 Okay, so like many of us that are giving a sequence of three lectures, what I decided I would do was start the first one with a slightly more technological or techniques approach to explain, and this would also explain some of the background for the subsequent lectures. And for me, the technologies are going to be genetics, so what I'm going to do today is to talk specifically about, go through genetic experiments that were directed at understanding the mechanisms of development, and then I'm going to build on those tomorrow's lecture by using the results from those screens to set up a system that allows us to study how transcription is patterned in the early embryo, how the information content ingredients specifically relates to cell fates, and we'll see how, and then also how eukaryotic enhancers respond quantitatively to distinguish subtle levels in concentration to activate genes. And then on Friday what I'd like to do, ooh, Friday I'd like to talk about the actual downstream mechanics that produce morphological changes in the embryo. You know, all of that is going to be based a bit on genetic analyses and the analysis of genes, identification of genes that have been carried out in Drosophila. What I, rather than just focusing on these particular problems, what I decided I would do is try to take a slightly more general approach to genetic mutagenesis experiments, into address, you know, basic questions of genome organization, how many genes, how one goes about identifying those genes, not just specifically with respect to embryonic patterning. And obviously we all know the genomes are sequenced. You can identify from those sequences based on your knowledge, based on your sense of your ability to identify open reading frames or functional units in the genome. You can make calculations for the numbers of genes in the human genome. You can base that on molecular biology. You can base that on bioinformatics. What I'm going to talk about today is really estimates, give you a sense of the, as how one approaches that particular problem with genetic approaches, it obviously becomes more interesting if one watches, focuses on a particular project, and this, what we're looking at here is obviously Drosophila embryo, from the early stages of development, which you can see, what we'll be able to follow in this movie is morphological changes in the embryo. Here you can see this layer of nuclei on the surface of the embryo. There are about 6,000 nuclei at this stage. These nuclei become partitioned into cells. The embryo begins the process of gastrulation, where you can see individual, the sheets of cells moving, changing their morphology. The process, this real time here, so I've sped up the movie, you have a sense when you do that, you visually have a sense of movement and a force that you don't, and that gives you a certain interpretive insights into what's going on in the embryo. The processes are going to continue, and I'm actually showing you this movie in part because it's, I sped it up here, just to give you a sense of how extraordinarily complex this process is. This, the forming intestines, the region of the embryo, the anterior and posterior intestines now fuse. This is the future brain of the embryo. You can see very small cells moving around that begin to move around. These are hematocytes that are actually phagocytizing dead cells and cleaning up debris. You can see now this internal gut in the embryo. You can also, we'll begin to see muscle movements in the embryo. These become more vigorous. This is the mouth, this is the anus, this is the intestine. The embryo breeze through openings at the posterior, and you will see these now, you suddenly fill up with air, and the embryo can hatches and goes off into its happy life, one assumes. You look at a process, and it's actually extraordinarily complex. I could look at that movie, and I have actually looked at that movie many, many times. I've made many movies that are similar, and I've looked at all of those. I can actually never lose the enthusiasm of watching those processes, and I send you all a copy and you can spend your time in the lab looking at the movies. But what one really wants to know, though, when you look at processes, you want to know how they happen, how they're governed, what is it that sets up spatial pattern? Why does a fold happen here? Why do these, why does a fold happen here? Breaks down into two questions. What makes this region different? What are the gene products specifically involved in programming cells to, to a specific region to make a specific fold? But there's also then, why does the fold form here? What's the relationship between those programming decisions and the actual mechanics to produce the movement that you've just seen in the movie? So how do you actually mechanically transform the embryo? And we're going to try to approach that problem using genetics. One of the major challenges, though, for almost all, almost all truly beautiful embryonic developmental systems is that they are multicellular and almost all multicellular organisms are deployed. And so what I wanted to, I guess, I guess one stands up here and advances, just to put us all, put us all on the same page, what I'm, this is going to start out, this lecture is going to start out fairly simple, but it may get, it may or may not get more complicated, who knows. One of the features, though, is that multicellular organisms are deployed. That means that we have two copies of each chromosome, one we inherit from our fathers, one we inherit from our mothers, and we know, and especially in flies, we know that from the number of genetic experiments that invariably one of those two copies is enough. And even though you have two copies, if you have a single wild type allele, you will develop normally. What that means, we would write that as m over plus is viable, even if the gene m is essential. And what that means is that in the geneticist parlance, loss of function mutations are recessive to wild type, or you could say that wild type is dominant to most loss of function mutations. Now that feature has two, that feature has two features. One, I would argue that it makes the genetic analysis of multicellular organisms much more easy than bacteria or yeast or organisms that are haploid, because you can make easily knockout mutations, destroy those mutations, and in a bacterial population, if a gene is essential, you destroy the mutation, that bacterial cell is lost among the million or so cells on your plate, and unless you develop clever selection protocols or special kinds of mutations that allow you to detect and work with these, you're going to lose, it's much more difficult to obtain and maintain loss of function mutations in a haploid organism than a diploid organism. The bad side, of course, is that diploids make organisms, make genetics difficult, because if any new mutation you produce is recessive, what that means is that you will not see the consequence in the individual in which that mutation arises, you will only be able to see the consequences by making an individual what we call homozygous. To make homozygotes, you have to cross heterozygotes, if you cross two heterozygotes, you produce one quarter homozygotes, and so what that means is you'll see, and I'm going to walk you through a mutagenesis experiment in Drosophila, you'll see that the major complication is setting up a series of crosses that allow you to reproducibly produce homozygous, both to maintain a new mutation as a heterozygous, but also to cross individuals to produce homozygous individuals that you can recognize phenotypes. And I'm going to do this experiment in Drosophila, and I'm going to start with one particular chromosome. Drosophila has four pairs of chromosomes, that's eight chromosomes, it's a dip blade, the half blade number is four, the second chromosome is one of the largest of these chromosomes, it represents about 40% of the total genome. So that's a good opportunity, what we're going to do is we're going to try to ask how many essential functions are localized to the second chromosome, we're going to do that by a mutagenesis experiment, and the essential idea is that we're going to start this set of series of crosses with two different strains that are essentially normal, the normal wild type females and flies which are carry males, which are homozygous for some eye color markers. And these will just help us follow the homozygous for this individual through our crossing scheme. Now what we're going to do is we're going to take these males that are already mature and have produced lots of haploid sperm and are waiting to mate with females, we're going to treat them with a chemical mutagen which induces single base pair changes. And this mutagen and the procedures that have been developed in flies are strong, basically what they do is they increase, they are random mutagen, so they're going to randomly change bases in the DNA, and they change the frequency with which that happens from about one in ten to the sixth spontaneously, ah, no, better explain, that what this does is it increases the probability of getting a mutation in any gene, in the average gene, from something like one in a million, one in ten to the sixth, to about one in ten, one in a thousand. So we're going to threefold increase in the efficiency with which we produce mutations. This number one in a thousand is actually useful because if you think about it, it's the probability of you treat in sperm with this mutagen, and there are a thousand sperm, then if you can sample those thousands, there is a good chance that you have, you've induced a mutation in the gene, any gene that you're interested in. So one in a thousand is not, a thousand flies is a lot of flies, but it's not a lot, and it's that property, the power, the ease with which you can induce mutations that allows us to do these experiments. It's very strange to be up here giving this lecture and having most of you nod, and I feel you're kind of with me because I feel like I'm going really slow, but I'm happy to go slow. Do we have any questions? Yes. And get the temperature, get the frequency even higher than one in a thousand. There are two things, fly, what you do is you put lace, you make a little bottle, make sugar water, put some Kleenex in it, and you soak the water with Kleenex, you dump all these males in, you make them really thirsty, and if you add too much of the chemical, they don't really like to drink it. So you have to sneak it in, and there's a limit to how much sugar water you can add. There's largely, there's also a second day from that, even if they eat it, they begin eventually to get sick under sterile. So this is about as high as we've gone in terms of concentrations and efficiencies. More questions? Okay, so the basic idea is then that we take these males, we make them with females, and we obtain an F1, and these F1s are heterozygous for mutations. Are we going to be able to see any phenotype? The group answers loudly? No, because the loss of function mutations are generally recessive. What if we cross these F1s with each other and get an F2? Are we going to be able to see a phenotype? Can we say yes? How many vote yes? How many vote no? Okay, good. So there's a problem though in doing this. Remember we fed these males this chemical mutagen. Individual sperm have been affected and they've produced an F1. Each F1 fly is derived from a different sperm. And that sperm, by chance, may or may not have had a mutation in any particular gene. So what that means is that you can't just cross the F1s because each of these individuals is a single individual with a new mutation. So the big job that you have is to take each of these single individuals and convert them into multiple male and female heterozygotes that can cross with each other and produce homozygotes. You set up little separate inbred lines where you take individual males and make individuals by mating single males with normal females. Okay? Then we get another generation. And now what we have are males and females that are heterozygous for the same mutation. Whatever mutation was on the second chromosome of this individual will have individuals that are heterozygous. You'll also have some other flies here. We'll talk about those just a tiny bit later. We now can allow these flies to mate with each other. And in the next generation, which is the third generation, we will produce progeny, one quarter of which is now homozygous for our original white-eyed colored mutation. And if these white-eyed flies survive, it means that on that particular second chromosome, in that particular male, we did not produce a mutation in the gene that was essential for viability. If they die, we can get excited. We look at this tube in which this little family has been living for two generations. We see no white-eyed flies, and we're happy because we know on the second chromosome we've induced a mutation. Now, that's the basic idea of the screen. There are two flaws, two problems. One, and this relates to a problem that Mayan introduced this morning, is that we randomly potentially produced a mutation on the second chromosome anywhere. In this F1 or this F2 individual, you could have recombination. What recombination would do would be to yield a chromosome that had the cinnabar brown markers but didn't have the new lethal mutation that you induced. What that means is that in this generation you'll see some white-eyed flies, and you'll throw out the tube even though you've produced a lethal mutation. And the probability of this recombination is high enough along the entire X chromosome that you're only going to keep any genes that happen to be induced very close to your markers. So what you need to do is to have some way of keeping this chromosome intact. And the way that that's done in Bresafla is to prevent recombination. You prevent recombination. Recombination cannot productively occur between two chromosomes which have different sequences or inverted sequences that are inverted relative to each other. So in Bresafla we have a number of such chromosomes. They're called balancers because they can be used to balance chromosomes or keep those chromosomes intact. And we're going to have to redesign this cross to introduce these essentially wild-type chromosomes into our background. So whenever we have a wild-type, many of the wild-type chromosomes, they will be overbalancers and then if we produce a new mutation here, we won't see that mutation will remain coupled to the Cinnabar Brown. And so here if the Cinnabar Brown individuals, if we have a mutation, all of the Cinnabar Brown individuals will die. This allows us then to focus our mutagenesis on this entire chromosome intact. And that will be important later for the math when we go through what the actual results are. There's one other problem is that if we take these individuals, even if they're balanced, we have this is the new mutation, this is a balancer individual over a wild-type chromosome, we're producing these wild-type flies that we don't want. And if you're a good geneticist and there are enough other markers floating around, you could dump out the flies from each tube and sort out the males and females before they've had a chance to mate with each other, throw these guys out and keep these guys. If you want to do this experiment on a big scale, you have to figure out some way of killing these guys off and the way that is done is that you introduce dominant mutations that are temperature-sensitive and will kill off any heterozygote that has that mutation. So we're going to introduce it here and then in the next generation, we'll produce individuals that are heterozygous, we'll also produce individuals that get this dominant temperature-sensitive mutation over the balancer, but these all die. And so now what we've been able to do is take a single male, put it in a tube with some females, and carry it through two generations, and then come back and look at the tube and say, are there any white-eyed flies in the tube? If there are no white-eyed flies in the tube, because it's a successful mutation, we will keep that tube because the only flies that will be in that tube will be the heterozygote that will continue to carry the mutation. That's the basic strategy that is used to identify mutations, essential mutations that are essential for viability. Now, I've told you that the mutagen we're using produces mutations with the probability of about one mutation, a fairly given gene, about one mutation per thousand sperm or per thousand chromosomes. That means that if we want to do a mutagenesis screen where we say we want to identify mutations in every gene, and we want to be sure that we've looked at enough tubes that we've actually examined or knocked at every gene, we need to do this cross on a scale of about four to five thousand chromosomes. Calculate that more precisely with the Poisson. Yeah, question? The chances are that you will only one the most. One of the things that worried us was if there are only a thousand genes on the second chromosome and you use this mutagenic dose, you will have some chromosomes that have no mutations, some that have one mutation, some that have two mutations, some that have three mutations. Yeah, you can maybe deal with that. If there are ten thousand genes on the second chromosome, then you can do the calculation and most of these second chromosomes will have many mutations. So before you do the experiment, you don't know. We made some good guesses, but essentially what we were hoping was the answer that you pointed out, that even if there were many, many genes, that ultimately for what we were going to be interested in, there would be a small number that we could sort those out. Okay, so this is the experiment for the second chromosome, and this is how the numbers actually look. We set up 5,764 little tubes with males and females in it, in the F1, and this is an efficient dose of mutagen, and so after two generations we checked, 4,217 had no white-eyed flies, no surviving. And what it tells us is that we had induced at least one mutation in these lines. So you'd like to really know how many mutations, and if you assume that the lethal mutations that you've induced are Poisson-distributed in equal probability, then you can calculate that in these 4,217 lines, there are something like 7,500 lethal mutations. Next question you'd like to know is how many of these genes are... these are all genes, these are mutations in genes that are essential at some point during the life cycle of the animal, because they die before you get to be the adult. You'd like to know how many are essential in the embryo itself, and what we did there was just set up these lethal lines, collect embryos from them, and ask what fraction of the lines produced 25% dead embryos, and we counted those as lethal... and what we found was that the first big result was that if you think of these 7,500 mutations as sampling the gene population, essential gene populations in the individual, what we found was about 20% caused death to embryos, and 80% even though the animal is going to die sometime, that that gene that we had eliminated was not essential in the embryo's survivability. So we're reducing down the number of genes that are essential. So it's only one-fifth. If we did the next step, though, and looked at these dead embryos and say, how did they die? Or do they die? Was this gene essential for the embryo to achieve normal morphology? The number falls even more, and 272 of these lines, essentially 272 lines by this point, have lethal mutations produce embryos with distinct phenotypes. What that meant was that they looked different enough from wild type that we could say, ah, there is something repeating here in this collection. Sometimes these embryos didn't have any skin, sometimes they didn't have any head, sometimes they had multiple duplicated structures in the wrong places, a whole range of possible bad things that could happen. But the nice, this was a limited subset, though, of all the bad things that you could imagine would happen. So you could then say, these are 272 lethal mutations. Say, how many genes are affected? All 172 could be mutations in the same gene, or many of them could be, there could be a thousand different genes, and we've sampled and we have 172 of them and we have one mutation in each. I'm going to stop and say, how would you solve this problem? How would you decide how many genes rather than mutations? What we want to know is we have a tube A dead embryos, a tube B dead embryos when the chromosomes are homozygous. You'd like to know if the mutation in tube A killed the same, knocked out the same gene as the mutation in tube B. What would you do in that kind of test? You would cross them with each other. So I'm going to ask you really simple questions that are going to go really, really slow unless you immediately shout out the obvious answer. Don't be shy, it will speed us up. You do what's called a complementation test. If line one and line two are both lethal and homozygotes, but viable and heterozygotes, you cross these two together, you produce an individual which is M1 over M2. If this individual dies, it dies because you have knocked out that M1 and M2 have knocked out the same function. If M1, if this individual survives, it means that the two mutations are in different genes. So the one chromosome can supply what the other chromosome needs, it will complement. So this idea of complementation is the standard kind of thing that geneticists do to define gene. So actually one of the words that we use for genes, we don't talk about genes, we talk about complementation groups. We arrange mutations in terms of complementing functions. Everybody with us so far. So when you do that, you find that these 272 mutations can be assigned to 61 complementation groups. There is 61 different genes on the second chromosome have activities that are essential for embryonic morphology. And you can also say something else, that the average number of mutations or new alleles for each complementation group is 4.5. So you could say, what we want to know is, are these the only genes that will pass this test? Are there really only 61 genes on the second chromosome? An argument in favor of that is, well, we have hit the average gene four times. What's the probability that we've hit that there's a gene that we haven't hit at all? And if you make the assumption that all genes are on average about the same mutability, and try to math your way through that, it's a really small number. Another way of thinking about it, though, is just to look at what was happening in the experiment. This is actually a historical, this is actually like a diary of our life during that time when we were doing the experiments. And there's really plotting two things here. We're setting up more and more crosses, and we're plotting in the dark spot, the frequency, the number of new mutations that we identify. And because we're keeping the dose the same and this is kind of linear, and we just get more and more mutations with every tube we set up, we can also ask how many new genes do we identify with each set of chromosome. And you can see that initially it was very exciting because we were getting lots of new genes and lots of new phenotypes and everything. And then at some point this curve begins to flatten out. And right at about here, you might decide that you had something better to do with your life. And so we stopped. Because there may be another gene out there, but you would have to set up many, many, many more tubes to be able to identify it. Okay, so we did that for the second chromosome and we did that for all the other chromosomes. And this is the end result, the characterization of the fly genome, which you can see is that ultimately we end up with lots of lethal... We end up with 566 mutations that kill embryos and produce distinct phenotypes and they can be assigned to 121 complementation groups. Now, you can do a couple of things with that number. Since we know that if we ask of these mutations, they represent about 3% of all of the mutations that we got. And if we take mutations to be representative of gene, essential functions, we can say that genes that lead to embryonic morphological defects represent 3% of all genes that are essential for viability. And that allows us then to calculate the total number of genes on the second chromosome that are essential for viability. That number is 378. It's a calculated number rather than a real number. And what's interesting here is that of course if you look at the sequencing data, you have 13,000 open reading frames, 13,000 proteins that are constantly being selected for function and maintained as open reading frames in flies. And yet when you knock these things out, only about a third of them result in homozygous knockouts in lethality. It turns out that we got this number for flies, but it's not unusual. It is probably comparable to what has been observed in mice and in humans. Now, you don't have another... mice and worms and fish has not been done on humans. But what it's telling us is that when we look at this genomic data and we recognize proteins, the very, very large fraction in code functions that for some reason are being selected for, but do not have obvious consequences on viability when we remove them. It's an interesting challenge for us. We'll come through that a little bit in a lecture. Do we have questions? Yes. A complementation group is a... If you have mutations, and you want to know if mutation A is in the same gene as mutation B, you perform a complementation test and you ask whether the function that's eliminated by A, mutation A is the same function that's being eliminated by mutation B. And when two mutations fail to complement each other, that is, they die, we say that they both are mutant in the same function, they both are mutant in the same gene. So a complementation group is a way that geneticists define gene. And so we say that there are 21... 121 complementation groups on the second chromosome, 121 genes whose function is essential for viability, or for normal embryo. Another question? Yeah. Okay, so the practical aspect here was that a 500 by 500 matrix would be pretty horrible. We already knew which chromosomes they were on, and so that kind of knowing where they were would reduce the number of complementation tests. And actually what we did was we did two things. We looked at them, and we first complimented everything... did complementation tests between everything that looked similar. And we... and that allowed us, once... our rule was once something failed to complement... once A failed to complement B, we no longer had to do complementation tests with B, we only had to do it with A. So by initially screening through, based on morphology, we could reduce the potential number of complementation groups. And then we genetically mapped all of these mutations using the... the protocol described this morning to map the monchromism, and then we mapped... we did complementation between any gene that was in 10 to 20 map units of any other gene regardless of what its phenotype was. Other questions? Yes. Say that again. It contains... we would say it contains a single gene. It is the single gene... it is the... because we have two point mutations which have removed that function. We're going to assume that this mutagen mostly works by changing bases, and we know that's true. And so if you change the bases in DNA here and here, and you put... and they fail to complement... the only way that they would fail to complement is if they are disrupting the same function. And so a complementation... the failure to complement is, for us, the definition of a gene. So A complementation group is one gene. Yes. Ah. So... Ah. So here, these were the ones... so all that we... here we're doing a slightly different map. We are assuming that mutations sample the genome equally. And we had 18,136 lethal mutations. That is 18,000 genes, or 18,000 mutations that affected genes that were essential for viability. And we... 566 or 3% of those vital... of those essential genes were essential for embryonic patterning or embryonic morphology, and they constituted 121 genes. So if you take this ratio of mutations to be the ratio of genes... each mutation is in a gene, and then the ratio of embryonic morphology genes versus total viability genes is 3%. And if I told you that there were 121 embryonic morphology genes, you could calculate the total number of genes. That's what we did there. That's where that number comes from. Ah. Because the markers that we're working with, this eye color, cinnabar brown, are the mutations on the second chromosome. If we... And we certainly did induce on these sperm mutations in the third chromosome, on the X chromosome, on the fourth chromosome. And they were floating around independently of all the other things that we're looking at in the background of these stocks. But they're not impinging on the survival of the cinnabar brown white-eyed flies. So our only test was, why did the white-eyed flies survive? Because we had... Let's go back one. Going forward. That's what happens when you stand in front. Okay. If you divide... If you have 566 mutations divided by 121 complementation groups, that's 4.5. Well, actually, maybe the 4.5 was specifically with the second chromosome, but you can get the idea that you have so many complementation groups. You take all of these mutations and assign them to complementation groups, and you can ask, what's the average number? Okay. Then we can ask this question, is 121 enough genes to build an embryo? Is this a sensible kind of result? I was just going to help us think about this. And then you have to ask, well, where do embryos get the gene products that they need? And they get them from two different sources. The mother, when she makes the eggs, supplies lots of RNAs and proteins in the egg. And the embryos, once they're fertilized and begin transcription, make gene products themselves. Those are the two sources. Sperm don't bring in gene products. So those are the two sources. And so... And then you can ask, what kinds of... So we have these two sources, and the genetic screen that we've just done actually distinguishes between these two sources. It distinguishes between the two sources because we've taken heterozygous normal females and males, mated them with each other. One quarter of the embryos are homozygotes, and the others are hetero... They're brothers and sisters that come from the same mothers and the same father are heterozygotes. The heterozygotes survive. The homozygotes die. And that means that the determinant for viability is in the embryonic genome itself, rather than in any maternal... So we're only able... So what we're really asking here, this makes this experiment even a little bit cooler. We're saying how many genes have to be... Well, being molecular biologists at all, and without doing expressionaries, we could say how many genes have to be transcribed in the embryo for the embryo to survive. And that's the number 121. Now, we can... Let's say this doesn't make sense. That's a good thing to ask yourself, lots of times. And then you can say, well, what if the number is small, what is it? The embryo needs all these gene products. Why doesn't the mother just supply everything? That's... When you're a younger child, I can see some people smiling. Why wouldn't the mother wouldn't supply all gene products that the embryo needs? Why do we... Clearly, there are 6,000 RNAs detectable and they're all supplied by the mother. What's the special feature of the 121 that need to be supplied by zygotic transcription? What does zygotic transcription do for you? And the answer is if you just need a gene product, you might as well get it from mom. Even if you don't need a gene product, but somebody else in the egg needs the gene product, or some other... Get it from mom. But if it's important that the gene product be expressed in these cells, but not these cells, and if those differences come down to single cell differences, if the lack of the gene product has as much meaning as the presence of the gene product, then supplying it by transcription is great, is the right strategy. So then in a way... Later, these genes were cloned and what you can see is it's really true. What characterizes this subset, with the exception of about 5-10%, is that they all show patterned, temporally and spatial patterns of transcription. They produce extraordinary patterns in the embryo. And the interesting thing to think is that each of these patterns, then, represents a point in development where having and not having the gene product is reflective of a... has meaning, is reflective, if you will, of a developmental choice. So what we've done in this screen is by looking for embryonic lethals, and probably we could say because of features of the evolution of flies where they tend to supply everything they can maternally, by looking for mutations that cause homozygotis to die, we zeroed in on a very special class of genes. Those genes whose presence and absence conveys information that makes decisions. So we can now then begin to describe the division hierarchy of development in terms of a sequence of genes' expression patterns being on and off. So we'll re-describe rather than just simply look at the morphology in that movie, lovely as it is, we can begin to translate that sequence into sets of decisions and choices that involve specific expressions of genes. Yes, it depends on both. So each of these genes comes on at specific times and off at specific times, and they come on in specific places and they will sometimes do this multiple times during embryonic development. So on the other hand, we said that mom supplies everything except for these 121 genes, but can we get a handle on what the mom is actually supplying? You see here this will become slightly genetically more interesting than what we thought initially, but to do this, if you go back and think about the mutagenesis screen and I'm not going to go through this in great detail. Yeah. A parsimonious way to add it is you are managing the regular transcription to be this buffer, right? Because if I myself, and I need to add the genes, and I could let them regulate by the same transcription factor, then those factors should mainly be transcription factors, right? I mean, it's the reason why you think that you mainly got transcription factor from that. We'll actually come to that, but the idea of what decisions are and whether decisions are always at the level of transcription or to the extent that decisions are always at the level of transcription, ultimately, your logic is right. Okay, so what we wanted to say can we get a handle on what mom's contributing? We could think through this screen and we realize that in spite of all those four lines there and this is my messy PowerPoint work, come back to this figure, there is this class where we got the tubes and the white-eyed flies were surviving where we knew that we hadn't induced a lethal mutation. We could still ask whether now if we took these individuals which are homozygous for this mutagenized second chromosome, whether the females in these tubes are themselves fertile. We have destroyed a gene that is essential for their ability, for some essential function that they contribute to the egg. Now, just to do that experiment with these leftover flies, we started it that way, but actually, Trudy Shupach and Niani Nuslan who did the majority of these kind of experiments basically meant having to go back and start all over again with another selective system to get enough flies to do this partially because you don't want to manually sort out the red, the white-eyed flies and put them in a tube. You'd like to have some magical thing where at the end you end up with a tube where you only have homozygotes and you can shake them out and see whether they're fertile or not. They figured out a genetic screen that I decided to spare you of and just presents you with results. This is just Trudy's results from the second chromosome, but they're representative and they're interesting because they show you what happens if you really try to get your hands on a maternal gene. Trudy set up 18,000 lines. She had to do that because she was using a large amount of mutagen and producing lots of lethal, so many of the lines the homozygotes didn't survive and there were 777, she had among her testable line she had 7351 and 528 of these lines the homozygotes females were sterile. Now, and I'm going to leap through a whole bunch of little minor analyses to just let you know what the classes are. 178 of these lines had mutations that we know now were partial loss of function mutations in genes that otherwise would have caused lethality. So as you knock out you don't always knock out total function of a gene and if you have a gene that's essential for viability you could imagine that you kind of tweak the activity get it down enough but still some homozygotes survive but they're sterile. That's 178 and that tells and that number so we're going to forget those for a second because they're not really specific for oogenesis there are about 350 mutations that are specific for female sterility and maternal effects and actually the way that we know the difference between these is that these mutations give you can be assigned to complementation groups with multiple hits per complementation group that is whenever you hit the gene you get the same phenotype. Here for mutations in viable genes to survive they have to be a very special mutation just tweak the activity enough to get it down enough to knock out oogenesis but not down enough to knock out viability this is a very special rare kind of mutation but you have a very large pool of genes that can mutate that way so you get lots whenever you do the mutagenesis this kind of mutagenesis you get a lot of genes a lot of mutant lines that contain mutations that complement all other lines that can't be assigned to complementation groups each of which are unique for particular genes because you're just sampling the viable populations again but at a partial loss the 350 mutations could be assigned to 110 genes so you're getting multiple hits for these and that assures you that these genes are probably specific for oogenesis many of them just didn't make didn't make any eggs at all or were blocked at some point but 28 produced morphologically normal eggs I think I'm out of genes you have 28 genes and among the... I messed up the numbers this should be 11 this should be 28 and 11 anyway we're going to end up with a small number of genes which when you mutate them the female makes an egg that looks perfectly normal everything's fine but when that egg is fertilized the embryo is unable to develop a normal pattern because evidently those genes and those products that the mother is putting into the gene are products that are instructive in their nature so mother is putting 6,000 gene products in the egg and there's a small number that determine either the anterior, posterior or different regions of the embryo of the egg and therefore the regions or the fates of the cells that form in those regions of the egg genes specific genes that are essential... I just said that okay and so one of these we'll talk about tomorrow is how these genes work we know that they produce localized cues we'll focus on a gene called bicoid that determines the anterior, posterior pattern of the embryo based on a maternal RNA that's deposited in the egg so how are we doing with other questions? yes okay so the question is what is the function of if there are only 12 that are essential for making a normally patterned embryo all of the other... everything that you know about from biochemistry oxidative phosphorylation any... RNA polymerase DNA polymerase all components forming cytoskeleton membrane synthesis all of those are supplied to the embryo by the mother those are all of these other RNAs that are there and they are uniform because all cells do need to be able to do those things it's the rare maternal gene that actually supplies... it will see supplies information the embryo would die but if the mother... think about it if you make a mutation in an essential ribosome or protein the homozygous individual will not be able to make functional ribosomes and she will die now she won't die as an embryo because embryos get their ribosomes from their mother but after the embryo hatches out and starts growing up and increasing in size and volume suddenly it becomes... it depends on its own ability to make ribosomes its own ability to make cytoskeleton its own ability to make actin and it's at that point that the embryo dies for the individual homozygous dies but they score in our screens as homozygous lethal and we cannot easily detect them as maternal effects because they are dead by the time we would have adult females that we could test for eggs are there other questions? okay so almost halfway... more than halfway through what I wanted to do was to say well, yeah, so far the picture that we have is that the genomes are made there are certain numbers of genes and whether you detect them or not depends pretty much on who is supplying them and when they're required when their expression is required so that in the embryo for normal morphological for events you have a small number of zygotic genes because all the machinery and the cytoskeleton is supplied maternally you have a small and a small number of maternally supplied genes that are specific for embryogenesis and you can mutate them and homozygous females survive and are happy and then you have a large group of genes which are essential for viability are supplied by the mother but we can't study them easily mutationally because when you knock them out you eventually die when the maternal sources run out so everybody here but I want to say no it's unfortunately more complicated than that there are other reasons why you might miss genes so I want to go through two of them because they turn out to be interesting and intriguing from the standpoint of understanding morphology and morphological transformations so I'm going to get into this though from a historical kind of standpoint just to remind you this is the movie that you saw at the beginning they start off this is an unfertilized egg about an hour into development forms this nuclei divide actually it's drawn on the next slide fertilization is followed by nuclear replications that unlike most other mitosis mitotic events followed by cytokinesis so you have a single cell with multiple nuclei they continue to replicate until the number is about 6,000 and then they stop dividing they enter they build they increase the plasma membrane the 30 fold to subdivide the embryo into an epithelium each of these nuclei into individual cells and cells begin to move around these this event is the first time in development where different cells are behaving differently from each other where you would maybe need to program different cell behaviors or different fates when we look through and we so we wonder the earliest time when any of these mutations that are active in the embryo the first set is like 121 when do they become visibly abnormal and I actually set up little 121 cups and looked at each of these and all of them were normal until they started to gastrolate and then depending on how good my eyes were I could say they're abnormal this one becomes abnormal at this stage so I had the conclusion was the embryo really only needs new zygotic transcription at this point when cells begin to behave differently from each other that's a great model I like it it's not true but it's a good model and what killed me I mean it didn't kill me the experiment that made me change my mind was you can pharmacologically disrupt RNA transcription there are different drugs one of them is called alpha-manitin it's derived from mushrooms you put it in it knocks that all polymerase 2 activity and some people had done experiments like this where you injected them into fly embryos and you block all transcription and we would predict that the embryo would come to to gastrolation and then become abnormal but the terrible thing was that the embryos became abnormal here rather than here and this meant one of two things either you can't believe pharmacology which was my favorite interpretation at the time or that we had missed important genes that were active in this early in the earliest active genes were the ones that we had somehow missed and you know you can have your preferences in science for what you think things would like to be but there's also you want to test it so I was trying to figure is there any way that I could test and show these alpha-manitin people these pharmacologists that they were wrong and so the other possibility was that somehow when you do screens for point mutations you miss genes and so it occurred to me that in flies there's a way of testing this idea because and the way that you if you could eliminate a whole the whole second chromosome for example and you made an embryo that didn't have any second chromosome the results from our mutagenesis screens would say that that embryo would develop normally to the gastro-lestate because I just told you there are no genes on the second chromosome that are active before gastro if we really have identified it but if you eliminated the entire second chromosome and the embryos died here you would have to say well there must be something else on the second chromosome that you miss so I mean one more complicated genetic experiment is this what we're doing today let's just go through the logic the earliest acting genes are affected by alpha-manitin so how do we know this okay so this is the most complicated genetics that you're going to see but it's really cool okay now normally for the second chromosome it has a they look kind of like this the diploid they have a right and a left arm on a centromere and during meiosis that one of these two arms go to the eggs and one or the other of these chromosomes go to the sperm and after fertilization you restore normal diploidy are there any questions about this there are chromosomal rearrangements that are essentially like what we would call translocations in Drosophila where the two right arms are fused to the same centromere and the two left arms are fused to the same centromere that's kind of like if you were to break here and here and refuse and those are just accidents of nature but fly people keep accidents of nature in their labs and call them stocks and so these stocks exist and now if we and they exist not only do they exist but they can mate with each other and so now what's going to happen is this two centromere and during meiosis the two centromeres will segregate from each other and give you two right arms or two left arms in the egg and what that means is if you work through the little pundit square here the total amount of DNA in all these squares is pretty much the same but if you look at the actual identities of the chromosomes this little square here has four right arms and no left arms and this little square here has four left arms and no right arms what that means is that there are any genes in the left arm of the second chromosome that these embryos will develop normally these embryos will develop normally until the first gene that's required to be transcribed on that arm are there any questions get the idea I bet you can guess what the result of the experiment was this is what happens when you induce alpha-maniton this is what happens when you don't have this chromosome arm a couple of catastrophic things happen they don't organize the actin cytoskeleton they don't form cells they remain a sensation they can't transport lipid there's a set of very sad things that happen to these embryos but not to any of the sister embryos to tell you that this is specific for this arm there are some genes there there's at least one gene that does all these terrible things or they're multiple genes so what are these genes can we identify them and now we're going to use a slightly different technique in addition to these kinds of chromosomal rearrangements in flies you also have ones where the second chromosome is been broken and part of it has been hooked onto the Y chromosome that's right there and part of it the Y chromosome has been hooked onto the end of the second chromosome these individuals are also perfectly normal diploids they have all of the genes that they normally need they're just kind of rearranged and rearranged means that during meiosis when sperm or eggs are made you will get sperm that have different contents in particular there's let's see where are we here yes well in particular sometimes you will get one eighth of the time you'll get embryos that are missing every gene that's distal to the chromosome breakpoint so since there are hundreds of these translocations available what you can do once you know there's a gene on a particular arm you can set up these crosses and kind of like march through the embryo making smaller and smaller deletions until suddenly your phenotype disappears and then you've localized this mysterious gene and by refined versions of that you can identify and clone so when we did these experiments what we found is that the entire alpha-amanitin phenotype the bad things that happen to the embryos could be reduced down to eight different regions scattered throughout the genome fairly good meaning that other people had mapped based on chromosome morphology, banding morphology down to three or four bands a feature of Drosophila is that they have chromosomes which you could build a map of the genes based on recombination you can also build a map of the chromosome based on morphological densities of DNA before you could clone poly-team properties and so you could build a map and so people had these translocations and they knew where the breakpoints were so one, two let's see if I get the numbers right one, two, three, four, five, six, seven, eight and so once we figured out where these genes are we could actually clone them and identify and they define a whole new class for us they are expressed, they're not expressed in the mom they're not there during early cleavage and they come on right at this point where the embryo is beginning to stop dividing and go on into trying to transform itself they come on uniformly they kind of go off in patterns we don't know whether that's interesting but the most interesting feature of these proteins is that they all encode small rapidly evolving proteins with poorly conserved amino acid sequence and therefore no easily recognized human homologs they are even within the their function and they are essential for cellularization but their function does not depend on obvious specificities or sequences of amino acids they have you know I now tend to think of it this is probably where evolution works when you suddenly at the end of embryonic development you realize you haven't divided the embryo into cells and you have to make cells and you have to say oh what do I have to invent a new and you have to fix this thing it's kind of like the way I fix things that go wrong in my house which they go out and I buy some plaster and squirt it up there and kind of fix things and in a way which is evolutionarily which is all that you need to do you need to do things whatever these proteins are they do these jobs and the I suspect and this is not unusual it would be useful to have somebody who does evolutionary sequence that having rapidly evolving proteins that don't have homology is not a peculiar feature of just flies most organisms if you look at the sequencing data there's a class of proteins that fall into this and no one really knows what their function is and this is probably giving us a handle on the function now if we go back and we ask well these genes exist why could we only identify them in this kind of screen why didn't we identify them in a conventional mutagenesis screen the reason for that obviously is that well if you're using a point mutagen and you're changing bases what the doing is changing amino acid sequence and that's not going to knock these proteins out and they're small and so the probability of getting stop codons is small I think curiously to this day we still don't have any point mutations in any of these genes we do in a certain sense because we've cloned them out and tried to work our way out from melanogaster to other fly species we've identified the proteins that are 40% similar in this region we've taken them and cloned them and put them back into melanogaster and they function perfectly fine so we do now have mutants of melanogaster but simply by taking the Drosophila virulis gene and putting it in melanogaster that's the only kind of mutants that we've been able to obtain so interesting class of genes okay one more what is our timing 15 okay one more set of genes that was hard to identify and I'm going to call this little last five minute section why is analysis of patterning of morphology more difficult than analysis of patterning sulfate okay to do that I have to give you just a little bit of the biology here in Drosophila we look at a cross section of the embryo the cells on the ventral side are going to form mesoderm and to do that the cells over a 15 minute period form a fold these cells are going to be the mesodermal cells and they form a fold that brings these mesodermal cells that are going to form muscle and internal structures into the interior of the embryo you can maybe watch that process here and you can see that it's just a simple fold bring these cells into the interior of the embryo you can look more closely at what's actually involved here and this is interesting you have at the end right before the embryo begins to gastroate here you can see the sequence of shape changes that are producing this fold in the interior you can cartoon it basically what happens is the apices of the cells constrict and they ultimately become triangular in their morphology and we can associate that change in shape with a change in the distribution of a motor protein cytoplasmic myosin and produces can be localized to the surface of these cells and only to these cells at the beginning of gastrolation most of the myosin sitting on the base of the cells but these cells here that are going to be mesoderm localized myosin and that contraction mechanically you can imagine we'll talk about that on Friday produces the forces that drive cells into the interior we're going to try to understand that process in greater detail but what's so what we would like to do though is geneticists is ask whether we can identify genes that affect this process and so in the original screens that Christiane and I did in Heidelberg we identified two genes sets of mutations two complementation groups that when we looked at that during gastrolation they didn't make this ventral furrow cells didn't move in it cells just look like this rather than like that so these are psychotically expressed genes that must be transcribed to allow this process to occur we know now that those two genes are part of a patterning sequence where maternal protein called dorsal defines the ventral side of the embryo and these two genes which are called twist and snail I'm showing you twist here are kind of a complementary interacting cell programming group of cells that say bimesiderm you express twist and snail in cells that's what the decision to bimesiderm is like the cells that don't express this gene are ectoderm these are the cells that are mesoderm an expression of this gene drives this morphological change within about 15 minutes now that's a nice story but the complication of course is that both these two genes are what we call they encode what are called transcription factors they control the fate mesodermal fate they define the cells of being mesoderm by being expressed in these cells and their activity is in the nucleus and they drive so they drive expression of drive the mesodermal fate and part of that fate must be to control expression of other genes some of which would account for this morphology morphological change we haven't none and these were the only two genes in the entire screen among the 121 that produced this failure to form ventral furrow and yet the screen was designed in this major features to identify transcriptionally active genes and we're arguing that there has to be a transcriptionally active genes downstream of twist and snail that actually accounts for the morphological transformations those yet those downstream targets of the transcriptional activation could not be identified in the screen with one partial exception so what we did is we went back to this complicated translocations and by once you had other genes cloned the genes that were early active we could add them back as trans genes into these stocks and gradually ask, can we identify are there regions of the genome that are required for mesodermal cells to move in not for mesodermal cells to be determined to be mesodermal but the actual mechanics of their moving in is there are regions these combine works from the leptin lab Rogers lab and our lab now identify 1, 2, 3, 4, 5, 6 they have different effects some of them seem to coordinate cell division and cell shape changes others involve actual assembly of actin or recruiting of niacin to the surface what is interesting about all of these genes is that if you what they actually do when you remove them is that they don't eliminate the formation of a furrow or the internalization of mesoderm they slow it down they make it irregular and abnormal it probably costs a certain fraction of it doesn't look very pretty but these embryos are actually viable with one particular this particular case it turns out that we had identified the gene in the earlier screens because of a lethal effect later in development in this region here make abnormal furrows or abnormal but they cells eventually get in and they survive so the picture that you get the picture that you suggest is that we have these genes and they are in a certain sense functioning redundantly none of them is absolutely essential but they are downstream of twist and snail we know that transcriptionally and their expression is activated and that activates myosin but you can activate this contractile process by a variety of different steps and interestingly if you take and these genes even though none of them is necessary if you mis-express them you activate now myosin over the whole surface it is sufficient to drive myosin but they are not the only way and therefore not essential ok so that class of genes in general the picture we have then if we go back and say what do we get and this gets close to Stefano's question if we look back now at the original screens those screens define genes that must be supplied transcriptionally transcription puts products here and not here we look at what those products are the vast majority are transcription factors or signaling molecules that control the expression control the localization or activation of transcription factors so the things which are local and control self-faith their functions are unique and they produce unambiguous phenotypes that means that they were really easy to identify genetically downstream targets that affect cell shape and change are substantially redundant they are sufficient to cause cell shape change but not essential they are not essential for viability in general and they require sophisticated imaging to recognize and understand their actual function you cannot recognize these in screens that are based on lethality because the disruptions that they cause are not sufficiently lethal you only identify these functions by looking at an embryo during the process of gastrulation and seeing what is recognizing the abnormality and then mapping the functions so that's the general picture that we have exciting things that we these genetic experiments have kind of established not quite a hierarchy but an overall cartoon view you have maternal reacting factors that supply positional information to the egg the embryo responds to that positional information by activating transcription control genes that determine cell fate and ultimately determine cell behaviors but this step here as we've seen probably substantially redundant in nature now in the next lecture what I'm going to do is talk a little bit about this first step using bicoid is how you actually go from graded information to transcriptional responses and then on Friday what I'd like to do is talk more about the mechanics what we've learned about the mechanics the transfer of information from here to the actual mechanical processes of producing cell shapes my desire in giving this lecture was to kind of do something where you had a sense not just where these pictures come from and where the knowledge comes some sense of what's possible with these classical genetic strategies in thinking over the this morning progression actually the whole course I had to stop and think just a little bit whether there's something special about embryonic development that it was this genetic approaches were particularly useful there are other questions that have been even raised today that where you could ask can we to what extent is a this kind of genetic approach applicable you could ask what are elements that intervene between phenotype what was really interesting in this trans this relationship between genotype and phenotype to what extent would it be helpful to identify the genes that intervene and is it appropriate or is it the best way of thinking about them as intervening between this transfer in your talk this morning there were a couple of intriguing examples that were identified through genetic analyses essentially and but have interesting histories where they could one expand that approach or is it possible to do an organized genetic approach we've talked about control and the kinds of feedbacks that are built into circuits that interpret or utilize information I'll argue tomorrow and I'll probably agree maybe that what we've identified in these patternings is kind of an open system without much obvious feedback or control in the initial patterning processes that we're going to look at but also it's true that we may not have seen them with the screen design the way we did it have identified any aspects of control or feedback that's something for you to think about or can we redesign or redo the genetics in this way and similarly if one thinks about hematopoietic lineages you can reconstruct a pattern a potential a proposed lineage based on expression patterns and overlaps of expression patterns but it's an intriguing task particularly if you're doing this in an organism like the mouse where genetics is possible whether there are genetic insights that would really allow you to test specific ideas that emerge so I'll stop there and take any more questions thank you to the attentive audience