 Hello, everyone. Thanks for joining us. It's an honor to be introducing our speaker today, Dr. Nipam Patel, the director of the Marine Biological Laboratory. So Nipam's work is in the area of evolutionary developmental biology, which aims to understand development. That is to say, the process of how a single celled zygote turns into a multicellular organism to understand that process from an evolutionary perspective to say, how has that process changed over time? How has it gotten to where it is today? By looking at many different organisms and understanding all of their evolution of their developmental processes. So Nipam's strength in evo-devo, as it's called, evolutionary developmental biology, in large part has come from his willingness to study non-model organisms to say, identify really important points on phylogenetic trees and say, these are organisms that will tell us really important principles. And that, in turn, use of non-model organisms has forged ties between Nipam and NHGRI through his advocacy for sequencing the genomes of some of these evolutionaryly important species. So through his work in evo-devo biology, Nipam has made important contributions to our understanding of body plan development and also germline development, some of which he'll be telling us about today. And then he's also done very fascinating and beautiful work in understanding butterfly wing patterning and coloration, which I think he'll also be telling us about. So Nipam, I wanted to say it's a real treat to have you here today, because in NHGRI, we have a lot of talks that have a lot of bar plots and scatter plots and disease phenotypes and sequences. So I'm really looking forward to seeing some beautiful developmental videos and butterfly wings. So it's a treat and looking forward to your talk. Thanks very much. Thanks. Yeah, thank you all for the opportunity to come ahead here and speak. And so as you heard, my background is really as a developmental biologist, but with a strong interest in trying to understand evolution and diversity as well. So I think you'll agree with me that life is really amazing in its diversity. And what I'd like to start off with is a sort of simple statement really that you can go back and forth on, but I think you will find is largely true, that all the questions that you're interested in biology, evolution's already solved. There's some organism out there that's already figured out how to do that or deal with this problem. And so it's great to work on the standard model systems. And of course, I am a big fan of that. My own training was with Drosophila. And so I understand the importance of those systems and how incredibly useful they are. And I still work on flies to this day. But in fact, what I'd like to argue for is that, to really broaden the scope of the organisms that we work on and to see that there is really interesting biology out there in the world. And so as a way of explaining that to you, I'm gonna talk about two different projects that we work on in the lab. And so really the first is to, is really in this kind of evo-devo realm to understand what we see in a model organism, how that actually evolved, how that got there. And in particular, as I said, my background was really on working on Drosophila and fruit flies. And, but I've taken over a couple of decades now this approach of really trying to understand the evolution of pattern formation in arthropods by looking at a variety of them. And more recently, we've really focused on crustaceans in part because they really have a lot of variation in their body plant. But when we started this work, we really realized that what we needed though was sort of an anchor species, as it were sort of a model, a non-model. So we needed an organism out there that we could start to develop the tools to really do some more deeper experiments and to manipulate gene expression to do those kinds of things. And so a number of years ago, we started working on this crustacean here called parhala. So it's a tiny little sand flea. So if you've ever been on the beach anywhere in the world and you see gray things jumping in the sand, it's this group of organisms, particular species we work on actually is found all over the world in tropical water. We don't know where ours actually came from because we isolated it out of the filter system of the Shed Aquarium when we were in Chicago. And it's a great lab model because no one took care of it and it just ate garbage. So it's a good animal to keep in the lab. So we started raising it and started studying its development. And this just shows you its embryonic development. It takes about 10 days to complete embryogenesis. It comes out looking like a small version of the adult. And it also helps to tell you the sort of three different things that we were interested in studying. So one that we started with really was this establishment of regional identities. We were very interested in how you got the right pattern of limbs on these animals. And again, taking advantage of what we knew in Drosophila. So in flies, we have this set of genes called the hox genes or the homeotic genes that are expressed regionally down the axis of the animal. We cloned and characterized all of those in Parahiala. But our question was is that this is an animal that has to make a lot more different types of limbs than Drosophila does. But it has the same number of hox genes. So how can it use those genes to actually make a greater diversity of appendages? So fortunately, we were able to not just look at the expression but with the development of RNAi and eventually CRISPR technology very easily make mutants in these animals. So this just shows an example of knocking out one of these hox genes, one of the hox genes called the abdominal A and transforming limb identities. And so I'm just gonna, I'm not really gonna go into these today but just tell you a little bit about what we did. So we're able to knock out most all of these genes now. And what I can tell you the bottom line is, is that what we've shown is that they can make more different types of legs because they can use these genes in a combinatorial way. So in flies, there's a process called posterior dominance where the more posterior gene always overrides. We can show that doesn't apply in this organism and that it's able to use them in a combinatorial way. So that was one of the reasons we developed the animal. The other was to look at segmentation because it makes segments just like Drosophila does but it never has a syncytium so it can't freely diffuse transcription factors. And so again, one of the amazing things here is the organization that the embryo has. So you might be able to see here that the cells are organized in these perfect rows and columns. So it's really quite a remarkable animal the way it does that. So the cells actually because they're organized into perfect rows and columns are actually cubes. And so they have a very interesting arrangement and they get into this interesting arrangement because of the way they're made. So it turns out that they actually start off the ectoderm condensis together and organizes itself into perfect rows. And then each of those rows undergoes two very specific divisions to make a four row organized grid. And so that's how you get this very organized pattern. And we can watch that in live embryos. So you're seeing here all of the cells of the ectoderm and we've false colored in a couple of the cells in these rows and you can see that the rows also undergo these very regular divisions going from the midline out. So we kind of joke this is a Swiss version of an embryo. It's got this very precise way of setting it up. And so we're very interested in the molecular parallels of differences to Drosophila. And so we've been cloning all of these different genes that are involved in the process looking at how they might be used and now making mutants and all of these things to try to understand in detail. But what I'm gonna tell you more about is something that we hadn't really intended on studying but got very fascinated by. And that was the early lineages in these embryos. So we right away notice that these embryos form these eight cell embryos after the first three divisions. And so of course we were curious about whether these different cells, the micromeres and macromeres reproducibly gave rise to different parts of the embryo and they did. So there were three cells that gave rise to different parts of the ectoderm, three cells that gave different parts of the mesoderm and an endoderm precursor and so on. And so of course then we also set out to ask how committed the cells were to those fates. So we did the kind of typical ablation experiments to ask what happened. And what we discovered was that for the ectoderm and mesoderm, if you killed one of these cells before gastrulation, the embryo would pause right after gastrulation, it would in a sense figure out what was missing and it would undergo a replacement response and it would replace those lineages. And so you would always end up with a normal embryo with at least with the ectoderm and mesoderm. And then we worked out where those replacements were coming from. So we put lineage tracer in one cell, ablate another cell, ask if that's the replacement came from. And from that we could do experiments like this. So this is the cell that normally makes the visceral mesoderm and you're seeing it a later stage embryo where that's located. And then if we go and do that, but we've now ablated the left somatic mesoderm precursor cell. Now that visceral mesoderm cell steps in and it goes ahead and makes the somatic mesoderm on that side. So we could work that out, right? So from that we actually discovered that yes, they can do replacements, but it's still committed to germ layer. So ectoderm could replace other ectoderm, mesoderm could replace other mesoderm. If we killed all three mesoderm precursors then, for example, no one would step in and you would end up with a mesoderm free embryo. Okay, so that was fine. But one of the other cells we were interested in was the germline precursor. So in this eight cell embryo at the eight cell stage, there is one cell which we call little g which makes the germline of the animal, okay? So, and of course we know a lot about how germline is set aside in various organisms, especially the models that we work on. So there are kind of two main ways that it works. There's sort of a pre-formation system, right? Where you have maternal determinants that become localized in the oocyte in the early embryo and whatever cells inherit that maternal determinant becomes the germline. And that's what we see in animals like Drosophila and C. elegans and zebrafish. And then you have an inductive mechanism which is what you see, for example, in mice and in humans and so on. Where the decision is made early but by it's an induction between germliers and that sets aside the mesoderm. But in either of these cases, the distinction between germline and soma is made very early and for a variety of important reasons such as protecting the germline from genome damage and avoiding having transposable elements cheat and get across into the next generation. Now there is some complexity to that. There are other organisms that have a little bit more complex way that they're setting up the germline that they actually have a multipotent cell that can continue to give rise to both soma and germline at the same time. And often these are also found in animals that have great regenerative ability and are able to regenerate all sorts of things. So what happens in parhyala? So parhyala seems to fall into that category electrosophila, C. elegans, zebrafish. There is a specialized germline. So you might be able to see this little specialized area of cytoplasm and this is in a one-cell embryo. And if we put this into motion now you'll see that as the embryo cleaves that gets swallowed up into the embryo but it's going to reappear actually on the surface in this cell right here which is the G cell, okay? So it has a specialized cytoplasm that segregates to G and in fact kind of fortuitously we found that maternal beta-catenin is in that specialized cytoplasm. And you can see what happens is it segregates asymmetrically on the centrosomes at each division so it's only at one of the two centrosomes in the first three divisions and only in G. Okay, so it seems like you have this sort of maternal system that you have in a lot of other animals. And sure enough if you follow the G cell all the vasopvasa being a good molecular germline marker all the vasopositive cells of the embryo come from G and all G cells are vasopositive. So that all fits, all right. So of course one of the experiments we did was to ablate G and ask what happened. So we killed G at the eighth cell stage, okay? And then this is a normal embryo here about a third of the way through development and you see where the germline is located again with vasas the marker. And so if we look at these ablated animals there's no vasopositive cells, right? And we've had other germline markers we can use and actually you can see the germline itself because the cytoplasm has a different birefringence to it. So you kill G, you get no germline. So at hatching these animals have no germline, okay? So the expectation of course is that they would be sterile adults and this would be no fun if that were true, right? And they're not, they're always fertile. So as adults they always can make perfectly good sperm and egg and have progeny, right? And that was a surprise and this is all due to a graduate student, Olivia Price who did these ablation experiments and then she concluded that they were sterile but for some reason she just kept feeding the animals and then she came back and said, oh they're not sterile, they're having offspring. And so in fact it turns out they almost always, at least greater than 96% of the time or 95% of the time or so, they actually are perfectly fine adults, okay? And in fact you can take ablated males and females and mate them together. And we of course, anticipating all the criticism from the reviewers came up with four different ways to kill the germline, okay? To make sure and even physically removing the germline and they're always fertile when we do these things, and again as I mentioned then we were curious, well when do they regain fertility? And so they regained it about halfway to adulthood. So if we just dissect out the gonads at various time points, about halfway through the juvenile stage they suddenly vasopositive cells reappear in the gonads and so that seems to be then the time at which they reestablish a germline. And as I mentioned we can actually take ablated males and females and mate them to one another and they make perfectly good progeny out of that, right? So their mutation rate may be a bit higher but as far as the lab environment goes they're perfectly viable offspring. And we have in fact an entire tank that is derived from animals like this, okay? So of course what we wanna know is where are they getting this replacement from? They don't have what seems to be another germline cell. So where are they making the replacement from? So to do this of course what we first did was we looked at the ovaries and the testes and we looked at asked well what are the other cells there? So in red you're seeing the vasopositive cells. So you see a lot of non-vasopositive cells as they're all the somatic cells of the gonad that do things like make the follicle that makes the cori on around the egg. And so what we did is we have a transposable element that we normally used to make transgenic animals but now what we did is instead of injecting it at the one cell stage to get our transgenic animals we just injected it in the single cells at the eight cell stage to ask which of these actually gave rise to the somatic cells of the ovary and the testes. And the answer was that it was this ML and MR cell, a cell that gives rise to the left and right somatic mesoderm. So if we label that cell what we see is that it's staining all the muscle and embryos but if we follow, and if we follow to adults you see here for example the musculature of the claw, the ectoderm of course, ectoderm precursors give rise to all of that ectoderm but then if we look in the gonad what we see is that in fact ML and MR are giving rise to all those non-vasa positive cells that are part of the gonad. So that's where mesoderm is really giving rise to all of that. That's great, okay. So and what you see here now if we look at the, if we've done that lineage in the mesoderm and we're also staining for vasa you see that those are mutually exclusive. So the germline is giving rise to those vasa positive cells and then the mesoderm cells are giving rise to all the other somatic components of the gonad. Okay, so now what we're going to do is we ask, okay if we inject that into the mesoderm does it pass it on to the next generation? And of course the answer is no because we put it in the soma. All right, but if we inject our transgenesis cocktail into G of course it passes on to the next generation. That's the germline. So now we do the experiment where we put our lineage tracer into ML but we kill G, okay. And ask now does this pass to the next generation? And the answer is yes. So now you see in this ovary you see that the vasa positive cells also have the lineage tracer even though that was put into the somatic mesoderm but more stunningly these are the offspring. So even though the transposable element was moved in the mesoderm it is actually transferring that transposable element to the next generation because those cells became actually the germline when we ablated the G cell. So this animal is fully capable of doing that of taking somatic tissue and turning it into a germline if it needs it, okay. So, oops, sorry, let me go back there. Oh, sorry, wrong button here. Okay, so it's fully capable of doing that. So this is really odd, right? If you think about, if you work on plants this is totally normal, right? Plants can make germline out of somatic cells whenever they need to but animals aren't supposed to be able to do that, right? But increasingly, there are observations suggesting that our model systems which can't do this may be the exception to this rule may be the exception and that other animals fully are capable of doing that. I have an interesting take on it. So it's like, well, okay, we know that the reason that we say you shouldn't do this is because of the higher mutation load that this won't be the same quality cells and also the chance that you can move things like transposable, omicidal, and extrinsic. But if your choice is to be sterile or to have really bad offspring, I would argue it's better to just have bad offspring than be sterile. And so it may be that a lot of organisms can do that. So we're very excited to continue this kind of research in this organism to understand mechanistically how is it able to do this? What is the signaling that goes on in the animal to do this, right? And how is it able to do that? So that's one of the things that we're doing. So hopefully that gives you a taste for this kind of approach that in this case, we weren't interested in using this animal for this property of germline replacement. But in studying a novel creature, we find novel biology that it's able to do things that other animals we don't think about can do, right? And so hopefully that illustrates that. But I'm gonna switch now to the other example of doing this, and this has to do with butterflies and structural color. So a completely different set of organisms, a very different question. But a similar sort of thing, we're going to now look at a phenomena that I'm gonna tell you about, structural color, where none of our model species that we normally work on are very good systems for studying this, but something like a butterfly is, but it's not normally what you think of as a genetically tractable system. So we have to work at it to really figure out how we can apply the tools we know from other systems to really tackle this problem, okay? So I think you'll agree that butterflies are a great system to work on and talk about because they're very charismatic, right? So it's very easy to give a talk to the public about butterflies, because they have that kind of intrinsic beauty to them because of the colors and patterns that they have primarily on their wings as adults, and we're very attracted to them. And there's a lot of beautiful work that's gone on a lot recently into the genetics of pattern formation, particularly in this group here, these Heliconius butterflies here, which have become a great system for doing genetics in. But I'm gonna tell you something a little bit different. I'm gonna tell you how they actually get the color that you see in a very specific example of colors, particularly green and blue, which are not made by the way you normally think about ways colors are there in organisms. Okay, so first of all, where do you get color in the wings of butterflies? So you know you're not supposed to touch the wings of butterflies, right? And if you do, what looks like dust comes off on your fingers, and those are the scales. So butterflies and moths belong to this family called Lepidoptera, which means scaly wing. And if you look at the wing of a butterfly, it's covered in these tiny scales. But what these really are are giant cells. So they're not alive in the adult wing. The adult wing can't regenerate, the scales can't regrow. But during the chrysalis stage, this was an epithelia, and these cells started to send out an enormous outgrowth. Okay, and then late in development, that outgrowth became covered in chytin, which is the sugar polymer that the exoskeleton of all arthropods is made of. It's a very durable material. Then the cell died right as the butterfly emerged. And so this is the dead remnant of the cell. But before it died, the scale cell in these cases got filled with pigments. So the browns and the blacks and the yellows and all those colors are pigment molecules that filled the cell before the cell died. But then it died, and it's still stuck in here into the adult wing as a scale and a socket. So a typical scale is about 300 microns long, about 50 microns wide. So it turns out that we use pigments like that to make colors that are in long wavelengths of the rainbow. So colors like yellow and orange and red and all of those things are generally pigment colors. What do we mean by that? So pigments are these individual molecules that have particular light absorption properties that give the color. So a nice way to illustrate that is to think about the ink in your highlighters. So for example, the yellow highlighter has a yellow color because it has this organic compound in it. And that ring structure allows it to efficiently absorb blue light. So if you absorb blue light, then yellow and red are what are being reflective as far as our eyes are concerned. And that gives you a yellow color. And that's how pigments work. They're individual molecules that absorb particular wavelengths of light. What's left to be reflected is what gives you the impression of color. Okay, and we know a lot about how pigments are made in certain systems. So for example, pigments that are black and brown and so on are from melanin. And that's a very ancient pathway for making those kinds of molecules starting with tyrosine as the starting point. But other ones are more specific to different lineages of animals. So for example, in insects, we know a lot about making the oranges and reds that come through a particular pathway that's well-studied in Drosophila and butterflies use that same sort of pathway to make those pigment molecules. It turns out however, that it's rare to make a pigment then it can absorb red light. And that's what you need to do if you wanna be green or blue is to absorb in those wavelengths of light. So for the short wavelengths of the rainbow, right, you have to find a different solution to this problem. Not always, and there are some cases where pigments can be made that give you green and blue. The most famous one is the reason that the world is green because chlorophyll, which is very good at absorbing red light, okay? And there are even butterflies that have figured out how to make a few pigments that will do this. But by and large, the solution is not by pigment. It's by a phenomena of structural coloration. Or instead, we're gonna use light refraction or the butterflies, gonna use light refraction. And this is not unique to butterfly. So for example, in the iridescent blues and greens of feathers in birds, it's the same thing. It's a light refraction phenomena, not a pigment phenomena. Even the blue faces of monkeys, the erythrophores of zebrafish, and even human blue eyes, there's no blue pigment there. It's an actual light refraction phenomenon. So how does this work, right? So the easiest way I think to explain it is to think about something like a soap bubble, right? So the soap has no color, but the soap bubble can have all the colors of the rainbow, right? So how does that work? So you remember back to what you learned in physics, right? That when light hits a material of a different refractive index, right? The light is going to change speed. It's going to change its direction of travel. But another important thing is that there's going to be some reflection at that surface as well, okay? So when you have a very thin material of a high refractive index, like the bubble of the soap bubble or a layer of chitin, the polymer that you make the scale out of, what's going to happen is you're going to have one reflection at the top surface and another reflection at the bottom surface, okay? And then you remember that light has these wave-like properties. So that means these light waves are going to interfere with one another, right? And so if they're in phase, they can constructively interfere, double in amplitude, look four times brighter. If they're out of phase, they can cancel out and that color will disappear. And then again, remember that white light is really a spectrum of colors, each of them of a different wavelength. So what that means is that when this phenomenon happens, certain colors will be enhanced, certain wavelengths will be enhanced, others will be diminished or destroyed. And so you're going to get a spectrum of response that's going to give you a color. So it's quite easy to calculate those wavelengths that undergo maximum constructive interference from this equation, which is just dependent on the thickness of the material and the two refractive indices and the angles of the lights coming in, okay? And if you remove this one half term that's in the equation, you will get those wavelengths that are going maximum destructive interference, right? In between you have a sine curve shape. All right, so that gives you a spectrum of color and that's how you can create the color that you want. You can get this to be even brighter if you repeat those layers over and over, right? And get reflections off of all those surfaces. So the next picture I'm going to show you is a moth. And in this moth, the only pigment in it is black, okay? But the moth can make all the colors of the rainbow. And it does that by just having scales that are different in the thicknesses of their chitin layers. So it can tune them to be whatever color it needs to be. So it can make short or long wavelengths of the rainbow this way. So it can make the entire spectrum of color simply structurally without any pigment, all right? Another great example comes from these bright blue morpho butterflies. Probably many of you have seen these like either if you've been in Central or South America or if you've been to a butterfly house see these things flying around. They're a brilliant bright metallic blue. Okay, so how do they do it? So one way we can prove that it's a structural color is play a little bit of game with the refractive index. So normally the light's going from air the refractive index of one into the chitin with the refractive index of 1.5. So in this movie we're going to drop a drop of acetone on the wing. The acetone has a refractive index of 1.3. It's going to replace the air, change the math of the equations and now the wing is bright green instead, okay? But we haven't really changed anything. We've just changed the way the light is interacting with the material. So what do they do to do this? So a typical scale actually has amazing architecture to it. So it has these long ribs that run down the length of the scale. It has cross ribs that go across. It has this lamina on the bottom. So it turns out that butterflies can actually manipulate any of these parts of the structure to create the interference it needs to create these colors. So what do morphos do? In the case of morphos, it's those ribs. So you see the ribs on this. They look just like any other scale. But if you cut a cross section across these fine ribs you get will look like these Christmas trees when you look on them. So they've really elaborated these ribs into these tall structures. And you can see how small they are. So that's a one micron scale bar. So these structures are far smaller than a half wavelength of light which they need to be for this phenomenon to work. So the thickness of those branches of the trees and the spacing between the trees is just right. Then you get this massive constructive interference of blue light. And so you get this great blue color even though there's no blue pigment at all in this structure. So, and then again, now we can just let the acetone evaporate off of this wing. The air goes back into that space and the wing will go right back to being its blue color. So that's how they do it. They create these Christmas tree nanostructures to do that. Okay. As I said, green often is created this way as well. And there's a lot of evolutionary pressure to be green. So green is a really good camouflage color for butterflies. So it's evolved over and over and over again. So these are these little green hair streak butterflies that live all over the world and they're this nice green color and they blend in really well to vegetation. So if you take one of their scales and you shine white light onto it, what you get are these facets of green coming back at you, okay. So it's not the ribs, the ridges that are creating the color, it's something in the scale. If you break open one of these scales, you see what looks like this honeycomb material inside of the scale, okay. And that ends up being a very specific shape called a gyroid. And this is a great story because in 1970, there's a guy, Alan Shown, and he came up with a mathematical equation that defines an infinitely connected, triply periodic surface that divides space into two non-overlapping domains indicated by if you fill in those spaces you get the red space and the blue space and they never intersect. So it was just a math equation, okay, with this particular property. And then people found, lo and behold, it exists in the real world inside the scale of these butterflies. And when it's made just at this size, it creates perfect constructive interference of green light. And so that's how they're green. And butterflies have used this solution three times independently in different lineages to create green color, okay. Another example, this is the emerald green, or the emerald swallowtail. And so it lives in the Philippines and it actually does sit like this with its wings open in the rainforest. And it's this nice green and black color, but it works really well as a camouflage. So it sits in the shadows of the vegetation and that green and black actually blends in really well. So they make green in a completely different way. So if you look at their scales, they have dimples on them that are reflecting the light, okay. And these dimples are these curved structures that are layers of chitin and air. And so, and they have a particular curved geometry to them. So what happens is that when white light hits the bottom of the dimple, the spacing is just right to get yellow constructive interference. But when white light hits the edge of the dimple, it sees a different angle, so the spacing is different. And it retroreflects blue light comes out the other side. So it's yellow plus blue, which you perceive as green. These dimples are only five microns across, so you can't distinguish that there's different spatial, spatially, the colors are coming from different areas. So you see this as a green color. But the beauty of this is that the blue light, because it's retroreflected, is polarized. We don't see polarized light, but they see polarized light. So they don't see themselves as the color of the vegetation. So they can see each other against the vegetation, but their vertebra predators see them as being camouflaged instead. Okay, so those are some of the examples that we have. And so there is a lot of interest then from, so a lot of that, all those examples were discovered by optical physicists who do the same thing. They find some dead butterfly that has some interesting color. They go ahead and do transmission EM and scanning EM. They work out the geometry, then they work out the math and they tell you this is how it makes a structural color. And then if they have enough money, they try to make it out of man-made material. So this is an example of making that Emerald Swallowtail dimples, but they're using beads to create the dimples originally, and then they're using layers of titanium dioxide instead of chitin, and then they show that they can make the same thing, and they've spent about $10,000 making the square millimeter of it, okay? So, right, but there's a lot of interest in these kinds of materials. So these nanoparticles and nanomaterials have lots of uses, for example, in medical applications, in all sorts of technology. So there's a great interest in figuring out how to make these things. So we would argue, and this is where as a developmental biologist, I come in, as well, the butterfly seems to be able to do this without a lot of problems. So maybe we should figure out how the butterfly does this instead, and then use that as the way that we think about how to make this material. So the big problem with that, of course, is that the butterfly is doing it while it's a chrysalis, okay? And so we can't normally see in to ask how it's actually creating these structures. So one of the things that we did, at first, was just to fix the wings and to look at them. And one of the things that we really, we spent a little bit of time on is showing how the general scale architecture is created. And the big driver of that is an internal actin cytoskeleton. So this is the filamentous actin inside of those scales. It's incredibly well organized into these giant rods. And in fact, what happens is that the animal is actually, the actin is shown here in yellow, and the chytin that it secretes is shown here in pink, and it's gonna secrete that chytin in between those rods. So that's how it's gonna first set out the spacing of the ridges and everything. Okay, and I show you that because it helps to understand, in fact, how you create these dimples. So in most scales, you just set up these rods of actin, then you start to secrete the cuticle and then you break down those rods of actin and get them out of the way. But in this butterfly, this emerald swallowtail, what you do is you have the same rods of actin, but then you do something quite remarkable to the actin. So this is the membrane of the scale. And you're gonna see the actin now, and it's turned into hexagons instead. So this is a single cell that take the rods of actin, reorganize them into hexagons, only on the top surface. The rods are still there on the bottom. And it uses those hexagons to create a basket of actin and it pulls the membrane down in order to make the right geometry. So we're real interested then in understanding how it can vary the geometry of that because there are other related species that can make other colors by changing the shape of that dimple. So that's a, and it's a kind of a crazy looking thing to create these hexagons, right? So one of the problems that we ran into is that we tried to dissect pupae of different ages and asked what's the intermediate between the rods and the hexagons. And we really weren't able to convince ourselves that we could find intermediates, okay? So what we wanted to do was find ways to live image this, to watch this happening in the living animal. As I said, that's not easy because they're opaque. So we developed ways to create windows into the wings of the butterfly. So here you're seeing a chrysalis of this particular butterfly called the buckeye. And this window now is there from the start of pupil development. So now we can see all of wing development. So this is eight days of development. You see the wing, the wing surfaces are coming together, that you see the veins have formed and now you'll see the scales as these silvery things, the silver color. And then right towards the last day and a half or so of development, you'll see all the pigments come in. But now the beauty is we can watch all of wing development, okay? And then we've been able to do things like use Surat to actually watch the individual scales growing and to see the cytoskeleton forming and inside individual scales in the living butterfly. And then, because this is no easy feat because these things are alive and their heart is beating and they're moving up and down and things like that. So we've had to develop a lot of tricks to do this. But now we hope to be able to see how those hexagons get made, for example. And we have some preliminary data and I can tell you why it's so difficult to watch the transition because the transition from the rods to the hexagons, right? Development is about 10 days and all of that transition occurs in one and a half hours. So finding that exact period of time, that's why we could never find it because but we can see it if we film it instead. So that's one of the things we're doing. The other approach that we're taking is a genetic approach. So it's to try to understand how you make structural color. And for that, we're using these butterflies, these buckeye butterflies which are common throughout North America. And you might say, well, this is a terrible choice because these butterflies are brown and yellow and orange. All the colors I told you were made by pigment, okay? But they're closely related to butterflies that live around the world that actually have really nice, beautiful, structural green and blue, okay? And if you look at a phylogeny for these butterflies, so the North American buckeye is the coenia which is right here, but you can see it's in the middle of a whole bunch of other species that have really nice structural color to them, okay? But it turns out when you collect North American buckeye as a small percentage of them have a little bit of structural blue in their wings, okay? Just a tiny bit. And so there was a woman named Edith Smith who lives in Florida and she owned one of these companies that raises butterflies to sell to the big butterfly houses where you go and you walk through and there's all these flying butterflies. And then she also raised them because I guess people buy them to release at weddings. So she had the butterflies for that reason. And she also noticed that occasionally she would have a butterfly with a little bit of blue. So she thought to herself, what happens if I take the bluest ones and I save them and I make them to each other? Okay, so over the course of a year which is about 12 generations, she mated the bluest butterflies to one another and she eventually got them to be really, really blue, stunning looking blue color. And we found out because we were buying buckeyes from her and then we had some of clothes and they came out this way and we called her up and we said, well, what are these things? And she said, or do you like them? I made them. And so of course she did a selection experiment, right? So she selected for this and then she got them to be really bright blue. And Rachel Thayer was the grad student that then it endeavored to understand what had happened to these butterflies. And so what Rachel discovered is that how they're blue is that that very bottom lamina of the scale is where the color comes from. So when you look on the top, you can see the blue color but it's much brighter if you flip the scale over. And if you rub the ridges off, then you can see the blue really well. So what had happened is that bottom lamina of the scale had become just the right thickness to be a perfect reflector of blue light, okay? So it had reached that about 180 to 200 nanometer thickness it needed to do that. But where did that come from? So she found that the brown butterflies actually were gold. So they have brown pigment, but they also have a lamina that's reflecting these sort of long wavelengths of yellow. And so that was a thickness of about 100 nanometers. So what had happened in this selection is that that bottom thickness, that lamina had gone from about 100 nanometers to about a little under 200 nanometers. So it about doubled. And that's what allowed it to change color, was that, okay? So then Rachel went on to show that the normal species that have that blue and green color, that's exactly what they've done. So the selection recapitulated what evolution had done or at least the genetic variation in those was the same as the mechanism at least that's going on in the naturally occurring blue species, okay? So the beauty of this now is that of course now we can use QTL mapping to find the low side that actually create this because the brown butterfly and the blue butterfly only separated by 12 generations so we can easily make them back together. So we make them back together. So this is stuff that Rachel had done. She'd create an F1 generation. The F1 looks intermediate between the two parents, okay? But then of course you do brother-sister matings and you get the F2 and of course because of recombination now you've got all different combinations of chromosomes that come from either the brown grandparent or the blue grandparent. So we phenotype all the F2s and then we sequence them and then we look for the QTLs. And Rachel was able to generate two very large families, 501 full sibs and 270 full sibs. I didn't know female butterfly could lay that many eggs much as anyone could raise them all to adulthood but that's what Rachel did. And so the variation in the F2s is really beautiful. So in terms of not just the percentage of the wing that has the iridescence but the hue of the color as well, okay? So if we actually just look at wings that have the same iridescence but different percentage of the wings with cover and we use it as our phenotype, that maps really well. So there are two major effect loci. So these are the different chromosomes. There's two major effect loci. They have a huge genetic effect on the percent of the wing that has the iridescence. We actually already know what one of them is. So one of the genes ends up being a gene called optics which is a transcription factor which was discovered because it controls the red color in Heliconius butterflies but in buckeyes it controls the blue color. And sure enough, you knock optics out by CRISPR-Cas9 and the buckeye suddenly turns blue, okay? So you knock that transcription factor out, the scale lamina doubles and you get these blue iridescence suddenly. Okay, we don't know what the other locus is which actually has a much bigger effect on the phenotype. So we're actively working to narrow down that interval to find that gene. But to me what's more exciting is the variation in hue because this is the precise thickness of the lamina. So in these F2s, we go all the way from an indigo color through to a green color. So that's a difference in thickness of the lamina from about 100 nanometers to about, or about 140 nanometers to about 240 nanometers. So what loci can precisely control that thickness of the lamina? So when we do that, there seems to be four or five loci that seems to do this, right? And some are in the same between the two families and there's some differences between the two families. So we don't know yet what any of these are but these are gonna be very exciting to us to try to figure out what some of these major effect loci are that control this because they're able to make that very precise control on the thickness. The nice thing about doing a cross like this is that there's also all sorts of other phenotypes that you can look at. So of course she's selected for the butterflies to be very pretty in her eyes. And one of the things she did is selected for the eye spots to get really big. And so that we can also map and that maps really well as to a couple of very big effect loci that control eye spot size. And that's great because there's been a lot of published work on candidate genes that are expressed in the foci of the eye spots but in fact when you knock them out you get things that are as I would put it sort of statistically significant but don't have much biological effect. But these are a loci that obviously have a huge effect on eye spot size. So we'll be very interested to see what these genes actually are. All right. And the nice thing is is that as we narrow down these candidates the CRISPR-Cas9 genome editing works really well in butterflies. And this is just showing a different gene. Wintae which was identified as one of the color pattern genes. And you can see that it's very easy to make completely mutant butterflies using CRISPR-Cas9 editing and actually just score in that immediate generation for the phenotypes. In fact, that way even if a gene is essential you can get small clones in the wing that let you see the effect. So we're gonna use that as one of the ways once we've narrowed down the candidates to be able to test them. And I'm gonna, and so hopefully from this I've shown you that there seem to be two major effect loci that control this big switch in lamina thickness and then probably about four to five loci that seem to control the precise thickness. And those ones are the ones that we're most interested in pursuing is understanding how you really, to me a fascinating question of how a cell can actually pattern something to plus or minus 10 nanometers when it's making it, right? And so that's I think what we're most excited to narrow down. And I'll just end by telling you the one other thing that we've been studying in butterflies which is transparency. Another obvious way to camouflage that should just be transparent. And so many butterflies or moths have evolved transparency. And so these are just a few examples of totally unrelated butterflies that have transparent wings, okay? So if you look and you ask, okay, so the wing itself has an inherent transparency. So you basically just have to get the scales out of the way. And so if you think about how to do that, basically everything you can think about some butterfly or moth is tried. So all the way from actually making transparent scales to doing things like weird things like turning the scales vertically so you can see them. But the most common solution is to turn the scales back into little bristles, right? And so then you can see between them. You rarely actually get rid of the scales because if you do that, the wing loses its hydrophobicity and if it gets wet the wings get stuck together. So the bristles allow you to maintain that hydrophobicity. So we've actually been doing quite a bit to understand the transitions and we think we understand a key transcription factor now that allows you to go back to these bristle morphologies. But one of the things I just want to end with is to show you one other thing which is that as you can see there's another interesting problem that as I mentioned, right? Anytime you have a change in refractive index you get some reflection. So if you think about it if you have a piece of glass, right? That works perfectly fine in the shade. You can see through it. But if you put it out in the sun you can see where the glass is because you get a reflection off of the glass. But you can see that so some butterflies actually then that's a problem. Well, it's just what happens. So this butterfly is transparent but it actually glares pretty badly. It's interesting. This butterfly is actually unpalatable to birds. So why is it being transparent? But if you see it in the wild when it's in the sun you can see it 50 yards away. You know where it is. As soon as it goes into the shadow of the rainforest it disappears, it becomes ephemeral. So there's probably two different predators that's trying to deal with one which it's signaling that it's unpalatable but another one that it's actually trying to hide from. But other butterflies you see in fact even with reflected light there's very little, I mean with transmitted light there's very little reflection. So they've actually been able to deal with this. So if you wear glasses you can pay extra money and you can have a coating on your glasses so that they're anti-reflective. So as humans we think it's great that we figured out how to do this. So butterflies solved this problem 50 million years ago. So they've actually evolved anti-glare coatings. In between those bristles if you look they have a whole different series of nanostructures which stop reflections and they've also evolved these many times independently. So they've come up with many different solutions to that problem. So we're actually very interested in understanding some of these nanostructures and how they're manufactured. So with that I will end and take any questions that you have and thank you for your time. So if there are questions from the audience there are microphones. Those are some really beautiful slides. My question has to do with the melanin content within butterfly wings. Is it contained within the cell in vesicles like it is when we think about mammals or is it just free-floating in the cell? Yeah so it's in many cases in butterflies it seems to get incorporated into the chitin. So it's actually embedded as tiny particles but within the chitin itself and often into the lamina and the structures. So there's not like a vesicle that the melanin's contained within. Like in mammals it's like deposited on a protein so. But remember that so the question the answer is is that I don't know what happened when the scale was alive. Okay. Right because remember the scale is not alive when we're looking at the adult and so it's just in this matrix of the cuticle. But yeah it would be great to know when it's alive how it's actually doing this and there's very little studies of all of that in living scales. That was really cool. I was interested in the ordering of the cells in the embryo of the sand hopper. You were saying that the ordering was randomly defined random cells seemed to join up to each other. Kind of two questions. Do you know for sure whether that's random and if so do you know how they align with each other? Yeah so we think it's random and so what we've been doing is so that it random in the sense that originally the ectoderm is essentially hexagonally close packed. It aggregates together and then the cells get aligned in a way of going from the head down over time into rows and columns. So one of the things that we've done is just lineage trace obviously sort of backwards. So we ask so when you watch the cells join do they then have any regular relationship to rows or columns and the answer is no as far as we've been able to see. They just clump pack in together and then because they're still dividing in random directions they can end up in a column or a row or any sort of arrangement that you could imagine. But do we know how they get together and form that we don't? I mean it's one of those things like you look at and it's just like magic they move and then you can even do things like a blade a cell after it's formed the row and everybody will move around until it reestablishes the rows and columns again. That's really cool, thank you. Hey I was interested in the shared use of the optics transcription factor. Is the reg pigment also structural or do you have the same transcription factor controlling pigments? Yeah I know the red is a pigment color. So it looks like I mean one of the things that people have really seemed to have stumbled upon in the last decade or so is that so three major effect loci have been studied in Heliconius. So it's winter which controls where the black color is optics which controls the red patterns and then cortex which controls the way the black windows yellow and red and surprisingly then those three genes explain much of the color patterns which are totally different in other species of butterflies. So they seem to somehow be master transcription factors that control different aspects of patterning and so they control basically a type of scale in a sense and that type can vary in color and things like that and they happen to control a different aspect which is whether or not it's a structural scale or not a structural scale. Nice, very cool, thank you. And there is an online question. Do the pigments play any role in meat attraction or is meat attraction in butterflies all driven by scent? Yeah, good question. So I mean butterflies are very visual animals so the color is part of the solution. They definitely also use hydrocarbons for smells but they are very visual animals and so they will use the colors to do that. So a very good example, well it's not necessarily the mates but like the morpho butterflies that are the bright blue so one of the ways you can collect them is just to put a blue piece of paper down on the ground and the males will come to fight it so they are very attracted to that color too. I really like the comment about the butterflies that can see each other but no one else can see them. You may have said this, have plants co-opted this to make themselves more visible to certain butterflies? Yeah, yeah and I should point out one of the things that I don't mention but is important is that of course I've focused on what we see, okay but other animals see things very differently and so one of the things for example is butterflies and other animals use a lot of UV coloration which we do not see but yeah, that's a beautiful example. Often plants, their flowers will have elaborate UV patterns that we can't see but insects see. And so the butterflies have the same thing, they actually do have UV absorbing pigments and UV structural colors so they can make UV patterns that we don't see but they see and possibly and we know for sure other animals may be able to see. So that's something also to think about and that makes for a complicated problem because the butterfly is dealing with many things that are looking at it both it's mates and a variety of predators all of which have tuned different photoreceptors to different wavelengths and so who knows what they're, who knows all the things they're trying to balance out. So I had a question about the germline development. So maybe this is kind of philosophical maybe one reason you want the soma to stay the soma and never contribute to the germline is you don't want like a cancerous situation where cells that are part of the soma might choose to become part of the germline even if the germline hasn't been ablated. So do you ever see that now that this pathway does exist does it ever get hijacked where sometimes you see some germline contributing from the soma when it's not supposed to be? Yeah, so great question. And I think that's very parallel to this transposon idea too that again the transposon that drives it to become germline to cheat, to take over, right? But one of the things that we're curious about is like what's the normal function of this mechanism? So why does the animal have this ability? It's not because some person was gonna come around and use a laser beam or something to kill off the germline, right? So we tried to do the experiment of if we just put the marker into the meseter and then keep the animals around. Do they ever end up making germline that's instead derived from soma? We haven't seen it in the lab but our end is very small and our time is also relatively short. And I'll tell you why the time is important. So these are very little animals but we have females that are three years old that are laying more eggs than they ever laid and they have very, very healthy offspring. So they are able to stay furlough. So they lay when they're full grown mature adults so they like two dozen or more, up to 50 eggs every two weeks, okay? And they never seem to go sterile. So it may be that one way to avoid that is as your germline is replenished you just take soma and you convert it into germline. But we in the lab have never actually seen that happen. So we're still left wondering but I will tell you we did one experiment which is, you know, so another question is well is it restricted to being a phenomena that only occurs as it goes through juvenile development that it's able to do that replacement or can adults do it? So there was an experiment. It was one of those experiments that after you do it you pretend you didn't do the experiment because it gives you a result that's very disturbing and it was that I had a student and I said, you know, at some point we might want to do mutagenesis this is very early on when we're working on them. And I said let's work out what the X-ray dosage is that they can withstand. And so how much radiation will kill them? How much will make them sterile? And we'd have to be below that dose to do mutagenesis. So he goes away and he on his little side project and he comes back after a while and he says, well, you know, I can kill them with X-rays but it takes a lot they're pretty resistant but he says I can't sterilize them. And I was like, what does that mean? You can't sterilize them. He says, well either they're dead or they're fine infertile. I'm like, well, okay, we'll pretend we didn't do that. That doesn't make any sense. But maybe it makes sense that basically you have to kill them before they're sterile. I understand. There are a couple of more questions. Is there a reason evolution has not invented chlorophyll in animals to make green color if that color is so advantageous? Yeah, so the answer is that they have invented green pigments. They're just rare. So in butterflies, there is just one genre of butterflies that's invented a green pigment. So you know, and you're probably familiar with where that's gonna come from. So bile is green. And so you can take that pathway and make a green pigment out of it. And a blue pigment in fact. And there is one South American butterfly that has green and blue and it's pigment-based. So it's not that you can't make a green pigment. It's just that for whatever reason that's a very rare solution to the problem. And that by and large, the solution is always structural. Thank you. And does temperature play a role in species-specific butterfly wing color? Does what? Does temperature play any role in the color of the butterfly? I still miss the word competition. The temperature. Oh, temperature. So the answer, does it play a role? It can. So in the sense that there can be very genetically programmed responses. So there are butterflies, I wish I could show you nice examples, but there's some fascinating ones. Like there's a butterfly in South America that was originally classified as two different species. And it was one that was there in the dry season and it was orange and there's another species that was there in the wet season and it's blue. And it turned out to be the same butterfly. And it's just two different variants depending on the temperature. But it's the temperature that the larvae sees not when the wing is developing. So it controls hormone levels which then subsequently control the color of the wing. But otherwise you can, within a range, you can raise butterflies at different temperatures and the wing colors will still come out the same. If you shock them to extremes, you can create variation. The most common one is if you cold shock them, they'll melanize and they'll just be much darker. Thank you. Great, great talk. I'm wondering like in cases, so for example in your bristle example, or in cases where the butterfly like changes or the thickness of the kiting layer, does it compromise the strength in their wings or some other, and how do they compensate that? Yeah, no, very good question. So in fact, some people have suggested that one that there's an added advantage to structural color that these architectures, these thickened cuticles and everything, they actually also make the scales tougher. And the other thing is like morpho butterflies, extremely hydrophobic. Those wings, if you throw a bead of water, it just bounces right off. So there may be other advantages that we don't think about to having those nanostructures, but the scales do give flight advantage to the wings. So it's estimated that having scales gives about a 15% extra, sort of 15% advantage in flight. And so not having scales, for example, is actually not just the coloration problem, it's actually an aerodynamic problem. And so all of those things may also factor in together. And again, we focus on the color, but yeah, if we think about the evolutionary pressures, there's probably a lot of other functions that we should think about too. All right, that's all we have time for it. Thanks, Nifon. Thanks.