 So, thanks very much, and it's a huge pleasure to be here at NHGRI when so much has happened and is happening in the area of genomics. I'm actually a product of the eighth grade museum experience that you heard about from Kirk Johnson. We all know animals differ in all sorts of very interesting traits, and I'm very fortunate to get to work on old problems related to what are the actual molecular changes that produce those interesting differences that you see between animals. So that's a very hard problem, although there's lots of organisms that have been sequenced. We still can't look at a sequence and tell you what the animal looks like. And that problem is related to a whole series of other issues, including how many genetic changes actually underlie the interesting differences. If you see some interesting trait, is it lots and lots of little tiny changes that have produced it or a smaller number of changes in major genes? What are the most important genes? What types of mutations occur in those genes? And if you give evolution some problem to solve, are there lots of different ways of doing it, or does evolution tend to use particular mechanisms over and over again? So about 15 years ago, we got interested in trying to study those old problems by trying to bring a genetic approach to the study of evolutionary change in vertebrates. We went looking for some organism that undergone very dramatic and very recent and repeated evolutionary experiments in nature. We chose the small fish, the three-spine stickleback, which lives in the ocean, but they're like salmon, they migrate into freshwater streams and lakes and coastal areas to breed every spring. That migratory life cycle set off a huge evolutionary radiation when the glaciers melted, created all sorts of new environments in North America, Europe and Asia. They got colonized by marine sticklebacks and have since had 10,000 years or approximately 10,000 generations to adapt to the new food sources and water conditions and predators and salinities and watercolors, etc., and all these new environments around the world. So if you go around freshwater lakes and streams, tens of thousands of new environments created since glacial melting, you see really dramatic differences between these newly established populations, huge changes in jaws and teeth and spines and bony armor and pelvic apparatus and color and physiology and behavior, all of which have evolved in just the last 10,000 years. So although many of these forms look as different as you would see in different generative animals, it's only 10,000 years of evolution and the isolating mechanisms between the forms tend to be behavioral and mechanical incompatibilities between populations. Those can be overcome using artificial fertilization in the laboratory. So if you squeeze out eggs and sperm from very different looking sticklebacks and do fertilization in Petri dishes, you can actually raise fertile F1 hybrids. That makes it possible to then raise large families and to treat evolutionary change in natural populations as a genetics problem. Can we actually map the key chromosome regions that control the differences and eventually get down to the genes and the mutations that are responsible for these major changes? I have to say, when we first got interested in this, sticklebacks had great biology that had been studied for decades, thousands of papers, several full length books written about the traits and ecological significance of different traits. But they weren't an organism that had attracted interest from molecular geneticists, so there was essentially no sequences, markers, maps, transgenics, none of the things that you need for a real model organism. We spent a lot of time with support from NHGRI over the last 10 years building four or three sticklebacks exactly the same sorts of tools that we'd found useful in mouse genetics for trying to track traits all the way from morphology down to chromosome genes and mutations. And so what I'll do today is illustrate how we're trying to use many of those tools to study some of the cool, interesting differences that have evolved repeatedly in these natural populations. So let's start with limb formation, a great example, limb modifications. Of course, one of the major changes that you see in organisms adapted to different lifestyles. One of the biggest changes in limb patterning in different vertebrates is the complete loss or reduction of limbs that's evolved in many animals, including loss of both forelimbs and hind limbs that's evolved in snakes, loss of only the hind limbs, which has evolved in marine mammals, including both whales and manatees. And that selective loss of the hind limb has also evolved in both fossil and many living stickleback populations. Here's a reconstruction of the skeletal structures of a marine stickleback heavily armored. I've colored the pelvic hind fin of the fish here in red. Sticklebacks are like most fish and land animals. You have two sets of paired appendages, pectoral fins or arm homologues, pelvic fins or leg homologues. And the pelvic fin of the stickleback consists of this bony spine that articulates with an underlying pelvis. The fish can raise that bony spine as a defense against soft-mouth predatory fish that try to eat sticklebacks in the ocean. So although ocean sticklebacks always have a robust pelvic apparatus, many of the ocean fish have colonized environments where there are no other predatory fish. Some of those lakes are shallow, full of insects. Insects actually prey on sticklebacks by grabbing onto the dorsal and pelvic spines and reeling them in and eating them from the sides. And in that sort of environment, there's actually a selective advantage to losing the pelvis, which has occurred in a couple of dozen populations around the world. So that actually gives us the chance to try to look at what's the genetic architecture of this sort of dramatic limb modification that's evolved repeatedly in natural population. So to study that, we cross marine fish with a robust pelvic apparatus to a lake fish that's completely lost to pelvis, raise a couple of thousand offspring, isolate DNA from the F2 animals, type them with a genome-wide set of linkage markers and compare it to pelvic size. And when we do that, what we find is pelvic reduction isn't the simple Mendelian trait, but it's not a miserably complicated infinitesimally tons of regions trait either. There's a single chromosome region that controls about two thirds of the variation in pelvic size in the cross, as well as a series of unlinked modifier genes. Well, one of the nice things about a map is it allows you to quickly evaluate any candidates that are known. There's a lot known about limb development. And when we map the stickleback homologs of a whole series of limb genes, we found that this particular gene called PITX1 maps directly to the major chromosome region that controls the size of the pelvis and the cross. That's actually a very interesting candidate gene because it's a homeodomain transcription factor that plays an important role in regulating the expression of lots of other genes. It has this name PITX1 based on a group that isolated it studying pituitary development. But the same year it was isolated by another group who named it the back foot gene. It was named the back foot gene because it has this very striking expression pattern where it's found in hind limbs, but not the fore limbs of a whole series of animals all the way from fish to birds to mice and humans. The gene's been knocked out by two different groups. The knockout mice have small hind limbs. They actually die at birth with pituitary defects and jaw abnormalities, cleft palate. So in many ways, that doesn't look like a very promising basis for evolving new traits in natural populations, which brings us to the view of the neo-Darwinian synthesis about the likelihood that major genes are going to underlie adaptive traits in nature. The quotable Ernst Mayer here talking about the fact that most mutations found in laboratory organisms that have dramatic effects on phenotypes are actually deleterious and reduced fitness. He said that to believe these sorts of drastic mutations would produce viable adaptive phenotypes is equivalent to believing in miracles. And you can see the problem. I mean, this is a good example of a mouse that has obvious viability problems, although also a component of the phenotype is something that we see in nature. So what's actually happened in the stickleback? Well, when we sequence PITX1 in marine and freshwater fish, what we find is that the coding region of the gene is identical in both types. However, when we look at expression patterns, there's an interesting difference. Larva from marine populations show PITX1 expression in the lips and inside the head in the pituitary. And then this little spot of expression along the side, that corresponds to the site where that pelvic hind fin would normally develop. Freshwater fish still show expression in the head and inside in the pituitary. They've completely lost this little site of expression along the side of the body. That isn't just because they've lost the pelvis. You can cross the marine and the lake fish. The F1 hybrid actually forms a pelvis. But in the pelvis, the only allele that's expressed comes from the marine fish, not the freshwater fish. So that suggests there's been a cis-acting change in the regulatory apparatus of the PITX1 gene. The freshwater sticklebacks have preserved the coding region. They've conserved expression for jaws and pituitary, but they've lost the ability to turn this gene on in one particular spot in the body. We think that could happen by the loss of a modular regulatory element that drives expression at that location. We've been looking to see whether that sort of model can be worked out in detail. Is it true? High resolution mapping in lab crosses and natural populations has shown that the genotype in a 20 kb region upstream of the PITX1 gene can predict in a natural population the presence or absence of a pelvis. We built a whole series of transgenic constructs using marine sequences from that region, hooking them up to GFP reporters. And Frank Chan injected them into fertilized stickleback eggs, looking for little magic pieces of DNA that would drive expression specifically at that site in the body. And they're there. I hope you can see this. It's a fairly specific expression pattern, but there's a small fragment of DNA in that key candidate region that drives GFP reporter expression specifically at the site where the pelvis will normally form in the fish. This is really the right gene, and that's really the right sequence. You also ought to be able to do a more ambitious experiment, and that's to try to reverse the evolutionary change. So for this, we hooked the marine control region not up to a GFP reporter, but to a PITX1 cDNA. And we inject that into the eggs of an evolved, pelvic-less population that hasn't made an external pelvic spine for thousands of years. And we were thrilled to see that the introduction of the marine information will actually put the pelvis back on the stickleback. Here's the original vestigial pelvic apparatus that's seen in the evolved freshwater population. And here's one of these transgenic fish where the introduction of the marine information stimulates the formation of a nice serrated spine that articulates with a restored pelvic apparatus on the ventral side of the fish. OK, so that then, with our hands on the right gene and piece of DNA, we could finally look at what's actually happened in different populations around the world. One of the nice things about the stickleback is the phenotype evolves over and over again. Series of complementation and mapping and in situ experiments implicate the same major chromosome region again and again when pelvic reduction evolves at different locations. And when Frank Chan looked at DNA sequences from a whole series of different pelvic-reduced populations around the world, what he found was a whole series of deletions of a few hundred to a few thousand base pairs that completely eliminate this pelvic enhancer region. OK, so I went through that in some detail to give you some idea of the actual genetic and molecular basis of a major skeletal alteration that's evolved in these natural populations. For pelvic reduction, if you actually look at the results from the crosses, you see that relatively few chromosome regions can have big effects. The chromosome region with the biggest effect corresponds to the location of a key developmental control gene that's required for the formation of multiple different tissues. Despite its essential role, regulatory mutations in that gene provide a way of producing a very large phenotypic alteration at a specific site in the body. And that's such a good mechanism for evolving this phenotype that when pelvic reduction evolves over and over again in different populations around the world, exactly the same mechanism as being used over and over again with a repeated series of deletions that eliminate that regulatory element. Well, we've been interested in using similar methods to study a whole range of traits. One of the reasons we thought it was justified to build a genetic toolkit for these fish was because of a large range of different phenotypes that have evolved in different populations. I'll give you another quick example from armor plate patterning. So marine fish covered from head to tail with armor plates. Freshwater fish are lightly armored and fast. They have greater body flexibility, usually only retained plates at the anterior end. And that dramatic difference in anterior posterior patterning of skeletal armor was used to give different species names to marine and freshwater sticklebacks back in the 1800s. Same sort of experiment. Here's a cross between fish that have a 30-fold difference in the number of armor plates along the side of the body. And there's a single chromosome region that controls three quarters of the variation, an armor plate number, as well as several unlinked modifier genes. We've also looked at some non-skeletal traits, like pigmentation, sticklebacks, colonized, water that's either t-stained or clear or different colors in different locations. So you can get dark sticklebacks and light sticklebacks. If you cross the darks and the light ones, there's a single chromosome region that controls 50% of the variation and pigment score in particular body regions, as well as unlinked modifiers. So what we're seeing then for a range of evolutionary traits that have been selected in these populations is not a Mendelian architecture. All of these traits you could call complex in the sense that they're controlled by multiple chromosome regions. However, for armor and for pelvis and for pigment, the genes of biggest effect control half or more of the variation in the trait. And that's a very nice result from geneticists standpoint because you can explain a lot of the phenotype by studying these major gene regions. So we've spent a lot of time carrying out positional cloning projects to isolate the genes for these other major chromosome regions controlling armor and pigmentation. The armor trait is controlled by a secreted signaling molecule called ectodisplasen, actually named after a series of clinical phenotypes identified in humans. This same developmental signaling pathway is required for the formation of hair and teeth and sweat plans. And mutations either in the signal to receptor, the intracellular adapter molecule will give that phenotype in sticklebacks. The signal has been altered to produce this major armor phenotype. The pigmentation changes are actually due to a very well-known signaling molecule, one of the most famous ones of all of the million mouse development called kit ligand or stem cell factor. This is a secreted signal that's expressed in the skin and the bone marrow and a variety of migratory paths and the general ridge of mammals. The signal binds to a receptor that's expressed on pigment cells and blood cells and germ cells. And the signaling is required to direct the migration of those cells to stimulate their proliferation and their maintenance once they take up residence in different places in the body. So mice with null mutations and kit ligand are white. They're also dead. They die of absence of blood cells. There's no germ cells that make it to the germline. Another example of sort of a hopeless monster from the standpoint of completely losing function. And yet that's exactly the gene that has been selected in sticklebacks to produce pigmentation alterations in particular body sites. So I think you can see the similarity then in the themes that have come from the studies of pelvis and plates and pigment. In each case we get big phenotypic differences from a small number of loci. The genes that have the biggest effects turn out to be these absolutely essential key developmental control genes. And although those are required for the formation of lots of different tissues, in each case we have evidence that there's been regulatory mutations in the stickleback populations that has made it possible to produce a big, advantageous phenotype confined to a particular body region in the natural population. And then finally, and I think very interestingly, we also, for every one of these traits, have found that when the same thing evolves in different places around the world, exactly the same gene is used, just like you heard from Sarah Tishkoff for the lactase tolerance gene. So I think that raised an interesting question about if it's used over and over again in different populations, how far the reuse of those evolutionary mechanisms might actually extend. And so we've also been interested in trying to generalize from results that we find in sticklebacks to characterize variation in other organisms, including humans. So humans show all sorts of interesting traits, as you've heard about, including changes in pigmentation. When we found the kit-legged gene as the basis of skin color changes in sticklebacks, we also found that the kit-legged gene shows one of the strongest signatures of selection in different human populations around the world. So sticklebacks migrated out of the ocean into different water environments. Modern humans migrated out of Africa into different environments. That migration is associated with very strong signatures of selection in the non-coding regulatory regions of the kit-legged gene, which we show were associated with levels of skin pigmentation in the study a few years ago. Let me show you what the selection signal looks like. So here are these big peaks of molecular signatures of selection centered not on the coding region of the kit-legged gene, but the megabase of non-coding DNA that flanks the gene, very high peaks seen in both Europeans and Asians. So we've been very interested in trying to now track those changes down to the alterations in DNA sequence, identifying candidate regions based on molecular signatures in humans, and then carrying out studies very similar to what I told you about in sticklebacks. So here's surveys now of genomic fragments from the stem cell factor gene hooked up to laxie reporters instead of GFP reporters and injected into mouse eggs instead of stickleback eggs. But again, we're looking for the magic piece of DNA that confer expression patterns at locations related to the evolution of a phenotype. So let me show you what this looks like. Here's a series of different constructs in the non-coding regions of the kit-legged and stem cell factor gene. If you put those into transgenic mice, the left construct doesn't do anything. The right construct doesn't do anything. The one in the middle drives expression in both skin and kidney. This is the fragment that contains some evolutionarily conserved regions. And if you break it into two pieces, the left-hand piece drives expression specifically in the kidney. The right-hand piece drives expression specifically in the skin and the hair follicles. So even in this small region, two different regulatory elements for expression in two different places in the body. This one, obviously, a great expression pattern for skin pigmentation. The endogenous kit-legged gene is known to be expressed in the keratinocytes of the epidermis and the hair follicle here staining endogenous kit-legged antibody. And that's the expression pattern that we're recovering in these laxity constructs with a small region of non-coding DNA upstream, several hundred kilobases from the kit-legged transcript. Well, what's happened to that hair skin enhancer in different populations, there's a single base pair change that separates the sequence seen in Africans and in Europeans. That single base pair change when cloned upstream of a luciferase reporter doesn't eliminate the activity of the enhancer. It produces a small quantitative change in the level of expression that we see. Is that quantitative change actually a big enough alteration to produce a significant phenotype? Well, to test that, again, we're trying to look at whole animal phenotypes. And this is challenging because you're trying to see what does one base pair do. The way that we're testing that is, again, to hook those base pair changes not up to GFP reporters but to the gene itself, either the ancestral or the derived hair enhancer that differ in one snip. We make transgenic animals using a special line of transgenics that has been modified to have an insertion site at a defined locus on chromosome 11 if we inject our two versions of this construct together with an integrase, then these things will get integrated at the same locus, same copy number, same orientation. So now you're scoring the two alternative base pairs when it's the only base pair difference in the mouse. And when we do that, here's the ancestral human snip. Here's the derived human snip. And I hope you can see this is a darker mouse than this one. That small base pair change is actually enough to produce a significant difference, both in hair color and in the non-pigmented skin areas of the mouse as well. So pelvises and pigments are obviously very different, but I hope you can also see the similarities between these two traits. In both cases, we've got an absolutely essential developmental control gene that's surrounded by huge regions of non-coding DNA. Those non-coding regions are chock-full. We think of regulatory elements for driving expression at very particular locations in the body. And in both cases, alterations have occurred within specific enhancers. The deletion of the hind limb enhancer in the PX1 gene, a single base pair mutation in the hair enhancer of the kit ligand gene, that produce these dramatic phenotypic differences that have been selected in natural populations. So key genes, but you can get viable, advantageous phenotypes by making regulatory alterations. Many of the people in the room actually are carrying a mutation, a regulatory mutation, and one of the most important signaling molecules for mammalian development. OK, last point I'd just like to end on is I know these are case histories. We'd like to know general patterns. And this is one of the areas where the repeated nature of stickleback evolution has been particularly useful. So because the same things have happened over and over again, you can try to use genomics to recover all of the loci that are contributing to repeated evolution. So we published a reference genome of the three-spine stickleback and re-sequencing of 21 different stickleback populations around the world with the Broad Institute last year. And the strategy that we used for looking for adaptive loci was to have repeated examples of the marine ancestral form and the freshwater form. We line all those up with the reference genome, and then we window the genome, looking for those places where all of the marine fish look one way and all of the freshwater fish look the other way. So that's a site where the same things happening over and over again. We were very interested to see when we just did that lineup and windowing of the genome-wide sequencing data, you can make an index of how different are all the marine fish from the freshwater fish. You get this big peak here on chromosome 4. That sits right on top of the ectodysplasen gene, which we had spent six years positionally cloning using high-resolution genetic mapping and transgenics to rescue the plates, et cetera. You can recover exactly the same locus just by doing the whole genome sequence comparisons of a whole bunch of marine and freshwater fish. You also see a whole series of other peaks, both on this chromosome and on other chromosomes around the genome. In aggregate, this is identified 84 regions. The median size of those regions is actually defined pretty precisely because the break points in the 21 different populations help narrow a minimal interval that's being repeatedly selected. So it's a relatively small fraction of the overall genome, but a very interesting fraction to us. And so I'll end then by trying to use now not just a few examples, but this larger set to come back to an old question like whether evolution is controlled primarily by coding or by regulatory changes. This has been a major debate in the evolutionary literature. The actual answer from a whole series of these regions in sticklebacks suggests both mechanisms contribute. We do recover, in this overall set of loci, a series that affect the coding regions of proteins and produce consistent amino acid differences between marine and freshwater fish. However, a large fraction of the regions map entirely outside of coding regions in the non-coding intervals between genes. So those are regulatory alterations. And then there's a fraction of regions that have both coding and non-coding intervals, but the only changes that we see between the marine and the freshwater fish are in the non-coding regions. And so again, those are likely to be regulatory changes. Very last point is I think it's very interesting to compare these results to the results that are coming from the kinds of studies that you heard about from Saratischkoff looking for signatures of selection in different human populations around the world. We now have a ton of data from HAPMAP and other re-sequencing studies that make it possible to look for molecular signatures predicted to come along with a genomic region that has recently been selected. Part of Sabeti recently intersected a whole series of these different signals to define 178 loci that show strong evidence for positive selection during recent human evolution. And I went through this list and put them into the same category that I showed you for the sticklebacks. And again, there's a mixture of both coding and non-coding regions, but I think what's really striking about the two different pie charts is how similar the two distributions actually are. So these are incredibly different scenarios. Last 10,000 years, sticklebacks colonizing freshwater. Humans 100,000 years ago migrating out of Africa to different populations around the world. And nonetheless, in both of those scenarios, what you see is the overwhelming number of adaptive loci appear to be this sort of regulatory alteration in genes. So I'll end there and just conclude by saying, I hope I've shown you that evolutionary differences is at least a studyable, mapable problem. Major differences in natural populations can come from relatively few changes. We see similar mechanisms being used over and over again, and it looks like regulatory changes may provide the predominant basis for adaptive evolution in both fish and humans. Thanks very much. I'd like to end by saying thanks to a lot of people who participated in the studies and also major thanks to NHGRI who made an investment in doing this sort of comparative genetics and comparative genomics work to try to study basic problems like the molecular basis of producing new traits in natural populations. Thank you. So you've done a lot of great work looking at the sequences that changes between all these different populations. I'm very curious if the networks between these different populations change as well. So if a mutation in a certain gene actually creates new connections or loses other connections in the different networks of these populations. I don't think we have good data to answer that. I can tell you that if you change a gene like PITX-1, which itself regulates a whole series of other genes, then you have in essence changed a whole genetic program that normally, if it turns on in this part of the body, will make a hind leg. So yes, we do expect to see by the alterations here a whole series of downstream events that actually are the black box that connects the genotype to the phenotype. But we have not done all by all proteome interactions in the sort of network analysis that you're also seeing starting to come out in some other systems. One more question there. So one theory for the loss of a structure is that it has an adaptive value, a positive adaptive value. I wasn't quite sure why the loss of these pectoral fins and the stickleback had a positive adaptive value. Yeah, so the pelvic reduction alleles have been positively selected. So if you look at the signatures of diversity surrounding the mutations, that's an advantageous allele that's been swept in the population. The ecological studies that have been done on the trait suggest that the loss of a pelvis occurs in environments with low calcium and lots of insect predators when there are no predatory fish. Okay, so you need three interacting environmental variables, absence of a predator that's retarded by the spine, presence of a predator that uses the spine, and very low calcium levels, which also may create a metabolic cost for building the big bony structure. Thank you, David. Just before the break, we will have the first of several videos that we're gonna share with you, interviews and the like. So please enjoy the video.