John Carlson Transcript
SPEAKER 0um, let's just started a real pleasure to introduce. John Carlson's, Second lecture. John is a professor of molecular cellular developmental biology at Yale. He, is a very distinguished scientist He gave us a taste of his research yesterday when talking about gr proteins and One thing I could say about John's research is It always starts from a beautiful set of genetics and It progress is through, all of the things you would want to do to characterize large family um, the way has worked brings light to the underlying mechanisms is by being really beautiful, really complete and really thoughtfully done, yeah, as a great scientist, He is, of course, been recognized by many awards, including the Genetics Society. he's a member of the National Academy, the American Academy of Arts and Sciences, etcetera, etc. And it's a real pleasure having him here. Thank you.
SPEAKER 1
So thanks very much. Leslie I've had a really great time here. But she mentioned one thing that, uh, that I actually gave a taste of my research yesterday. There's actually sort of a literal interpretation. I think, uh, a number of people came up afterwards and tasted some of that noni juice and I have to say, I woke up in the middle of the night, sat bolt upright and wondering. Are all those people okay? Is there Anybody here who had some of that? noni juice. Who is actually sitting here today. Okay, So you didn't all get raced off the Beth Israel the middle of the night or anything? Okay, good. I'm glad to hear that Also yesterday, I mentioned how touched I was that. Well, I put it so looking around, standing there with a map on campus, trying to find where I was going, and no fewer than three undergraduates came up and helped me spontaneously offered to Help me find where I was going. I really very warmed by that. Well, this morning I showed up. I was standing in front of another place, holding the map, trying to find where Bassine building was. And it was It was really wonderful that a faculty member came up. And asked if I needed any help finding. All right, so I found it. Not only a BRANDEIS, So the students wonderful, very helpful. But also, the faculty are too. So thanks very much for that. Good. So my lab is interested in how animals detect and identify chemicals in their environment. And we are interested in the receptors, the neurons and the circuits that underlie chemo perception both in the fly but also in insects that transmit disease to hundreds of millions of people each year. Okay, so today I want to tell you two stories that concern two of the greatest of all biological imperatives. All right. And these are of course, eating and mating Okay, So, first, I'd like to tell you something about the problem of sugar detection and some of the mechanisms that underlie how animal regulates how much it feeds. And then in the second part of my talk, I'd like to tell you a bit about the problem of mate detection. This is a classical biological problem. Very, very fundamental. How an organism finds a mating partner of the same species of suitable mating partner of the same species. Okay, so basically, the fly has a number of chemo sensory organs. It has an antenna up here which senses odors. And it also has taste organs which uses to detect taste in some of which are sweet interest indicative of nutrition, others are toxic um, and taste bitter. These taste cord organs include the low Belem here, which we talked about a bit yesterday. Also, there's the pharynx right here, and then they also have taste organs on their feet, so flies actually walk on various food sources so they could taste through their feet, which actually makes a lot of sense. All right, so flies have big, big, big families of chemo receptors. There are the odor receptors or OR genes of which there 60. These detect odors. There are 60 gustatory receptors, or gr genes, and there are also 60 ionotropic receptors or IR genes. Yesterday we talked a lot about gustatory receptors. Today I'm gonna talk mostly about these ionotropic receptors. Among these, there are many. There's been some just beautiful work on the antenna, a group of them called antenna Liars, which map in here in blue Um, the some of these are expressed in the antennae and where they detect acids and a means. And there's been some really spectacularly beautiful work done right here at BRANDEIS, showing that some of them are important in detecting humidity and temperature. There's another big clade of these genes, Members of this big family that we know virtually nothing about and those are the ones that want to talk about today. Okay, so since we know virtually nothing about, um, a post doc in the lab tongue, Waco first wanted to figure out where they express. So he systematically went through and made promoter gal4 drivers for all 35 of these genes in this clade And he found that many of them were expressed in taste organs. Here's one that's in the l labellum that taste organ on the head. Some of them are expressed in the leg and some of them are expressed in the pharynx. All right, now that raised questions, what do these receptors actually detect? What are they doing? What role do they play in behavior? So we're very curious about that, and I'd like to tell you first about some work we've done showing a role for some of them in feeding regulation in the fly. So feeding regulation is a tremendously important in the fly. How much to eat? It's also really important problem in us, okay? And especially in this day and age, where we have a ramping worldwide obesity epidemic. Okay, so if we could learn something about the basic mechanisms that control feeding regulation on the fly, some of them could be useful in understanding, perhaps controlling, feeding regulation in humans and also an insects that transmit disease to hundreds of millions of people. This is a tsetse fly here. We've actually done some work on these in our lab. They're much, much bigger than drosophila Okay, that's just an example of one kind of insect that's terribly important in transmitting disease. Both the people and to cattle in Africa. Okay, So when an animal of fruit fly feeds on something, food can pass up through this pharynx here. And there's various taste organs along the pharynx, of which one is called the Labral Sense organ or the L so shown right here. Okay, it's got a number of neurons and Cem console, which I'll show you. Here. We found that eight of these IR's from this clade that we know virtually nothing about are expressed in the in these pharyngeal taste neurons. Okay. And actually, there's one of these eight which just leapt off the page, so to speak and grab their attention. And that's this one here ir 60 b And there are two reasons why we're excited about this one. First of all, it's expression is just exquisitely specific, all right, and the entire fly. We only find one symmetric pair of neurons, which expresses it. This is the cell body up here, a long dendrite, which terminates in a little since Ilham here, this is in the labral sense organ, So IR 60B is expressed with enormous specificity. The other reason why we were really interested in it is because of the rate at which it's evolving. So we by looking at how much sequence variation there is among in this gene among all these ir genes, basically in this, clade in different strains of Drosophila melodic Astor and in different species, you can get a new idea of what's called the direction of selection, which is to first order approximation, sort of the ideas of how fast it's evolving. And of all these genes there is exactly one IR 60 B, which is under a lot of pressure to keep its amino acid sequence. All right, so that suggests that the sequence of this gene could be a very good solution to a very difficult problem. All right, So, um, we were curious to know whether ir 60 b, by virtue of its expression and its sequence conservation, might be playing an interesting role in feeding regulation. So the first thing to do is find out where exactly, is expressed in this libersense organ. All right. And this little sense Ilham, here, there are eight neurons. Previous work by others had been done and shown that two of the neurons in this of these eight expressed gr genes. We talked a little about gr jeans yesterday and these are sensitive to sugar, and they seem to promote feeding. All right, so we wanted to know whether this IR gene was expressed in those neurons or perhaps another neuron. So we did a systematic double label analysis and found that these this ir 60 b gene was expressed in its own neuron on orphan neuron we know Nothing at all about on these gr genes are expressed in two other two other neurons. Okay, the I R 60 beaching was co expressed with two other IRS from that same clayde and I'll come back to that later. But no gr genes in this neuron. So it's a new neuron that haven't previously been, um, well studied. Okay, so we wanted to know whether this neuron played a role in feeding regulation. So, Ryan Joseph, post doc in the lab, Use differential pumping paradigm. Okay, what you do is you take a fly and you immobilize him in a, uh, micro pipette. Tip such that head is just protruding. And then you offer just a droplet of liquid food containing some sucrose. It also contains some blue dyes You can sort of watch it, get eaten. Okay. And you actually see some of this blue dye, which is actually already moved, started to move down the track. Right? So we do, Then is we advanced towards this fly this source of a blue food with a micro manipulator, and then you can see the fly gets wind of it on, then starts eating and eating. And pretty soon you'll start to see some blue accumulating down here in the abdomen. Okay, so it eats and eats, and we measure how long he eats before he stops. Okay, so then if you look at you, measure you measure the feeding time, so this is measuring the feeding time as a function of sucrose concentration. You find that greater concentrations of feeding, elicit of sucrose, illicit longer feeding times. And it saturates about 100 million Mueller. Okay, now, how about feeding time? We Look to see whether the total time that it feeds correlates with the number of swallows that the fly takes, and it agrees very, very well. We also asked whether it's a good measure of the total amount that fly eats. To do that, we took advantage of this blue dye, and at the end of the experiment, we would grind up the blue dye and use Inspector Fatima to see how much blue he'd eaten. Right. And you can see that there's actually a pretty good correlation between the total time of feeding and the total volume of ingestion. All right, so bottom line from this is we think that this total time feeding is a pretty good measure of the how much it ingests. All right. So in order to look at the function of this ir 60 b neuron, we silenced it. Using some very nice technology available. The fruit fly where we used this gal-4 driver to drive expression of tetanus toxin which basically block synaptic singling from this neuron. So we asked if we silence this neuron will just have an effect on the rate of feeding. And we did and had a very strong effect, but completely opposite to what we were expecting. So you find out when you silence this ir 60 b neuron, the feeding time greatly increases compared to the control parental controls. And at one point I wanna make throughout this talk, we were extremely careful. Um, every fly you're going to see here, all the different components the TNT the gal-4 is they were all back cross five generations to our control genetic background to minimize the any effects of any genetic background effects that before we took any behavioral data, any from a typical data at all. So the surprising thing here, then, is that we silence these neurons and we get more feeding. This is a 300mM sucrose, but we see the, uh, same effect across a broad range of sucrose concentrations. When we silence, this neuron will get more feeding. Okay, so that's the conclusion that the simplest interpretation is this neuron is actually acting to limit the intake of sucrose. Well, how specific is this is just sucrose. Is it all sugars? We tried a bunch of sugars and found that it didn't have any effect on the feeding for any of these others. Fructose trailers and glucose. Remember this treeless and then come back to this later. All right, so basically, if silencing it, increase the amount of feeding Well, we wonder what would happen if we activated these neurons. Okay, so for that we use opta genetics. We used we expressed in this neuron a channel called Crimson, which is sensitive to far red light. So if we on activate, if we shine, shine far red light on this neuron, it will actually activate it. So this is a frequency of light that we can see is humans, but the fly can't see. All right, so this is what the experiment looks like to us. Here's what the experiment looks like. The fly. And here's what the fly looked like to the reviewers of our manuscript. So sure enough Oh, I should mention we did this experiment with Trehalose because we wanted to use a sugar which would activate feeding, but which didn't operate through this path to the i. R. 60b neuron. So we wanted to see where the levels of Trehalose would decline when ir 60 b neurons were activated. And sure enough, they did. We shine red light, okay? There was no effect. And either the parental controls And if you just look at the results in darkness where there's no red light, they were equivalent for all three genotype. Okay, So basically from the experiments I've shown you, it shows that when you silence these neurons, you find that IR 60 b neuron is necessary for normal levels of feeding. And we learn from the activation experiments that this IR 60 b neuron is sufficient to reduce levels of feed into a nutritious sugar. Okay, so this basically have to say it was really surprising. We've been expecting exactly the opposite. It shows that this neuron is inhibiting adjust ingestion of the soup of the sugar, which is actually good for the fly. It's nutritious, you know. So that was very surprising to us. Um and next question was Well, this is a neuron which I showed earlier expressions not only ir 60 b, but two other receptors as well. Op priority. This effectively mediated through any or none of these three receptors. So to get the molecular basis of this, we made a crisper mediated deletion of ir 60 B back crossed it and then tested to see whether the mutant had the same phenotype. And sure enough, it did. If you mutate this IR 60 b gene, you get an increase compared to parental controls, we also get a hetero Zygo. And this was true across a broad range of sugar concentrations. All right, so we also wanted to check this was we increased the feeding time. But we also just wanted to make sure this was also increasing the amount of ingestion. So we used this measure when you measure how much blues actually on the fly. And again we found that the ingested volume was greater when we deleted the IR 60b gene. Okay, so the interpretation of this, then, is that this particular receptor is acting to limit sucrose. We looked at specificity. We found them way looked at a bunch of different receptors at higher 100mM sugar concentration. I'm sorry we looked a bunch of sugars, at 100mM and Only sucrose gave this effect larger on intake. Same at 300mM almost Onley sucrose when we went up to the very highest concentrations of you go up to 900mM Glucose. Then you can start to see a bit of a phenotype for glucose. Remember, Glucose is a breakdown product of sucrose. That could explain why and we looked at a whole bunch of other stimulate a bunch of bitter compounds solves different pH is amino acids. And in no case, was there a phenotype with IR 60b solution All right, so what All these What I've shown you now is behavioral tests, and it suggests that the 60 b neuron is a sucrose sensor that depends on the IR 60 b. gene But we wanted to confirm that directly. And to do that, we return to calcium in imaging. And then this took a long time to develop. But Ryan was successful in developing a lovely calcium imaging paradigm where he had actually image the cell bodies directly in the pharynx. So it takes a fly head and puts it on a microscope slide. And then he's able to diffuse in some sucrose solution. And then we can image from a microscope above activation of reason. Calcium sensitive reporter of the cell bodies directly in the pharynx wanted to look at this as directly as possible. And when we do this, when we give a some 500mM sucrose, we get a big calcium signal is a control. If we just use water, we don't get such a signal anywhere near that day. So the evidence then shows that this is not just a mechanical signal, that it's actually a response to the sucrose. And here you can see the dynamics, which is interesting. It actually is pretty slow. So you know, it takes a while to get the sucrose to, profuse into the system and reach the cells and the pharynx But once it gets in, it takes about 30 seconds to reach peak amplitude, and then it declines. Okay, that's slow, but there's actually some precedent for that from fringe ill neurons that have been looked at in the larva. Ther drosopholia larva. Okay, so we then looked at specificity again and found that no other stimulus elicited this kind of calcium signal except the highest concentration of glucose. Sucrose gives the best again. And then the highest concentration of glucose, we could get some. This is just quantitative. The same data, right? And then, lastly, is this dependent on the IR 60 b receptor? And the answer is yes. If you knock out the IR 60 b receptor, you lose this calcium signal. All right, So the interpretation of all this, then, is that sucrose is actually acting via the 60 b receptor. Its activating a neuron that inhibits feeding. Okay, so all these experiments we've done so far event in this poor, hapless flight is a mobilized in some horrible way or has been cut off. You know, way thought it was important to actually see if 60 b is playing this role in a healthy, happy fly that's running around so For that, we used a fly liquid food interaction counter, also known as the flic system, and this was developed by a couple other labss a few years ago. So in this system, there's some liquid food in this little well sucrose In our case for the fly to reach this liquid food. It has to stand on aluminum ring, which functions is an electrode, and there's an elected. There's also a second electrode at the bottom of this, also an aluminum plate. And when the fly comes over and makes contact with this food that closes, an electrical circuit generates an electrical signal, which is easy to quantitative account, and other people have shown that thes electrical signals actually turned out to be a pretty good measure of ingestion. So there's a lot of these signals. You get a lot of ingestion. Okay, so the interesting thing is that sure enough, in the mutant on the fly is spending more time in contact with sucrose solution. But if you look at the number of feeding bouts, so these flies, you know, they feed for a while and they go away and then they come back and they go away and they come back. The total number of bouts is actually very similar. There's no phenotype here, but the length of time for an individual bout is longer in this mutant, just to show you some raw data to get this idea across. Oh, I should just mentioned we did all these limited the duration of the experiment 15 minutes to make sure we didn't get post ingested effects. So we don't have to worry about what happens after sucrose and enters the system and starts having more central effects. Okay, so here's some of these bouts and you can see that in the mutant. They're just playing longer in the mutant than they are in the wild type. So what we think is going on is that this receptor is actually limiting the length of feeding bouts. You get the same number about when this receptors active. It keeps them shorter. The fly starts eating but stops faster. Okay, so just putting all this together, we think then that this this are 60 b receptor and the neuron that expresses it. Are new elements in the, uh, in the circuit logic of feeding regulation. So it's been known for 50 years that insects there are neurons that are sensitive to sugar but which activate feeding circuits. They promote feeding. All right. And I actually mentioned there was one of these in the pharynx that expressed gr genes, which seems to promote feeding. Okay, but what we think now is that 60 b is a receptor in this new kind of neuron that actually has the opposite effect. It inhibits feeding. We don't think it works by itself. We think it acts as a brake on, uh, this feeding promotion circuit. And one interesting thing is, remember, the dynamics of this was very, very slow. So one interesting model is that the sucrose activates first, this positive circuit And kinetics on that based on work from other labs, seems to be pretty fast. But our neuron here actually has pretty slow kinetics. So perhaps the sucrose first starts to activate this and you get a feeding bout. But then 30 seconds later or whatever this a some point, this neuron then gets kicked in and shuts this down, terminating that feeding bout earlier. Okay, so in an IR 60 b mutant, you lose this inhibitory circuit, and so you get over consumption. So just a couple of key points about this circuits. It's interesting that sucrose actually has two different valence is the same molecule, the same sucrose molecule as a positive valence. For some neurons like this one that has a negative violence via the other neuron are neuron. Another important Interesting point is that this is a peripheral mechanism. I'm sure you're all aware of central mechanism. You have some big ice cream cone or something on. Then 20 minutes later, it goes up through your blood brain barrier gets into your brain, and then the other mechanisms which make you want to stop eating. All right. But this is a peripheral one, right out in, out in the taste organ itself. And that actually kind of makes sense because it acts much faster. You don't have to wait for the get go down your gut and then your blood up through the blood brain barrier and your brains acting much faster, which makes it efficient . What this would be able to do is to moderate sucrose, intake and help maintain homeostasis All right. And so the bottom line here, that is, we think there's both dual. There's a dual negative and positive regulation of feeding, and by having two different arms to this pathway, we think that sort of enhances the ability to modulate feeding. Okay, So if you wanna have a precise fine tuning of feeding, you can now have on factors which modulate either the positive arm or the negative arms. So, for instance, internal state if you're hungry or or or full, you can regulate feeding, either by virtue of modulating this positive circuit or the negative circuit. And there's already some good evidence from a couple other labs for mechanisms by which the internal state can regulate this positive circuit. But now we are interested in the possibility of modulation of this inhibitory circuits as well. Good. So just to summarize this first part of my talk, I mentioned that there's this huge clayde of IR genes that we know virtually nothing about, and I showed you one of them is expressed with great specificity in a single pair of these fringe all neurons. I showed you that if you inhibit these neurons, there's an increase in feeding on sucrose. If you optigenetically, activate these neurons, you get a decrease are I showed that these neurons act as sucrose sensors that are actually dependent on this one particular receptor, and that in free moving flies that the these neurons several role in terminating the length of these feeding bouts. So we think this is a new mechanism then plays an important role in homeostasis and regulating the feeding of the fly. All right, so just want to thank Ryan Joseph Postdoc, who did the great majority of this work with some help from Jennifer Sun, who's just started a postdoc in Princeton right now. And Endric Tam It was a visiting student in our lab. We did some of the coding we needed to analyze the data. Great. So that's the first story I wanted to tell. Let's take a very quick thank terrific. And in the second part of my talk, I'd like to tell you about another very important biological problem, a very classic biological problem that underlies all kinds of biology. Which is the question of how an animal finds a mate. How do you find a suitable mating partner of the same species? Now, if you look at this drosophila melanogaster male, right, it needs to find a mate. These two flies look very similar to all of us in this room, but they're actually different species. This is melanogaster This is another species. So this fly has to be able to tell the difference between these two species. How does it do that? All right, well, we're very interested to find that three of the i r genes in this clayde actually expressed in the leg sensilla on the legs. and We had done previously a very extensive analysis of all these sensilla on the legs with a whole variety taste compounds, and most of them respond to bitter compounds. Yesterday, many respond to sugars, but there were a few really mysterious ones that didn't respond to anything we try and also these mysterious. Sensilla didn't express any of those gr genes which all the other sensilla responses, we didn't know what those sensilla work. They are actually located on the dorsal surface of the leg, which actually doesn't make all that much contact with fly food. But I actually borrowed a camera from my friend Rick prom. Have any of you guys read his books on birds? He's in the EEB department at Yale. I should got the short listed for the Pulitzer Prizes here for Bird. You know, he just did a lot of work on hummingbirds. He's a fantastic guy. So we borrow this incredibly high speed camera we could actually see the contact that a male fly makes with a female through this for life. Okay, All right. So we made an antibody against one of these 3 IR genes are 52 a, c and D, and we found that two of them c and d were actually sexually dime or fic in their expression. A lot of expression in the mail and little if any in the female. These IR 52 C and D genes are expressed specifically in the fore legs, not in the middle leg of the hind leg. And it's a four legs that the male makes to make contact with the abdomen of female. It's part of the courtship ritual that it goes through. All three of these shows sexually dimorphism Um, in their expression, these are the gal four lines this IR 52 a gene So this is the left half of the C. N s and the right half. There's actually this calm assure that you see in the mail that you never see in the female. If you just measure the total amount of labeling for 52 C and 52 D is much higher in the male than female. So the sexual dimorphism, um all right. And also, if you actually trace the projections of these neurons into the brain, you find that the projections are different for these genes. Here, so are 52 a in green from those that carry information about bitter or sugar. So they have different projections and are likely to form synapses different of post synaptic partners and activate different seats. Circuits. Okay, so taking all this together, you start to wonder whether maybe these genes have something to do with sex and consistent with that. Remember, we talked just a minute ago about how ir 60 b was the Only one of these genes that have a negative direction of selections under a whole lot of pressure to keep its amino acid sequence. Well, 52 C and 52 d are two of a very small number, which is the opposite. They have these very high directions of selections. Is if they are plane is there under pressure to change the amino acid sequence, and this is characteristic of genes that are involved in sex. Uh, there's genes that are involved in reproductive isolation that also have high directions of selection. So that also is evidence that these might be playing a role in, perhaps in speciation. So 52 c and 52 d are right next to each other in the genome. They've got a pretty high level of sequence similarity on, and they are co expressed in the same neurons. So we made a deletion. We got rid of both of them and tested them to ask whether they had an effect on sexual behavior. And the way we did this is we took a little plastic, um, dish with a whole lot of wells and in each well, here we put a single male fly the bottom. There's another set of wells in which put a virgin female fly. They're separated by a sheet of plastic, and then we yank out the plastic male falls on top of the female, and we record what happens next with a video camera. Yeah, okay. And then when you do this, you can see that here's here's a male fly, a female, and what the male does is he extends a wing and moves around. The wing can issue a vibrate if your courtship song. And if the female is receptive, then you get copulation so we can quantitative these parameters. And when we did, we find we measured the percent of copulation as a function of time. We took either a wild type male or a mutant male with a wild type virgin female, and we measure how long it takes to get copulation And you see, there's a a delay. In the case of the mutant, it's actually pretty big delay. If you look how long it takes for 50% of the males to copulate, it takes twice as long. For in the case of the mutant, we showed that we could rescue this phenotype by supplying a wild type copy of either IR 52 c or 52 d suggesting they may be functionally redundant. We also measured the time that it takes before the mail starts initiating this wing extension, and that also is delayed mutant. So next we use the NFAT system. I don't know if you are familiar with NFAT systems, some of you are probably s O. This is a system for actually measuring the activation of individual neuron. It's nice because you could do it under you. Use it under natural circumstances supposed to under a microscope, and you could integrate the signal for a long period of time. So we did is we took this in NFAT protein affected, genetically engineered version of it, which was designed by Jing Wang ucsd, which has a lexa group on it. And so this binds calcium binds calcium. It moves into the nucleus of the cell. And because of this Lexa domain here it combined A LEXA and turn on G f p. So, in other words, the bottom line is, if this you express this in a neuron anarchist, some of these leg neurons expressing ir 52 c if the neuron gets excited. If it gets activated, this will bind and calcium move in and those neurons will will turn green. So we did that and first we put we tested a single solitary male all by himself in a vile and nothing happened. Then we added to the vile 10 virgin females from melangaster We got a big signal in these neurons. Next, Instead of adding virgin females to this vile, we added males. Nothing happened. But most interesting. We found that if we added virgin females of a sibling species of different species drosopholia, stimulants, nothing happened. So basically, these neurons appear to be detectors off for both, Uh, are detecting females, but specifically females of the same species. So we think they actually function is mate detectors. Okay. And just I guess I should have quantitative. Here's the quantification. It's actually pretty strong female. Yeah. Mhm. Okay, So we think then that ir 52 c and D could represent a completely new class of pheromone receptors that is acting in this process of species recognition. Jump in your video. Cross species interaction, male. Still mhm the female. Yeah, we haven't done that. Will be good to do yah. Haven't done that right. Good experiment. So at this point, the story took an interesting turn. All right, there's a grad student, a lab bob who was interested in the fly wing. All right, now along the edge of the wing, there's this whole row of chemo sensory. Sensilla, which are curved, alright. And remarkably little is known about what these do, Jen. A grad student, the lab Look at these under the microscope. They have this pore at the end. so they look a little bit like taste since l. A. And these have been speculated to be taste since Ella. But if you go through the literature, there's very little data to support that. All right, So what Bob did is he decided to do an RNA seek experiment. So he took advantage of a mutant called Pox Neuro where the's curves and still are displaying missing. So we handpicked 12,000 wings and did RNA. You know, he's a very patient guy Did RNA seek both from the wild type and this box neural mutant looked at 600 million reads He found a whole bunch of genes which expressed higher levels in the wild type that has these sensilla or these bristles and that are missing them in the mutant. Okay, so a lot of these genes and when you do one of these geneontology of analysis, you find that among the genes that are heavily enriched in the wild type, there's a lot of them that get this term of sensory perception of chemical stimulus or if you just look at all the genes in the fly genome. It's only like 2% of great enrichment, also, for terms like detection of chemical stimulus response to pheromone detection of pheromones. So it really made Sounds like these these sensilla on the wing could actually be playing a role in detecting pheromones. All right, And one of these genes in particular that was enriched was IR 52 a, which is right next to 52 c and d and has a lot of sequence similarity. So we were wondering, you know, why would you put pheromone receptors on the wing? It kind of makes sense to have them on the leg were because the leg touches the mhm, the female that makes sense to have them on the antenna because a lot of more volatile. But they're actually a lot of pheromones that are not volatile. And so we asked this actually work of a really clever rotation student. The lab Sweeting song was actually now a grad student. The lab and what she decided to do was to test this possibility that pheromones that big pheromones that aren't very volatile could be transferred from one fly to another fly to the wing of another fly, and the way she did that is she picked a large hydrophobic village you can actually see. So this is about the same molecular weight of one of these big fly pheromones. It's a very hydrophobic, not volatile. She picked one called Nile Red, and she basically impregnated a big filter disk with Nile red. Put it in the bottom of this chamber and let a fly. Wander around for half a now, er and the fly gets labeled. So whereas a fly that hasn't been to this chamber looks like this. Here you can see a whole lot of dye on the fly that's been wandering around the chamber so you can label them with this big hydrophobic molecules. And then you take one of these labeled flies and put it in a clean chamber with an unlabeled fly. And then he asked, Well, does any of this this fluorescent dye actually make it to the wing of the unlabeled fly? Yes, it does. Okay, so here you can see it's getting labeled. We tried to quantitative this, too. So the bottom line is that large hydrophobic molecules like pheromones can be transferred from females to the margin of the wing of males. And she also did the reciprocal showing that the these, uh, dyes can move from males, two females. So it kind of makes sense that at least it seems plausible. Then that pheromone could be transmitted from one fly to these things wing margin of another fly where they're all these of candidate pheromone receptors. All right, just to confirm just, you know, just wanna make sure that these things IR 52. Gene was really expressed out there. These Were the data from the RN any seek control of the missing? That doesn't have the sens silla We also did a whole lot of Q PCR just to show that in both males and females you confined ir 52 a out in the wing and Also we did. We found that that expression of ir 52 a on the wing has been conserved from millions and millions of years and fly evolution. You can find it out in the wing of drosophila Suzukii distantly related species. So there's IR 52 a in the wing. Um and we made a gal4 line for it and showed that it's expressed in the margin here and in high resolution, you can see that it's actually in neurons which innovate these sensilla you could thes um, they're dendrite go up into the sensilla a cell body and their Exxon's which go into the ventral nerve cords. The CNS here. So here, you can see them coming in from the wing. Also, this chain 52a is expressed in the fore legs, the mid legs and hind legs as well. All right. Okay, so we wanted to do some genetics to figure out what they're actually was playing a role in sexual behavior. So again, we used well, So this 52a gene is very near c and d the ones I talked about earlier on the high degree of sequence identity. Um and so we used CRISPR to make a deletion in of it, which we back cross five generations on, then tested in this sexual behavior. Awesome. So here we're measuring the percentage of population that you get when you take a mutant male and pair him with a wild type virgin female as a function of time. Here's the copulation you get with the control fly and each of three lines of this crisper mutants that was greatly greatly reduced. So this is copulation and We also saw the same with wing extension in two of the three lines we saw significantly significant decrease in on the extent of wing extension in males that are missing IR 52 A. Okay, so this was um, showing the loss of function, confer to Phenotype. But we want to make sure we could rescue that. So we express 52 a on found that we got a great elevation in both copulation and wing extension so that is deletion of the gene. We also wanted to silence the neurons for a couple reasons. So we used opt in genetics. We put in a, um G t a c r which then you shine red light and you silence the genes on and the neuron so sorry and so copulation was also greatly suppressed when we silence the neurons in males also wing extension. So this is important to do to show that this effect is actually physiological one and not a developmental one. So you could just turn on the light and see in effect right away it works with a deletion There's always the possibility there could be some sort of developmental effect So this shows its physiological. All right. Okay. Now the most of the stuff that really surprised us the most. So we showed them that these gene and the neuron were required for sexual behavior. We then wanted to see what happened when we activated these neurons again with a different kind of optic genetics. So we took a kind of a a scenario where there's very, very low levels of off male sexual behavior. We put two males together, which do show some courtship a very, very little. But we put two males together, all right, and these are the controls and then activated these IR 52 neurons. We got this dramatic increase in sexual behavior, okay? And we also did this something similar. We took a milanagaster male and put him together with Drosophila Suzukii female. Usually they show no sexual behavior whatsoever, But when we blast these neurons with light to activate them again, there's this huge increase in the and sexual behavior. So basically, what this is saying is that activation of these IR 52A neurons can drive a male over the species barrier, which really surprised us because when millions and millions of years to build up these species barriers, this will will override it. Right? So this Oh yeah, just just interesting control expert. We wonder what happens if there isn't any drososphila. Suzukii female around. What if you just take a single male sitting right here? You blast it with light? Is male gonna start joining sexual behavior by itself in the answer that was No. So we think that this male needs in addition to these probably some other kind of input. Either olfactory input or some kind of visual. All right, good. Okay, So all these data have been showing so far have been in males. How about females? Alright, because ir 52 a unlike CMD, is actually expressed in both males and females. So we found that these deletion mutants and made females much, much less receptive to copulation, so it decreases their sexual behavior. Alright, in all three lines that we tested and I have to say this is really unusual. There's very, very few receptors which are known to play a role in both male and female sexual behavior and We also showed this opted genetically. If you silence these neurons, you again get a decrease in female sexual behavior. So then we asked another question. This is something I never would have thought. Would have worked So when a female mates okay, she then becomes very, very un receptive to future mating Okay, she loses interest in other males. If a male comes along and tries to court her she kicks him and runs away. All right, so this is a post mating affair and it's interesting. It's been shown to be mediated in large part by factors that a male transfers to the female in semen. So the male mates with the female transfers a bunch of factors which suppress for interest in future, mating all kinds of interesting evolutionary reasons Why that happens. And a lot of the biochemistry that explains has been worked out by Mariana Wolf there and other people. Okay, so basically, there's this post mating suppression you see in females. So it is very, very strong. But we found that if we activate these ir 52 a neuron in post mated females Okay, we get this huge increase in activity. So now she will allow mating Okay, So somehow activation of IR 52 a is ableto override this circuit, which normally inhibits meeting, which really, really surprised us. Okay, so we think that then ir 52 is detecting a pheremone signal from males that's gonna make that normally makes females receptive, but it's suppressed by mating Okay, So what happens to all these signals? Where do they go? How does all this work? What's the circuitry involved? So for that, we took advantage of this new technique called Trans Tango. Okay, you read the paper on this this technique and it sounds really complicated. I read it and didn't think it was gonna work. It was gonna take ages to get it to work. But my grad student each end wanted to try it. You tried it? I worked first time perfectly right away. So I have to give a lot of credit. Philip Barnea Brown, for working this out. So what you do basically just over way over simplify. This is you can drive it and neuron not only gal four, but also a Liggan which sits in the membrane of the neuron you're interested in. And then