 So, what happens when we knock this gene out?
00:14:58.02 So, again, the expectation would be that the segments that lose expression should be transformed
00:15:03.24 to something more anterior.
00:15:05.06 So, remember that abdominal-B expression starts in A1, in these pleopods.
00:15:10.28 And so when we knock out... and in this case I'm actually showing the results from RNAi,
00:15:14.15 but you see the same thing by CRISPR... is that what happens is that those pleopods
00:15:19.21 are transformed beautifully into the reverse jumping legs.
00:15:23.00 So, a very, very different morphology.
00:15:25.08 And that happens for A1 through 3, those three pleopod... pleopod-bearing segments are transformed
00:15:31.00 into segments that bear these reverse legs instead.
00:15:34.06 Okay.
00:15:35.06 Here's a nice example where we've also seen the same result by CRISPR.
00:15:38.16 This is... you're looking at a single segment, and you're looking at the distinction
00:15:41.19 between the left and the right side.
00:15:43.07 Now, if I hadn't told you what segments this was, it actually would be hard to distinguish,
00:15:47.04 because this is, like, a perfectly good reverse jumping leg, and this is a perfectly good pleopod.
00:15:51.24 But this is... there should be the A1 segment, but what we've done is knocked out abdominal-A
00:15:55.26 only on this side.
00:15:56.26 So, you have a wild type pleopod on this side.
00:15:59.11 And then, instead of a pleopod, that's been transformed into this reverse jumping leg.
00:16:03.19 But when we look closely at these transformations, there was another surprise for us.
00:16:08.17 So, again, these are transformed into these reverse jumping legs, but the very most
00:16:13.16 posterior segments were not transformed into the reverse jumping legs.
00:16:17.07 They were actually transformed into the forward legs.
00:16:20.08 Alright?
00:16:21.08 So, we got... very surprisingly -- it's something that you really don't see in Drosophila --
00:16:25.10 which is we got a nonlinear transformation.
00:16:27.22 So, the very posterior part of the animal was transformed into something far more anterior.
00:16:34.28 And so, then, again, I've Illustrated that here, with this kind of diagram.
00:16:38.08 That... again, here was our wild type pattern.
00:16:40.25 So, what's happened here is we've lost the green fate.
00:16:43.16 We've lost those pleopods, right?
00:16:45.27 But we didn't continue that transformation all the way down the animal.
00:16:48.25 Instead, when we get to the last few segments, they are actually transformed to a fate
00:16:53.16 far more anterior.
00:16:54.19 And that's a very surprising result, not one that we would predict from anything we knew
00:16:58.22 in Drosophila or other organisms.
00:17:00.10 So, how might that be coming about?
00:17:02.14 So, if you think about it... okay, here again in this schematic we look,
00:17:06.07 and we've got this knockout of abdominal-B, right?
00:17:09.07 But what we're getting here is all the way in the posteriors we're getting these forward legs.
00:17:14.21 Well, if you think about it, the forward leg phenotype... that's really compatible with
00:17:18.20 just expressing Ubx.
00:17:21.00 Okay?
00:17:22.10 And not expressing abdominal-A or abdominal-B.
00:17:25.27 So, one hypothesis, then, is that what happens is that, in fact,
00:17:29.15 abdominal-A expression is not... you know, there's no effect on it
00:17:33.16 by knocking out abdominal-B. But you'd change the expression of Ubx, right?
00:17:37.21 So, the hypothesis would be that Ubx now extends more posterior, right?
00:17:42.22 And that means when it gets to these last few segments, you now have segments that
00:17:46.24 express Ubx without abdominal-A.
00:17:49.08 And of course, you've knocked out abdominal-B.
00:17:51.15 So, we can test that hypothesis.
00:17:53.12 So, we can take embryos, and we can knock out abdominal-B, and ask what happens to Ubx expression.
00:17:58.19 And that's shown... so, we can ask what... what happens, and that's shown here.
00:18:02.09 So, here's a wild type animal.
00:18:04.13 And in red is... so, the blue is just showing you the nuclei of the whole embryo.
00:18:08.26 And in red is shown the expression pattern of Ubx.
00:18:12.02 And so, as is what you expect, right?, from what we've characterized before,
00:18:16.20 that Ubx is expressed starting in T2, actually, and it goes all the way to the end of the thorax,
00:18:20.25 and it's not expressed in the abdomen at this stage.
00:18:23.15 But then when we look in our knockouts of abdominal-B, what you see, quite clearly,
00:18:28.13 is that Ubx now extends down the whole length of the animal.
00:18:32.00 So, that gives you this pattern, that you knock out of abdominal-B,
00:18:35.12 abdominal B normally acts as a repressor of Ubx expression,
00:18:38.25 and now Ubx extends all the way back in the embryo.
00:18:42.09 So now, these pleopods are transformed into the reverse legs, because they have that combination
00:18:48.03 of Ubx plus abdominal-A expression, typical of the normal reverse legs.
00:18:52.17 But these last few segments, now, are only expressing Ubx.
00:18:55.17 And we've shown that abdominal-A doesn't move when you knock out abdominal-B.
00:18:59.10 But these segments now are expressing Ubx, which means that they actually have the fate of
00:19:04.04 forward legs, the typical fate of segments that only express Ubx.
00:19:07.23 So, again, this is a result that's very different than what you might have expected in Drosophila,
00:19:12.19 that's very insightful into the way that these genes function.
00:19:15.22 And they really start to point to this fact that abdominal-A has escaped from this rule
00:19:20.14 of posterior prevalence.
00:19:22.07 That instead of being overridden by abdominal-B, abdominal-A acts combinatorially
00:19:29.10 with Ubx and with abdominal-B to give the fates of the reverse legs and the pleopods.
00:19:33.15 Okay.
00:19:34.15 So, how else can we test this?
00:19:36.19 So, another way we could test this is to keep... is to make double mutants.
00:19:40.07 So, in this experiment, what we're gonna do is we're going to knock out
00:19:43.16 both abdominal-A and abdominal-B, and ask what happens, okay?
00:19:47.20 So, again, in this... in this schematic, it's the same one where I'd knocked out abdominal-B,
00:19:52.02 but now in addition we're also gonna knock out abdominal-A.
00:19:55.08 So, you can think about what would happen, right?
00:19:57.15 So now, the expectation would be that Ubx still extends back, but there's no abdominal-A
00:20:03.02 to interact with.
00:20:04.09 So hopefully, then, your prediction would be... well, now given these new rules,
00:20:07.23 what we'd expect is that these forward legs would then extend all the way back.
00:20:11.19 And that is exactly what you see.
00:20:14.03 So, here's a mutant animal -- the wild type and the mutant -- and if you look closely
00:20:17.28 at this animal, it's actually... starting from T4, it has all forward walking legs,
00:20:24.05 all the way to the end of the animal, consistent with that model.
00:20:27.19 Okay.
00:20:28.19 So, again... and that's shown schematically here, that if you knock out both of these genes,
00:20:32.26 and now Ubx extends all the way back, all these limbs are now only expressing Ubx.
00:20:37.25 And the characteristic morphology, then, from that... specified by that is to just be
00:20:42.15 the forward walking legs.
00:20:44.03 So, now the next experiment we could do... think about is what happens if we knock out
00:20:48.09 all three Hox genes.
00:20:49.15 So, now we're gonna knock out... or, the three posterior Hox genes.
00:20:52.17 So, now we're gonna knock out Ubx, abdominal-A, and abdominal-B. Okay.
00:20:57.03 What would you expect?
00:20:58.07 So, remember that when we knocked out Ubx, Ubx was making that distinction between
00:21:05.26 the maxilliped and the locomotory appendages.
00:21:08.02 So, in fact, what we see is that you get this terrible looking embryo.
00:21:12.24 It's very hard, in fact, initially, to see what's happening.
00:21:16.01 But if you dissect these limbs off, what you see is that, all the way from T1 on back,
00:21:21.23 they're all transformed into maxillipeds.
00:21:23.16 So, this should have been the A1 segment and the T8 segment, but the limbs on them
00:21:27.23 are now maxillipeds.
00:21:28.27 You can see the chewing parts right here.
00:21:31.13 And so the whole animal from T1 back is all T1, is all maxillipeds.
00:21:36.06 So, this all fits in very nicely, then, with our model of how these genes interact
00:21:41.12 and work combinatorially to set up the pattern of the animal.
00:21:46.27 So, hopefully I've given you a little bit of a picture of how these genes work in Parhyale.
00:21:51.20 But the main message I want you to take home is that, in fact, you wouldn't have predicted
00:21:56.13 some of these interactions from Drosophila.
00:21:58.22 You have a slightly different set of interactions.
00:22:00.18 Most important, like I said, is that abdominal-A has escaped from this system of
00:22:05.04 posterior prevalence, so it's not directly... its expression is not directly affected by knocking out
00:22:10.08 either abdominal-B or Ubx.
00:22:11.26 And that... more importantly, though, it acts combinatorially with those two genes
00:22:15.28 to set these fates up.
00:22:17.02 That... in some ways some... you know, it also is insightful in that you have to set up
00:22:21.28 a lot more different segment identities, different distinct limbs,
00:22:25.09 in crustaceans than you do in flies.
00:22:27.22 And if you just had posterior prevalence, that would limit the number of interactions
00:22:31.17 you could actually have.
00:22:32.26 By using a combinatorial system, you can actually specify an increasing number of fates.
00:22:37.26 Now, there are other ways you could have done it.
00:22:39.15 You could have actually evolved additional Hox genes.
00:22:42.02 That might have been one way to overcome a limit in the number of different types of
00:22:45.18 appendages you could have.
00:22:47.04 Or you could have done something that flies do, which is actually they also have
00:22:50.06 some temporal control over when Hox genes are in different segments,
00:22:53.02 and that can give rise to some diversity.
00:22:55.18 But it seems that, for whatever reason, in crustaceans what you see -- certainly in Parhyale --
00:23:01.06 is that what you've done is changed the rule to allow for combinatorial interactions.
00:23:05.23 Okay.
00:23:06.27 So again, that's the... the summary diagram.
00:23:09.02 What I want to... the only other thing I want to point out is that there's some really interesting things
00:23:13.05 that go on the head, where the genes again seem to be acting combinatorially.
00:23:17.16 But what they do is they can also make kind of hybrid limbs,
00:23:20.15 that part of the limb shows one identity, part shows another in the mutants.
00:23:24.23 I won't go into detail on that.
00:23:27.07 But suffice to say that it's been really amazing to be able to do the experiments in
00:23:33.04 these other species and see that there are some really significant changes in the kind of
00:23:37.14 rules that the Hox genes follow.
00:23:39.26 Okay.
00:23:41.01 So, I've shown you, then... you know, hopefully I've given you a pretty good idea that,
00:23:47.22 now with our results, we can understand how you make each of these different types of limbs.
00:23:52.00 And again, that's great from the perspective of trying to understand the development of
00:23:56.02 this individual species.
00:23:57.25 But how does that help us understand the evolution of other crustaceans?
00:24:01.13 Well, it turns out that within this particular group of crustaceans that Parhyale belongs to...
00:24:06.04 it's a group called the Malacostracan crustaceans.
00:24:09.05 These are the ones that you eat.
00:24:10.07 So, these are things like lobsters and crabs and shrimps and so on.
00:24:13.18 It turns out that one of the ways that they vary their body plan is to mix and match
00:24:18.06 the relative numbers of appendages with these different morphologies.
00:24:23.04 So, this is the pattern, again, that you have with Parhyale, that we've introduced before.
00:24:28.26 But for example, if you think about the number of maxillipeds, Parhyale has 1,
00:24:32.21 but in Malacostracans you can have 0-3.
00:24:34.22 Okay.
00:24:35.22 Likewise, with the gnathopods, Parhyale has 2,
00:24:38.26 but throughout the Malacostracans you can have 0-2.
00:24:41.13 And so on.
00:24:42.13 So, you can have 2-8 walking legs.
00:24:44.10 You can have 0-2 of the... 0-3 of these reverse legs.
00:24:49.14 And then you can have various numbers of pleopods and uropods.
00:24:53.12 The thing is that this number always has to add up to 14.
00:24:56.16 So, you can mix and match appendages within this area, but you can't really change
00:25:00.21 the number of segments that you have.
00:25:02.06 Or you... at least in this group, you're not changing that number.
00:25:05.28 So now, hopefully, you can realize, well, this gives you some predictions about
00:25:10.22 how you would generate these other body plans.
00:25:12.15 That if we know the rules of how you set up these different limbs in Parhyale,
00:25:17.17 we can now make predictions about how you would make different crustacean body plans that had
00:25:21.25 different numbers of these different kinds of appendages.
00:25:24.27 Okay.
00:25:25.27 So... but we have to be careful with that, because remember in Part I of the talk,
00:25:31.07 I showed you the example where people thought, well, it would also be insightful...
00:25:35.15 these four-winged flies in thinking about where... how you made four-winged insects.
00:25:39.13 And then I showed you that... subsequently, that turned out not to be the case.
00:25:43.06 That at least in this particular instance, the changes were not in the Hox gene expression boundaries,
00:25:47.28 but were in downstream genes.
00:25:49.21 So, what about in crustaceans?
00:25:50.27 So, what kind of... can we see other examples, now, that you are changing Hox gene boundaries
00:25:55.27 in order to make different body plans?
00:25:57.18 So, I'll give you a couple examples.
00:25:59.16 So, I showed you multiple times now that Parhyale has the 3 pairs of the pleopods
00:26:05.08 and the 3 pairs of the uropods.
00:26:07.13 And really, it's expressing abdominal-B that gives you uropods, and the combination of
00:26:11.16 abdominal-A and abdominal-B that gives you the pleopods.
00:26:15.01 And Parhyale has this pattern of 3 uropods and 3 pleopods.
00:26:19.13 But if we look at another crustacean, here... this is a crayfish.
00:26:22.17 So, the crayfish has the same number of abdominal segments -- it has 6 abdominal segments --
00:26:27.13 but now it has 5 pairs of these swimming pleopods, and it only has 1 pair of anchor appendages,
00:26:32.23 only one uropod.
00:26:34.09 So, not the 3 and 3 pattern of Parhyale, but a 5 and 1 pattern.
00:26:38.21 Okay.
00:26:39.21 So, if you think about it... right?
00:26:40.21 So, now the pattern is such that you have more pleopods, fewer uropods.
00:26:44.07 So, now that you know the rules of the genetic interactions or the molecular interactions
00:26:49.03 that go to pattern this, hopefully you can make a prediction about what happens in crayfish.
00:26:53.13 And that prediction would be that what you do is you extend abdominal-A so that it's
00:26:59.04 expressed 2 more segments posteriorly, right?
00:27:01.20 And then, given our information from Parhyale, the prediction would be that
00:27:05.16 this would give you an animal that had 5 pairs of pleopods and only 1 pair of uropods.
00:27:11.02 So, the nice thing is, now, we can go and test this idea.
00:27:13.17 So, we can get a crayfish embryo, and we can ask where abdominal-A is expressed.
00:27:17.26 And lo and behold, what you see is that in fact abdominal-A is expressed all the way
00:27:21.14 through A5.
00:27:22.14 So, it's shifted two segments posterior relative to Parhyale.
00:27:26.11 And that fits perfectly with that kind of prediction.
00:27:28.21 So, in this case, then, this is another example where we can show that you've shifted
00:27:32.08 the Hox gene expression.
00:27:33.19 And that accounts for the altered morphology between species.
00:27:37.14 Here's another example.
00:27:38.20 So, Parhyale is called an amphipod crustacean, and it's called an amphipod because it has
00:27:43.03 both the forward legs and the reverse legs.
00:27:45.25 But the sister group to amphipods are the crustaceans called isopods.
00:27:49.13 So, probably the most familiar isopods... the isopods that are most familiar to you
00:27:54.13 are things like pill bugs and stuff like that.
00:27:56.02 This is an example of a giant deepwater isopod.
00:27:59.15 But they're called isopods because they don't have the two different types of thoracic legs.
00:28:03.16 Rather, they have just the one type of leg, the forward-facing legs.
00:28:07.06 Okay, in addition they also have the 5 pleopods and only 1 uropod.
00:28:11.20 So, now you can again make a prediction about what abdominal-A expression would
00:28:16.10 look like in an isopod.
00:28:17.12 Okay?
00:28:18.12 So, the idea would be that, well, since you only have one type of leg, and it's the forward type,
00:28:21.27 in fact, you would retract abdominal-A expression out of the thorax.
00:28:26.09 And that's, again, exactly what happens.
00:28:28.10 So, previously published research from other labs had shown that, in fact,
00:28:32.11 abdominal-A in isopods was not expressed in the thorax, but was actually restricted to the abdomen,
00:28:37.14 in, in fact, the 5 pleopod-bearing segments.
00:28:39.28 So, now I've given you multiple examples where, in fact, the predicted changes in Hox gene expression
00:28:47.22 actually do turn out to be real.
00:28:50.07 That... that is the way that crustaceans seem to be playing the game of altering their body plan.
00:28:56.10 So, I've given you these examples with abdominal-A.
00:28:58.09 And then, much earlier, I'd given you the examples of Ubx controlling the position of the maxillipeds.
00:29:03.02 So, we have, now, multiple examples where crustacean are actually... or, evolution is
00:29:09.08 altering the body plan of crustaceans by actually shifting around Hox gene boundaries.
00:29:13.12 And that's very different than the picture we have from insects, where there can be
00:29:17.00 some late modifications to Hox gene expression -- in fact, there are some quite striking ones --
00:29:20.23 but in fact the early domains of Hox gene expression in insects is very well conserved.
00:29:26.08 But in crustaceans, there seem to be major alterations that go on in the expression pattern
00:29:30.17 of Hox genes very early in development.
00:29:32.19 And those appear, actually, to then be responsible for the differences in morphology
00:29:37.06 that you see between crustacean species.
00:29:39.13 Okay.
00:29:40.13 So, what are some other things that we can look at?
00:29:42.26 So, what I'm showing you here, again, are the legs.
00:29:45.08 And the... and the... and the outline of the legs is in blue.
00:29:48.05 But in red are the muscles.
00:29:49.13 And one of the things I wanna highlight is that the muscles between a jumping leg and
00:29:53.23 a swimming leg are very different.
00:29:54.27 So, that's the T8 reverse jumping leg, the A1 wild type pleopod.
00:30:00.06 But when we transform the pleopod into this jumping leg, not only are we transforming
00:30:07.07 that external morphology, but lo and behold we're actually also transforming the musculature.
00:30:12.22 So then, this presents an interesting problem, because we don't expect that in evolution
00:30:17.15 there were these giant jumps in morphology.
00:30:20.06 We expect that... in evolution, that the changes in expression were gradual,
00:30:24.05 and that there was a slow change in the morphology of these appendages.
00:30:28.06 Something like that T2 appendage I showed you in the mysids that's somewhat in between
00:30:32.09 the maxilliped and the swimming leg.
00:30:35.00 But one of the problems in that kind of scenario is that you have to, of course, always keep
00:30:39.00 the limb functional, right?
00:30:40.15 It has to be doing something useful.
00:30:42.03 You can't suddenly evolve a function-less limb as some sort of intermediate.
00:30:45.28 But complicating that, then, is understanding, like, how you would also keep the muscle
00:30:51.04 in some sort of usable fashion as you were transitioning between different types of limbs.
00:30:55.16 So, one of the issues, then, is how do you actually regulate Ubx?
00:30:59.08 Do you need to regulate Ubx differently between the muscle and the... and the ectoderm,
00:31:04.03 between the mesoderm that makes the muscle and the ectoderm that's making the external morphology?
00:31:08.28 So, one of the things that we're actively doing now is actually manipulating Hox gene expression
00:31:14.20 differentially between the ectoderm and the mesoderm.
00:31:17.13 And again, we can that in Parhyale because these macromeres that you're seeing here make
00:31:21.27 the ectoderm, but these two micromeres, ml and mr, they actually are making the mesoderm.
00:31:26.27 So, what we've started to do is to actually genome edit, separately in the ectoderm from mesoderm,
00:31:31.25 to ask how you actually regulate the genes separately in the two tissues,
00:31:36.19 or do you need to, or does one actually follow the other?
00:31:39.08 So, those are ongoing experiments.
00:31:41.17 Another really interesting question is how does the brain... how do you keep the nervous system
00:31:45.06 wired up the entire time?
00:31:47.06 That's another interesting question that we're continuing to pursue.
00:31:50.04 So hopefully, now, I've shown you that if we follow this Swiss Army knife analogy,
00:31:55.25 that we can now use the mutagenesis of Hox genes to really understand how it is that you get
00:32:00.23 all the different types of appendages that you see on a crustacean.
00:32:03.21 And in our case, Parhyale is the crustacean that we've been analyzing.
00:32:07.17 And now we have a very good description about how you specify each different type of limb.
00:32:12.19 And that's great from a developmental perspective of understanding exactly how you do it
00:32:16.22 in this one species.
00:32:18.04 But then, at the same time, the beauty from an evolutionary viewpoint is that now that
00:32:22.25 we understand how you specify each type of limb, we can also now start to make hypotheses
00:32:28.06 about how you make different crustacean body plans, or how evolution has created
00:32:32.16 those other crustacean body plans.
00:32:34.12 We can make specific tests about the change in expression patterns that we would predict,
00:32:39.00 and we can look at these other species and tell if that's the case.
00:32:42.11 So, I'm gonna end by going ahead and acknowledging some of the people that have done this work.
00:32:46.23 I want to highlight that the original cloning of all of these Hox genes was done by
00:32:51.20 Julia Serano and Danielle Liubicich.
00:32:53.22 And then the single mutant knockouts were done primarily by Arnaud Martin.
00:32:58.05 And then more recently, the double mutants were done by Erin Jarvis.
00:33:01.20 And we've had a lot of really great collaborations with Michalis Averof's lab
00:33:05.21 in working on the Hox genes.
00:33:07.22 Thank you very much.

Videos in this Talk

Part 1: Patterning the Anterior-Posterior Axis: The Role of Homeotic (Hox) Genes
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 2: The Role of Ubx in the Development of Crustacean Body Plan
Audience:
- Student
- Researcher
- Educators of H. School / Intro Undergrad
- Educators of Adv. Undergrad / Grad
Part 3: Homeotic (Hox) Genes and Evolution of Crustacean Body Plan
Audience:
- Researcher
- Educators of Adv. Undergrad / Grad

Speaker: Nipam Patel

Total Duration: 1:42:47

Recorded: November 2017

All Talks in Development and Stem Cells

Talk Overview

Homeotic (Hox) genes are transcription factors that dictate the development and compartmentalization (regionalization) of body parts in animals along the anterior-posterior (head to tail) axis. Using various insects and crustaceans, Dr. Nipam Patel studies how alterations in the expression of Hox genes could explain the evolution of specialized body parts in arthropods. Patel describes the spatially restricted patterns of Hox gene expression, explains the effects of Hox gene deletions, and how these phenotypes help us understand the manner in which Hox genes act to control the insect body plan. Taking a closer look at the pattern of the Hox gene Ultrabithorax (Ubx) in different insects, Patel summarizes the discovery that what drives changes in the number of wings during insect evolution is the not changes in the expression pattern of Ubx, but the regulation of its downstream gene targets.

In the second lecture, Patel describes the work of his lab to expand the studies of Hox gene function to other arthropods. Patel describes the development of specialized body parts in crustaceans, and describes the transition between feeding to locomotor appendages. Using the beach hopper, Parhyale, his laboratory, in collaboration with the laboratory of Michalis Averof, showed that Ubx controls the boundary and transition between feeding and locomotor appendages during development.

In his third talk, Patel explores the function of additional Hox genes in the development of crustacean body plans. Using CRISPR-Cas9 genome editing, his laboratory has characterized the expression and function of six of the nine Hox genes in Parhyale, and describes the combinatorial role of Ubx, abdA, and AbdB in the development of specialized appendages in this species, and how changes in the regulation of abdA is responsible for several morphological transitions during crustacean evolution.

Speaker Bio

Nipam Patel

Nipam Patel is the Director of the Marine Biological Lab (Woods Hole) and a Professor at the University of Chicago. He obtained his bachelor’s degree in Biology from Princeton University in 1984, and completed his doctoral degree in Biological Sciences at Stanford University in 1990. His interest towards developmental biology started when he was studying… Continue Reading

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usman says

July 13, 2020 at 5:17 am

A supreme lecture FOR MY A LEVEL MIND.
IT WAS GETTING COMPLEX FOR ME.
BUT I HOPE I CAN UNDERSTAND IT.
SIR WHERE ARE YOU FROM

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Part 1: Patterning the Anterior-Posterior Axis: The Role of Homeotic (Hox) Genes

Part 2: The Role of Ubx in the Development of Crustacean Body Plan

Part 3: Homeotic (Hox) Genes and Evolution of Crustacean Body Plan

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Speaker Bio

Nipam Patel

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Nipam Patel

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