 Ladies and gentlemen, good afternoon. Here to present Dr. Bruce Baker is one of the veterans around here. I have to have a few of us to make this college go. It's William Hyde Camp, a professor of biology. Bill? In 1961, the National Science Foundation funded several dozen high school students to attend Cornell University for the summer. And in that group, there were a group of zoology students. And the first lecture that was given by Professor Essel Leonard started with Professor Leonard, who was then in his 50s, running into the room, taking a center position in the room and yelling, the penalty for sex is death. At the age of 17, this had an effect upon me. And my first reaction was to say, well, just like the praying mantis, if that's the price, so be it. But I learned three things that summer. I learned, first of all, that biologists were, first of all, and primary interested in two things, sex and food. That's been a good motto for all of my life, and I intend to remain a biologist for the rest of my life. Now, granted, we couch those terms in terms of reproduction and nutrition, but don't let them fool you. That summer, I also memorized, I should say, because this doesn't really learn, that entire table that Dr. Hood's last slide put up there, and while many of you were wondering what it even said, there was this immediate recognition of, oh, my God, there's my life. At that point in time, we had to learn all of the channels of embryonic development, and we could draw every tissue and cell, ectoderm in blue and endoderm in yellow and so on. We also introduced that summer to the beginning of an intellectual pursuit that was an introduction to the whys and wherefores of evolution and development, embryological development. And it started really a quest for knowledge into the field of what was the basis of sexual dimorphism along with the evolution of sex. And for those of you who don't know the penalty for sex for death is a long-standing biological law that only sexually reproducing animals have to die. All others simply divide and continue going. One year later, after this summer of mine, Bruce Baker graduated from high school in the south side of Chicago and moved on to Reed College. At that period of time in our lives, this stuff called RNA was just being discovered. Dr. Blackburn talked about the central dogma, and I can tell all of those young students in here that you could simply rip out all the material in your biology text that had to do with molecular biology. It didn't exist in those early days. We were just discovering that this stuff called RNA with poly... you could produce phenylalanine peptides. In other words, the genetic code was just in its infancy, and it hadn't reached the undergraduate level at that point. Embryology, the concept of the differentiation of sex or any other tissue, really was in a state of what Dr. Hood referred to this morning as idle speculation of how systems work. We filled our books with notions of inducers, organizers, germ lines, germ plasm, but these were really mystical terms beyond anything we could really comprehend. There was no way to really understand what was happening in those embryological systems. Well, Dr. Baker completed Reed College, went on to the University of Washington in Seattle, took a post-doc at the University of Wisconsin-Madison, and then short stood as an assistant professor at the University of North Carolina in Chapel Hill. He then went on to nine years at the University of California in San Diego before his current position the last 12 years at Stanford. He is currently a member of the developmental biology group there. He is a past president of the Genetic Society of America, a member of the National Academy of Science, and has received medals and awards for a distinguished career in genetics, most notably the Genetic Society of America Award medal and the National Academy of Science medal in molecular biology. As with many geneticists, Dr. Baker has a sense of humor. Yesterday you heard about the couch potato gene. Today I suspect you will hear about genes called sex lethal and male specific lethal, marking back to a good old Professor Leonard's first lecture back in 61. You will also hear about intersex and double sex genes. And the most prominent one that you will hear about is that one called fruitless, which prevents males from doing anything about their sexual drive. And one of my favorite, which I'm not sure that he will get to today, but there is a dissatisfaction gene in fruit flies. Dr. Baker's work has led him into uncovering for us the hierarchy of gene activity, the role of alternative splicing in post-transcriptional events, and the mapping of the male sexual behavior in the central nervous system of the fruit fly. His papers range from topics such as the regulation of fruitless sex-specific 5-prime splice site selection by transformer and transformer 2, and that one third of you who understood that, good for you, to topics titles such as sex in the single cell, and also my favorite, sex lethal, master and slave, which is about the hierarchy of germline sex determination in Drosophila development. Kind of tricked you into reading it. I am proud to introduce Dr. Bruce Baker with a comment that it is through individuals such as himself with their intellectual perception, the diligent pursuit of the data that's out there, and the doggedly hard work that we know must go along with any laboratory work that we now know a good deal more about embryonic development than we knew in 1961. We are on the verge of really truly understanding both evolution and embryonic development, but really probably the first time in our history. My highest esteem and accolade is to introduce one of the country's preeminent developmental biologists who also happens to be a fruit fly geneticist, Dr. Baker. So what I want to do this afternoon is to take off from some comments that Lee Hood made this morning. He is Craig Venture pointed out also yesterday, a lot of what's happening at the genome these days is the sequencing of many of the model organisms and humans, and also getting us the ability to identify which genes are expressed in which part of the body, what genes are expressed in the brain, which genes are expressed in the liver, what genes are expressed in a prostate cell when it's normal or when it's cancerous. And one of the things that Lee Hood pointed out is it's one thing to know which genes are expressed there, it's another thing to know what each of those genes is doing there, and the current way in which we most commonly find out what those genes are doing there is to make use of model organisms where we can take a gene out, where we can knock a gene out, and ask what is it doing, and from it what it does there, in many cases as you'll see, we can infer what it also does in humans. So by that as way of preface, what I want to say is I'm a developmental geneticist and there's many different flavors of geneticists as you've seen during these talks, and what developmental geneticists are interested in are, I can have the slides on, questions like how do you build an eye? This is a side view of the human eye. If you're a developmental geneticist with a lot of hubris, you might ask a question of how do you build a brain? The human brain is said to be the most complex organ in the universe, complex known structure in the universe. At a more finer level what developmental geneticists ask are questions like how does a single nerve cell make all of the proper connections that it needs to make every time in every organism in a species? And most generally what developmental geneticists address is the range of questions that encompass how do you go from a fertilized egg to a functioning adult? And in my particular case, what I'm interested in is sex. What makes a male a female? And what makes a male a male and what makes a female a female? So we're interested in all the differences that there are between the two sexes, not only the differences in how they look externally, but the differences in biochemistry, the differences in behavior. And, well let me back up before I say that. Before I get you too interested in the topic that I'm going to tell you about sex, let me say three things. The first is depicted here, which is a cartoon from the magazine article about the field I work in. What I'm going to tell you is everything else you wanted to know about sex, but what we were afraid you'd never ask. The second thing I need to tell you is in talking about sex, I'm going to be talking about sex in flies, in fruit flies, but what I hope you'll come away with from this talk is an appreciation that you and I are much closer to a fly than any of us ever thought, and that how much of a human body is built is the same way that a fly's body is built and the same genes do it. As you all know, the basis for sex in humans is the number of sex, the kinds of sex chromosomes you've got. If you have two sex chromosomes, you're female, an X and a Y chromosome, you're male, and that is true in flies as well as people. And what I'm going to be interested in and what we're going to talk about is not what these sex chromosomes do, but rather what happens after that. And so the question that we're interested in is how do you get from here to here? For those of you in the audience who are too young to recognize Bogart and Bacall, I reframed the question in this way, how do you get from here to here? And so that's the question that we're going to focus on before I start in detail on that, what I want to do is to give you some background, some perspective by which a developmental geneticist comes at a question like how do you go from this initial signal to be either female or male to the final form of that particular part of the body? And by way of background, I want to start with the title of this symposium, Genetics in the New Millennium, and I found that a very appropriate kind of topic to be addressing currently for three reasons which I want to say a little bit about. One is because of the changes that have happened in recombinant DNA technology in the last 20 years, which we've heard a lot about. The other two things we haven't heard as much about. One is a revolution in genetics in terms of genetics as a field changing what it does and going from a field that studies heredity to becoming a set of tools by which we can dissect any biological process that we want. And thirdly, what the role of model organisms have in this. And I'll go through each of these three things saying some background words that tell you where developmental geneticists, people who are taking apart how the various organs and tissues are built, come at things. The first thing I want to say something about is how somebody like me reviews recombinant DNA technology. And for me, often the important thing is the ability to isolate a single gene that I'm interested in, to know its sequence, to make changes in that gene, and to put it back in the organism and ask what happens when I make those changes in the gene. In other words, to be able to manipulate the genes of the organism that I study. And the state we're at today with respect to these tools is in principle we can do this to any organism, and in the model organisms we can do it superbly, and we can really get this up and running, I think, in probably any organism for which there's the willpower and the manpower to do it. And so that's where recombinant DNA comes in to let us study individual genes and what their role in building an adult. The second thing I mentioned is an important contribution to where genetics is and the revolution in genetics is what has happened to the field of genetics. And this slide and its left-hand side, put together with the right-hand side, is actually the whole genetics course that any of you have had if you've had a genetics course. On the left what I've listed is the basic questions that genetics is a field was founded to ask and address when genetics first started around 1910. The topics genetics was concerned with what are the units of heredity, where are they located, how are they inherited, what are they chemically, how is that information stored, and what do they do. And we saw the answers to those being gotten during the first half of this century so that we knew that the units of heredity were genes. They're found in the nucleus and cells on chromosomes and they're inherited as chromosomes are inherited during cell division. And what they are chemically is DNA and how the information is stored there is the base sequence in that DNA and what that DNA does is to make proteins. That's the genetics course all of you have had if you've had a genetics course and that stops temporarily around 1960. We knew all of this by the early 1960s. So genetics by the early 1960s had answered all of the questions it had posed for itself. But what it did is not die out as a field at that point but transform itself because of two things that happened. One, in answering these questions about heredity, genetics also showed that the things that were true here were universal and true for all organisms on Earth. The second thing that happened was an intellectual or conceptual change was the realization that all biological processes were in part and in many cases major part under control of genes and so that one could dissect any part of an organism, one could approach any part of biology by asking what role did the genes have in that part of biology. And so that's the way in which genetics has been transformed. It's no longer a field for the most part that studies these questions but it uses the rules of heredity and what we know about heredity to dissect a whole variety of biological processes so that genetics is now central to neurobiology, central to developmental biology, almost any area in biology that you wanted to think of. The third component that has gone into the revolution in biology and genetics is model organisms and there's really three major genetic model organisms, higher model organisms. The yeast, the Brewer's yeast, lives as a single cell organism, the fruit fly which I do my work on, roundworm and nematode C. elegans, and a distant fourth is the mouse. And these model organisms were all picked for study many, many decades ago because they had various properties that made it good to use them to try and work out the original questions of genetics, the original questions about the mechanism of heredity, what were genes, where are they, what did they do. And that's why these model organisms were picked. And they were picked largely for the reason that it was cheaper and quicker and easier to do experiments on these invertebrate microorganisms that it was on vertebrates and less of a concern then but very much of a concern today. There was many fewer ethical questions about doing experiments on a fly or a single cell yeast cell than there were about doing experiments on vertebrates. So those are the three components that to my mind make up the revolution that genetics is undergone and why genetics and its pervasions across biology is going to lead to a real revolution in biology to make it the major science probably in the next 50 or 100 years. And what I want to do to give you a feel for what's happened with model organisms is model organisms were originally founded, as I said, to study the mechanism of heredity. And up till about 15 years ago every practicing biologist would have probably told you that model organisms, the fly, the roundworm, the yeast were really very different developmentally than humans. They were not the same at all, that we'd been simply too far apart in evolution and things were done differently in vertebrates than they were in model organisms. Where we've come to today is a complete revolution in that idea where now it's very obvious to most of us that most of how many important things are done to build a human or another vertebrate is the same way that they're done in a fruit fly and a nematode and a yeast. And part of the recognition for that and part of the seminal findings that led to that were carried out by three fruit fly geneticists who just about four years ago, almost exactly this week, received the Nobel Prize. And I thought I'd tell you a little bit about their work to give you an appreciation for how much you and I are like a fly. And two of the researchers, Yanni Nusslein and Erik Wieschhaus, are pictured here. And what they were studying is how the early embryo in Drosophila just after fertilization is initially patterned. They were interested in the genes that patterned the embryo and among the many things that they showed was that there were a set of genes that were expressed in repeating patterns, one red and one blue, each depicting two genes here, along the anterior to posterior axis of the embryo and they divided the embryo up into a set of segments. And so initial development was dividing an insect up into a set of segments. And the second line of research that intersected with theirs was carried out by Ed Lewis, shown here together with his wife Pam. And what Ed Lewis worked on is what happened after the embryo was initially divided up into a set of identical segments and what he showed was that there was a set of genes called hox genes these days that functioned in each segment and told it what it ought to develop into. So that if you look at an adult fly here at the top is a normal adult fly, you can see the segmented nature of the body, the abdomen with each of these stripes denoting a segment. And so on the head segments, these hox genes directed the formation of an eye in the middle segment of the thorax of wing on each of the three segments of the thorax of leg and so forth. And so you built the basic body plan by dividing an embryo up into segments and then specializing each of those segments to become a particular part of the adult. What I've shown down here is one of Ed Lewis' famous mutant flies in which the gene that normally causes a wing to be formed here is expressed in a different segment and you now get a four-winged fly as opposed to a two-winged fly. And what was so important about these findings was not only did these findings hold true in Drosophila but having found the genes in Drosophila people could use those to ask did those genes exist in humans and other vertebrates? And it turns out that they did and it turns out that those genes patterned the human body, the vertebrate body, much like they patterned the Drosophila, the fruit fly body. So that if this is taken from a textbook where here is a Drosophila embryo with head wrapped around the tail and in different colors are where the different hox genes are expressed along the body axis and the only things important is that different colors are in different places from anterior to posterior. Here is a vertebrate embryo and you can see that there's also the similar colors repeating in a pattern from anterior to posterior along the long axis of the body. And so the same genes that are involved in building the basic body plan in a fly are involved in building the basic body plan in a human and although I can't show you a four-winged human I can show you a six-armed human indicating that those changes might be possible in a human as well. But beyond building the basic body plan other aspects of invertebrates, model organisms and humans are built much the same way. There is here a picture of an early Drosophila embryo with its heart which is a simple open tube a much simpler structure than the complex vertebrate heart that you see on the right. It turns out that there's a gene that's important for building a Drosophila heart that was found and called tin man and in flies you name genes for what happens when the gene's absent. So this got named tin man after the tin man in the Wizard of Oz who didn't have a heart because when this gene's absent a fly doesn't have a heart. And it turns out that there is a gene invertebrates closely related to tin man that's involved in building the vertebrate heart. Similarly, the eyes you see here on an owl and the eyes that you see here on a typical insect with its many facets look very different to us. But it turns out there's a set of genes involved in building eye development building eyes and insects that are also essential for building eyes invertebrates. So the basic take-home point of this sort of introduction is that model organisms in very many profound ways are good places to understand very basic and very important parts of vertebrate development. So what I want to do now is to begin to take you into how a geneticist would approach a process like eye development or in my case sexual development and understand how that process was brought about. What were the genes in it involved in that process? How did they function to bring about sex in our case? And genetics to most people, I think, is something mysterious. And it's also something that I don't think needs to be mysterious because what geneticists do in dissecting a process is very simple-minded. And I have up here an analogy for you to think about if I reframe this question in a different way, what you might do. Say you didn't know anything about a car and somebody asked you, you go find out what it takes to make a car stop and we'll give you that you've got no knowledge about a car, you've got a good set of tools and you're not all thumbs and you want to do it yourself and you don't want to look it up on the internet. So how would you go about proceed to find out which parts of the car were needed to make it stop? And let me give you ten seconds to get your thoughts running and I'll tell you how I would do it and we'll see how close we are. So if you've got some ideas, hopefully, well what I would do and what a geneticist would do with a car is our organism, is you'd go in and take out one part of the car and then see if the car would stop with that part missing and you'd do it again with a second part and you'd do it again with a third part and once you got through all the parts in the car you'd have an idea of which set of parts were needed to make it stop and those would be the parts of the braking system. And that is in principle with one change that I'll say in just a minute all that geneticists do when they dissect a biological process. We go in, make a gene non-functional, ask what the consequences of that are, did it affect our process or not, go in and make a second gene non-functional and so forth to identify all the genes involved in the process we're studying and then put them together to ask how do they make a brake system or sex. The one change I'd make in doing this and the one change that geneticists make is based on two things. If you're like me, you wouldn't be sure that you could take a whole bunch of pieces out of a car to get it one deep inside, remove it, put everything back together again and the only change you'd made was taking that one part out. For me, when I take apart my car and try and fix it, I never get it back together right. And so what you would like is a way to reproducibly take out the same piece from a bunch of cars and get that set of cars to show you really the defect you're seeing, the lack of ability to brake, was something that was reproducible, that every time you took out that part, you would get that. And if you think about how cars are built, there's a way we could do that with cars and we could do that with cars because what cars are built is on assembly lines. And so what we could in principle do, and this is much closer to what a geneticist do, is we could go into an assembly line, tell one station if that assembly line make us a whole bunch of cars that are lacking this particular piece but are otherwise normal, a different point in the assembly line to turn us out a second set of cars that are missing a different piece but are otherwise normal and so forth and then look and see whether those sets of cars and a factory and a car are not at all a bad analogy to a model organism or any organism because what an embryo is at fertilization is basically two things. It's the cytoplasm of the cell which is a factory which will make any and all the proteins that it's instructed to make and it's a set of instructions in the chromosomes and the genes that tell that developing organism when and where to make different proteins, when and where to make the different parts. And so what geneticists do is to go into the genes and change those genes so that they don't make particular parts of the organism just like we could have gone into an assembly line and made cars that didn't make particular parts that make cars lacking particular parts. In terms of genes and in terms of thinking about how you approach biological processes there's two kinds of genes that are interesting to geneticists for the purposes of our talk that's all we're interested in. There's most genes which we commonly call structural genes and they provide most of the building blocks for the organism if you think in terms of the factory analogy they're the genes that supply the electricity the steel, the plastic, the wiring and so forth that get molded in a whole bunch of different ways to make different parts of the car. The second set of genes and the genes will focus on mostly and they're much less common and what they do is provide the instructions for which basic building blocks to make when, how to assemble them and in what order to assemble them how you build the various parts of the car or the organism and so what we'll be interested in in terms of the topic that I'm going to talk about how do you build a male and a female is what are their regulatory genes that control maleness and femaleness what they are, what are they and how do they function to build the two sexes if you look at individuals among humans you notice obviously that there's differences between the two sexes externally there's also differences internally and behaviorally and just like people are sexually dimorphic flies are also sexually dimorphic this is a female fly so I could show you a live fly she's busy laying an egg as you can see on the right hand side of that and this is a schematic of on the left a male and on the right a female fly they differ in body size with female being larger they differ in a number of external characteristics the only obvious one you can see here is the pigmentation on the abdomen is different but they also differ in their bristle patterns they also differ in a number of internal biochemical things certain proteins are made in one sex or not the other and as we'll see in a little bit they also differ profoundly behaviorally in terms of sexual behavior and so if we were interested as we are interested in are there genes that control these sexual differences we can ask how would we go about finding those genes and if there were genes that controlled all aspects of sex then we'd expect the gene to have the property if we knocked it out and had a fly that didn't have one of those genes that it would cause changes in all the characteristics that distinguish females from males the genitalia, the other external differences the internal biochemistry and the behavior would all be abnormal when one of these genes was not functional and so if we think about looking for genes that to see if there's regulatory genes controlling sex and I wouldn't be up here if there weren't we would look for genes that simultaneously control all of these different aspects of the fly and when I initially started out studying sex and I won't say when I became interested in sex, but when I became studying sex in flies which was about 25 years ago I guess there were five genes known that seemed to be the kind of regulatory gene we were interested in that controlled sex and I'm going to focus in this talk just on these five genes and I'll tell you about them in a minute plus one other one I'll introduce in a few minutes but I should say in fairness to the field that where the field is gone is we now know that there are about 25 regulatory genes and probably others we haven't discovered all of which work to control sex in flies and as you'll see all of these genes work together so there's a single pathway of genes that controls every sexual difference in females from the behavior to the genitalia and everything in between so let me introduce the genes that we're going to talk about so you get a flavor for what these genes are the very top line here is what a fly is like if it's got a normal set of genes if it's got two X chromosomes it's female an X and a Y chromosome it's a male there's three genes here transformer two X lethal any one of these genes if we knock it out what we find is that what should be a female fly with two X chromosomes instead develops as a male and that's externally a male internally a male behaviorally a male in all aspects it's not distinguishable from a normal male those same three genes when they're knocked out in a male no consequence from which we infer those genes don't have any function of female development there's a fourth gene called intersex which also is needed for female development because when we knock it out in a two X individual we don't get a female but we instead get an intersexual individual that has characteristics of both males and females but like the first three genes intersex is not needed in a male when it's knocked out there that male's still perfectly good normal male gene double sex that when we knock it out seems to is in fact needed for both female and male development because either X or XY individuals develop as intersexual individuals when this last gene double sex is missing so given you've got a set of genes like this and I hope you will generalize what I'm saying I'm talking about genes involved in sex I could be talking about genes in eye development, heart development neural path finding or we'd use the same approach but given we've got a set of genes like this that we can use to study sex one of the things you obviously want to know is why do we have this whole set of genes how do they work together or do they work independently or what to control sex and I won't go through the experiments that led to understanding that but simply say we've done a lot of genetics we've molecularly cloned and sequenced all these genes and understand a lot about their function and the next slide will summarize how these genes work together as part of a pathway to bring about male and female development in the fly so what I have in this slide in the middle are each of these genes and what happens in terms of these genes in an individual that's XY on the right and an individual that's XX on the left so let's take normal male development first what happens is that these genes function as a cascade of genes and in a male in an individual that's XY the top gene sex lethal is not turned on it doesn't make a protein the genes transformer and transformer 2 don't function in a male either and we've seen from the previous slide that the gene intersex doesn't function in a male so the only thing that happens in a male is the gene double sex gets turned on and it makes the double sex male protein a particular form of that protein that's found only in males so males are very simple of these 5 genes one of them gets turned on the others don't females not surprising to me at least are much more complex what happens in a female is 2x chromosomes causes the sex lethal gene to get turned on that makes the sex lethal protein that protein regulates the transformer gene so now it makes a protein that protein together with a transformer 2 protein now regulate the expression of the double sex gene so it makes a different protein one that's found only in females and as far as we know the double sex female and male proteins are the last proteins in this cascade and what they do is 2 things in females the double sex female protein activates female development it calls on all the structural genes to build a female characteristics into the organism and it represses all the structural genes that are needed and used to build maleness into the organism and the double sex male protein does just the converse it represses the formation of female structures and activates the genes needed to supply the structural material to build male characteristics so what we have is a way of taking this initial signal that determines sex based in the sex chromosomes and spreading it down through a cascade in which genes have different activities in males and females to ultimately control different developmental processes in the 2 sexes and I've asserted that we knew all of that and I'll tell you just one experimental test of many experimental tests that let us know that we really know that and if I go back one slide what you'll see at this level in the hierarchy at the level of transformer the sole difference between a female and a male is whether the transformer protein is expressed or not this gene is normally not capable of being expressed in an XY individual but can be expressed in an XX individual so one could test our model if you clone the transformer gene and altered it so it could be expressed in a male then if everything I've told you is true if we express that protein in a male it should cause that male to get turned into a female and you can do experiments like that in flies and Mike McEwen as if the Salk Institute did that the way you do it is to take an embryo from a fly inject your modified gene into that embryo and grow that embryo up and what you hope will happen is that that gene you've injected the modified transformer gene will insert into one of the fly chromosomes and become a normal part of that chromosome then as this embryo grows up into an adult you breed it to see if you can recover a fly where you've put this gene back into the organism and having found that fly you then do a set of crosses to ask does this transformer gene which now gets expressed in a male can do anything and what you find when you do that is that if you now allow transformer to be expressed here male individuals are turned into females and a number of other tests like that show us that what I've asserted we know we really know very well so having said that about what I've said about sex in general let me turn now to sexual behavior and how it's controlled and let me first introduce you to a fly's sexual behavior and I'm going to look at this from the point of view of a fly male because male courtship behavior is very overt and complex and relatively easy to study and the things a female does are much harder to study and haven't been studied as much because they're much more subtle in terms of a male sexual behavior when a male senses a female's nearby he orients towards her he will go over and then tap her with one of his four legs to get her attention and there may be chemo sensory cues that take place here if she seems receptive and doesn't run away if she does run away he'll follow her and try to tap her again and get her attention but if she stays there and doesn't run away he'll go around to the side extend a wing and play a courtship song which is species specific to her to try and get her interested the rest of this I think you probably all recognize and I don't need to go through but suffice it to say male flies have a complex set of behaviors all of which seem to be born inbred in innate in the fly in that every male fly as soon as it becomes adult knows how to do all of these things very well and it's not changed much by it being around other flies where it could observe and learn there's some learning but it's mostly inbred it's mostly in their genes so one of the things we wanted to ask was with respect to the genes that we knew controlled sex determination in terms of the body of the fly did they also control the behavior of the fly and so we took flies that were mutant for each of these genes and asked what effect did they have on behavior the three genes at the top of the hierarchy sex lethal transformer and transformer two those controlled not only the morphology of the fly but also its behavior when these genes were mutant in an XX individual that developed a fly that looked like a male and thought it was a male but double sex the gene at the bottom of the hierarchy when we looked at it when we knocked it out and even when we changed it in ways where we could change a female into a male by making mutants in double sex those individuals showed no male behavior so what that said to us was the genes at the top of the hierarchy controlled behavior the genes at the bottom didn't and what that suggested to us was that we must be missing a gene and that the real structure of the hierarchy must be sex lethal controls transformer and that controls double sex which controls most of sex but some other gene X must be what's controlling behavior and so Lisa Reiner who was a postdoc in the lab wanted to find out what was gene X and what did it do and the way in which she was able to do that is schematically presented here we knew from Lisa's work a lot about how the tra and tra two proteins the transformer and transformer two proteins regulated the expression of the double sex gene in particular what she had shown is that those proteins bound to sequences in the double sex gene that were short sequences of DNA about 16 nucleotides in length that were present multiple times in the double sex gene and that was how double sex was regulated to make a female versus a male protein and so what Lisa argued was that if there was some other gene gene X the transformer and transformer two were controlling so that females normally didn't show male behavior and males did show male behavior then it also ought to have multiple copies of these target sequences for tra and tra two regulation and to make a long molecular story short she used these sequences to identify other fly genes that had those sequences and came up with gene X and so what is gene X gene X is a gene that was already known by mutants that had been around since the early 1960s in flies and it was a gene called fruitless it was initially given its name because males that carried the fruitless mutant didn't give any progeny they didn't succeed in mating therefore gave no progeny therefore they were fruitless and what fruitless was known to do from the mutant phenotypes is to affect many different steps in male behavior that mutants in which the fruitless gene is almost completely knocked out never carry out the later steps in courtship weaker mutants in fruitless may extend a wing but play the wrong song or play a defective song and so there's various steps in all stages of courtship that fruitless affects so it looks like it's not just affecting the start of the process but many of the different steps in courtship most dramatically in terms of phenotype is fruitless's effect on the first step in courtship where a male in wild type will recognize a female is an appropriate courtship partner and court her what happens in fruitless males is they can't discriminate between males and females as somebody you should court and so fruitless males court males just as readily as they court females and if you put a group of fruitless males together and don't provide them any females then what you get is those males making a courtship chain in which each male is courting the one ahead and being courted by the one behind it so the basic point here is we have a gene that seems to affect all the steps in a very complex behavioral pattern and what you'd like to then know is how does this gene lay down the potential for this behavior what is the role of this gene in this behavior and so one of the ways and I should say by interjection here the work I'm talking about fruitless is a collaboration between my lab and here's Lisa Reiner and Anurajan a post doc in my lab and two collaborative labs Jeff Hall at Brandeis here Jeff and two of the people in his lab and Barb Taylor at Oregon State so Jeff and Barb in my labs have collaborated on all this work and it's the result of all of this so in terms of going back to fruitless itself what we showed first of all was that fruitless was really a member of the sex determination hierarchy I told you it had those sequences that we picked it out with and it turns out that it's regulated just like double sex is by transformer and transformer 2 so that in a female where transformer and transformer 2 are functional they regulate the expression of the fruitless gene it doesn't make a protein and double sex causes the double sex female function to be on whereas in a male individual where transformer is off fruitless goes ahead and functions and controls male sexual behavior and double sex makes the male double sex product so the fruitless gene at a molecular level is part of the same cascade that controls every aspect of sex in terms of what fruitless does one of the things you'd like to ask is where does this gene make the protein that is involved in sexual behavior and I don't know if you can I can't see it maybe you can the purple here is a fly central nervous system this is the head up here this is the thorax these are legs and a fly central nervous system is fundamentally in a crude sense like ours there's a brain in the head there's a ventral nerve cord which is like our spinal cord and so one of the things that we've done is to ask where in the whole body of the fly is the fruitless products made that are involved in controlling male behavior and I should say in preface that a fly at a crude guess on my part probably has between a million and a few million cells where fruitless is expressed is only in 500 of those cells and those 500 cells one of them is in the central nervous system of the fly no place else in the fly so and moreover they're not just any place or just one place in the central nervous system here I've overlaid in yellow the places where fruitless is expressed there's basically nine regions which are present bilaterally symmetrical and I've only shown half of those nine regions on each side of the central nervous system in both the brain cord where fruitless is expressed so being and the central nervous system the fly has about 100,000 cells so the 500 cells involved in which fruitless is expressed in which we think are involved in controlling male sexual behavior are only about half a percent of the central nervous system and so we think we have a gene here that because it's part of the sex determination hierarchy is actually laying down controlling in a profound sense the ability to have sexual behavior are not and it's doing that by functioning in a relatively limited number of places in the central nervous system I can also say that we know something about sexual behavior from previous studies in the fly central nervous system that Jeff Hall who's depicted here on the right and is one of my collaborators many years ago made flies that were half half mosaics, half male and half female and in those flies asked the question what part of the brain had to be male to get particular steps in the male courtship pattern and what he found when he did that was that the early steps in courtship like orienting and tapping mapped to a particular place in the brain whether a male could play a song or not was determined by whether you were male in the thoracic part of the ventral nerve cord whether you copulated or not depended on things towards the back of the ventral nerve cord and for each of the places where previous workers such as Jeff had mapped particular parts of the central nervous system involved in male behavior we have fruitless expressing cells in each of those places we still have fruitless expressing cells in places not known to be previously involved in sex and we're very interested in finding out what's the role of those particular places in this complex behavior we can also say something about what fruitless is doing how a gene can control a behavior by looking at the kind of cells in the nervous system that fruitless is expressed in if you look at a central nervous system it has a if you look at a behavioral pattern a typical behavior pattern will have some sense organ like an eye sensory cues from the environment back into the central nervous system there may be local neurons that coordinate things other neurons that coordinate things in longer distances within the central nervous system and finally motor neurons that go out and tell legs or wings or whatever to do something in response to the stimulus that the organism received in a simple behavioral circuit what we see in terms of fruitless expression is it's not expressed in any of the sensory neurons it's not expressed in the motor neurons it's just expressed in the central neurons so in other words if you had to think about what this gene was doing what it's doing is acting in response to generic information brought in by the sensory system coordinating and bringing about various things within the central nervous system but it directs the behaviors that are comprised courtship by driving motor neurons that are generic so it might tell a motor neuron to a wing cause that wing to move but play a song don't fly and that's the kind of function that it has so to summarize on fruitless it controls all aspects of male courtship behavior is expressed in a very small part of the central nervous system and the most important thing I think for thinking about it is that it is a part of this hierarchy of genes that control all aspects of sex and so I think we can say reasonably well that what it does is to control the potential for a whole complex behavior it's not just something that's correlated with that or needed for it it actually lays down that and so what in closing moments what I want to do is to generalize this a little bit and first of all just comment on the fact that fruitless at all when we first found it we were very surprised that a gene could control a whole complex behavior when we thought about it a little more that didn't seem surprising at all because everything developmental biology has taught us in the last 20 years is that every physical part of the body in any organism we study has genetic hierarchies just like the one I've been telling you about to build those parts of the body precise and exact in every organism so if organisms are devoting all that genetic energy to building two legs with the right number of toes, two arms with the right number of fingers etc is it very surprising in something like courtship behavior where if you don't do it right you don't leave progeny and evolutionarily you're dead wouldn't an organism also devote the same kind of genetic energy to reproduction and making sure you reproduce by having the proper behavior that would to making sure that you had two legs and two arms and so by that sort of argument I don't think it's surprising perhaps in retrospect that there are genes out there that in complex organisms lay down the ability for complex behaviors and you can also ask well are there other behaviors you might think of that would fall in this category I think I missed this one and there are many other behaviors that in model organisms are very stereotype behaviors they seem to be genetically determined and they're certainly very important for the survival and I'll just run through very quickly a few of those in a kangaroo rat a kangaroo rat knows at birth if here's the rattle of a rattlesnake it jumps and if it doesn't have that ability it's going to be eaten and not reproduced if you've ever watched ducks or cats or geese nurture and mother a flock of young you know there's a whole stylized ritual of behaviors that any goose or any dog or cat goes through in raising their young and again if that's not done right those organisms probably are not going to survive because they're progeny are probably going to get eaten or die at other levels we see stereotype conflicts between males in courtship behaviors and establishing territories in many other organisms we see animals marking territoriality animals giving alarm calls that puts them at risk so that their siblings who are around them maybe will have a better chance of survival building a web building a complex nest all of these things are things that all members of these species know how to do pretty well and they're all important for the survival of that individual either directly or in evolutionary terms and so it shouldn't be perhaps surprising that fruitless which is the first gene we know of that can control a complex behavior will be just the first of many genes controlling many other complex behaviors or set another way laying down the potential in the central nervous system for other complex behaviors and to stimulate a little conversation what does this imply for humans and at two levels one is sort of theoretical I've told you that how many parts of a fly in a human are built are by the same genes and many of the evolutionary arguments I've made with respect to flies and these other organisms supplies to humans equally well so perhaps some human behaviors do have underlying them genes which build into us the potential to respond in certain situations with certain kinds of behaviors and empirically we'll just add to catalyze things the gene transformer 2 in flies one of the two genes that sits right above fruitless and double sex and controls their expression has homologs in people and you can, Bill Maddox at MD Anderson took one of the human genes put that into a fly that lacked the fly gene that human gene functioned perfectly well to supply the missing function of the fly gene so across the hundreds of millions of years of evolution between this sex determination in gene in flies and a related gene in humans the function has been conserved the gene exactly parallel with fruitless the gene double sex at the bottom of the hierarchy is known to be conserved across at least 600 million years of evolution because that's a related gene functions in sex determination in the roundworm and we know from work at the University of Minnesota that if you put the double sex gene into the roundworm that's missing the roundworm homologous gene the fly double sex gene will correct the defect in the roundworm and 600 million years in evolution is back nearly to the time when the evolutionary lineage from flies to people became separate so it's at least potentially possible that not only in theory will there be behaviors and genes involved in sex found in humans but also they may in fact be related as trot 2 is to the fly genes and by way of summary let me just add two things the first is the question of so what and to reframe the question I posed at the beginning as to what happens in here if you find yourself here does it really matter how much it's your genes or your environment that got you to here the second thing I want to share with you is just a little bit of pure fun when we first published this it got a lot of press attention and this is just a newspaper clipping from a little town you can in northern California where my brother lives and he sent it to me it says master male sex gene discovered in fly that's all you need to know maybe you already know what's following because readers wrote in and made comments on that and you can't see it up above but you can raise that up a little bit because part of it's missing so when one readers wrote in they say where else would they find it as long as he keeps it zipped tell me something I don't know and with that I'll stop it actually one or two reporters made it really uncomfortable but Stanford Press Office is great with great background material and they wrote us with questions got to give ten second sound bites any ten second thing you say long is what's going to be picked through this realness and realism as we're waiting for questions to come up from the audience we asked the people that I know that this late hour people have to leave but for those that are leaving to try and be quiet so that we can hear the answers from the participants I asked for questions and comments and Dr. Renner wanted to start so with genes that control behavior and determine behavior do you think the fruit fly is just a very complex genetically controlled robot fruit fly behavior as I presented to you the sexual behavior is mostly ingrained but there is an idea emphasized in the talk an ability of a male to learn in terms of sexual behavior he really knows how to do all of that but he can do it a little better if he watches somebody else do it first there's also learning has been studied flies can learn, you can teach them in the equivalent of a maze and people have found genes involved in that and are beginning to check what is memory and what is learning so that flies are not simply a robot though many of the things they do have that kind of property for the most part that was a beautiful story and obviously someone who works on sexual orientation in humans it's encouraging to see that it can be studied in such elegant detail in the model organism but I do want to make a comment and ask you a question which is you've shown that there is a master gene for sexual behavior in Drosophila but of course there are other genes that also affect sexual behavior in Drosophila for example one story is that the white protein causes very similar behavior inability to discriminate between males and females, courtship change and so on and of course that protein isn't really involved in sexual determination per se at all, it seems to be that if you change something perhaps in serotonin actually that might affect it so I wonder if you just comment a little bit on how many other genes or how many other pathways might be involved in this very complex behavior besides fruitless well I mean all fruitless what actually fruitless encodes and I didn't even talk about that is a protein that is what's called a transcription factor it controls other genes and causes other proteins to be made so at one level there's many genes probably maybe even hundreds of genes downstream of fruitless that are needed to build this potential into the central nervous system the other thing with respect to the first comment is that there are many other genes in flies that affect sexual behavior almost any impairment of any sensory system mutants that are blind, mutants that can't smell they don't behave that well sexually I mentioned couch potato yesterday it doesn't behave very well so what I guess I would say is whereas there's lots of genes that affect behavior if we could go back to a car sort of analogy where the car is the behavior there's lots of ways to make a car not function there's only one way to build a car and the only things you can do to a fly are probably any organism so this behavior will be impaired if it doesn't have any energy it's not going to do any behavior but there's perhaps few ways where you can bring about that behavior by laying down some sort of a circuit in the central nervous system that gets called up in appropriate circumstances to cause that behavior Dr. Fox? Bruce, I presume the human transformer two gene in the normal human male does not induce fly courtship behavior we don't know okay I will say let me say two things about that first of all there's three copies of the transformer two gene in the vertebra genome and nobody has any mutants in the human gene or has anybody knocked out the mouse genes so we don't know if they have any role in sex or not but let me go on to answer your question you haven't asked it yet no, no the rest of your question you said does it really cause male behavior does the transformer gene true gene lead to male behavior in humans well, let's look at what Gorsophora courtship behavior is you see somebody and you decide that's a female versus a male you go over and give them an open line you tap them, you get your attention if she pays attention you do a little courtship and play a song does that sound familiar? as you described it but the point and the point I'm making is that the behavior the fact that the human transformer two gene in Drosophila works to induce the specific behavior that's appropriate to Drosophila and not to humans suggests a much more complex causal story than no, because all we need the human gene to do in flies is to recognize the fruitless gene and regulate it appropriately and all we're saying is the human transformer two protein has conserved within it across all that evolutionary time the ability to appropriately regulate the fruitless gene I'm saying there's a fruitless gene in humans it may have a target, nothing to do with sex in humans but it's regulated the same way and so you've kept that biochemical function that says nothing about the biological function right, but that regulation that biochemical regulation may be employed to a very different functional end and the so the relationship between the gene and the function has become in that description much more context dependent completely, I mean I only met that last slide to say that there some of these sex determination genes have for whatever reason been conserved in evolution, it may not be the same function but the same gene is there and it carries out the same biochemical function even though it may not in the case of transformer two carry out the same biological function and I just wanted to make you aware that maybe if we're lucky in the next few years you'll hear that maybe these genes are or aren't in humans and what they do there and I think the odds would be on your side of relationships between fruit flies and humans to guess that the behavior is the same if you look at all the other molecular changes that have been adaptable from fruit flies to humans it's more an exception when something doesn't relate have you seen evidence for other insects related behaviors also being influenced by this same gene very good question I've told you that fruitless is expressed in the central nervous system it affects behavior but maybe it's just having fairly general effects in the central nervous system it affects the ability to fly walk well, learn and we put Jeff Hall's lab is actually the behavioral lab we collaborate has put fruitless flies through a whole number of different behavioral tests they look just like wild type they're completely normal it's completely specific to sexual behavior in everything we've looked at so if there's any difference it's something that can't be discerned in the lab from a lot of at this point one of the best labs going but are there other I think that perhaps the basis for that question is that in humans of course there are a number of different behaviors not related to sex that have been correlated with gender car driving ability communication skills et cetera et cetera and so the question is are there any behaviors like that in Drosophila are there differences in foraging or feeding or something like that I have to say that despite how well Drosophila is known as a genetic organism as a behavioral organism is abysmally known nobody has really looked very much at other behaviors in flies I mean I would dearly love to generalize this to one other behavior and show you there was one other behavior it doesn't matter what where we could say there's a gene laying down a circuit in the central nervous system that's responsible for that but flies just don't have many well characterized robust behaviors to do it with except sex and cause there's that foraging gene in C. elegans now isn't there no I mean there's certainly genes there's a nurturing gene in mice there's mutants in other organisms that affect behavior lots of them just as there are lots of things in flies the phototaxis and things like that but in all the other cases we don't know if those are just genes whose products are needed to have that behavior something ancillary to it as opposed to something controlling it and as far as I can see the only reason we can say we've got a gene that's controlling in this case is because it's a regulatory gene embedded in this regulatory hierarchy that controls all aspects of sex if we just had fruitless and it's phenotype and didn't have it embedded in that we could you could argue oh it's just a gene needed for a lot of different things in sex but it's really not laying it down and controlling it we have a question from the audience that gets at the double sex gene what they noticed was is that it coded for two different proteins and wanted to ask about this as a violation of the one gene one protein no it's only a violation to make it easier for this audience many genes in higher organisms make more than one protein and they do it in the following way the gene is very big it gets transcribed into RNA but then that RNA is cut down various pieces of it are cut out to put together a final RNA which is the one that's actually translated into protein and that's process is called splicing and it takes together the parts of the RNA called exons and gets rid of the parts of the RNA called introns and for any given gene in many cases you can use different exons from a given gene to put together the final message so that a gene depending on which exons are selected can encode one or more proteins and so that is what happens in the case of double sex different parts of the gene are used to make the double sex male and female protein and there's hundreds if not thousands of examples in higher organisms where genes make more than one protein in exactly this way in the most extreme cases there's 50 or 100 proteins made by one gene I have a question from the audience that gets at something that hasn't come up in the conference at all to this point about the commercialization of genetically engineered agricultural products and how that has resulted in corn pollen that kills monarch butterflies we put thousands of chemicals into the ecosystem without understanding their impacts how can we not make the same mistakes with genetic information I will tackle it it's a very legitimate question that we have put all sorts of chemicals out there for many decades without realizing there was any environmental consequences and only in the last few decades have begun to try and deal with that and rectify the situation and there are the same concerns there legitimately about putting genetically modified organisms in the environment and one has to take care and use reasonable thought and reasonable tests to do that maybe I'll add to that I think this is an issue that I've had to deal with a lot around the world one thing that's very clear is we can get answers by using science it's a question of trying to do the experiments before all the things are put in the environment to be sure about it the trade-off with some of the genetically modified organisms though has been a reduction in up to 100 fold the amount of chemicals that are put in the environment what's coming up is there's a trade-off you know there's questions about the monarch study but I think it has to be taken seriously because there are going to be side effects from any new chemical or an insecticide whether it's cloned into a genome of a species or we spray it from airplanes but there are trade-offs that have had a huge impact already on the reduction of chemicals going into the environment but that's not an excuse for not studying things more thoroughly before they get put out I want to call on Chaplain Elvie right now to make a few announcements this brings us to the conclusion of our conference except for a lecture tonight which will take place in this hall because of the tornado damage we were unable to provide for a dinner for more than 300 people and those tickets were sold out six months ago so we are going to gather for dinner in a small dining room over in the college but Dr. Evelyn Fox Keller will give her talk tonight at 8 o'clock in this hall and you're all invited to return for those of you who cannot come back at 8 thank you for again your attendance this year as I've always explained to people who come from elsewhere you've been a very faithful loyal constituency through the years you are what you make this conference and so may your genes carry you home and their gods and you know everybody has their own god and their own gene but that god is one and I wish you a good year next year we're doing a conference on globalization and we've got a great list of people coming so those of you whom we will not see this evening see you again next year good night thanks for that