WEBVTT 00:00:03.537 --> 00:00:10.497 This is the Convergent Science Network podcast. Leading researchers in the domain 00:00:10.497 --> 00:00:16.777 of neuroscience, brain theory and technology are interviewed by Paul Verschure and Tony Prescott. 00:00:26.537 --> 00:00:32.097 This is Paul Verschure with the Convergent Science Network. I'm here with Peter Mombarts. 00:00:33.357 --> 00:00:38.637 And Peter is a neuroscientist who has been specialized in the development of 00:00:38.637 --> 00:00:40.797 the nervous system, the wiring of nervous systems. 00:00:40.817 --> 00:00:48.237 And Peter, you were telling us this morning that you chose the olfactory system 00:00:48.237 --> 00:00:52.197 of the mouse as your target system. So why is that? 00:00:53.817 --> 00:00:59.077 At heart, I'm a geneticist, molecular biologist. And when these auto-receptor 00:00:59.077 --> 00:01:00.497 genes were discovered in 1991, 00:01:00.917 --> 00:01:08.517 I felt that there was a great way of approaching the nervous system, 00:01:08.577 --> 00:01:13.957 particularly development, in a genetic and molecular way because that's what I'm at heart. 00:01:13.957 --> 00:01:21.077 And indeed, with time, we have learned that using these organ receptor genes, 00:01:21.237 --> 00:01:23.957 of which there are a very large number in the mouse, 1,200, 00:01:24.197 --> 00:01:30.657 we can differentially stain and manipulate populations of olfactory neurons, 00:01:30.877 --> 00:01:32.397 each expressing one of these receptors. 00:01:32.537 --> 00:01:36.637 And that's a very unique experimental advantage that the olfactory system of 00:01:36.637 --> 00:01:38.597 the mouse offers at this time. Right. 00:01:38.737 --> 00:01:43.317 So that was really a big discovery in the early 90s, right? That the idea that 00:01:43.317 --> 00:01:48.577 actually individual receptor neurons would be, if you want, tagged by single 00:01:48.577 --> 00:01:51.497 genes or would express single genes. 00:01:52.317 --> 00:01:58.897 Yes, the history of research in olfaction, in my view, can be divided in a pre- 00:01:58.897 --> 00:02:04.437 and post-1991 era, with the paper of Linda Bock and Richard Axel, 00:02:04.557 --> 00:02:07.297 published in April 1991. 00:02:07.297 --> 00:02:12.057 One, and it was the discovery of these auto-receptor genes that really made 00:02:12.057 --> 00:02:18.657 a proper experimental investigation of this system possible. 00:02:18.797 --> 00:02:22.957 At that time, it was not known that there would be only one gene expressed per neuron. 00:02:23.437 --> 00:02:27.997 It looked like that from the beginning, that a small number of genes would be 00:02:27.997 --> 00:02:31.977 expressed by a given cell, and more and more the evidence is consistent, 00:02:32.077 --> 00:02:36.557 perhaps asymptotically, with this one neuron, one gene rule. 00:02:37.577 --> 00:02:43.897 So that in some sense, now given that you were exposed to this discovery, 00:02:44.257 --> 00:02:49.517 you saw this as an opportunity to now have a specific preparation to understand 00:02:49.517 --> 00:02:51.457 how this system might wire itself. 00:02:52.531 --> 00:02:55.351 But why in particular an olfaction? You could also have gone for, 00:02:55.391 --> 00:02:59.611 let's say, any other system where you might have some genetic label to look at wiring. 00:02:59.791 --> 00:03:04.031 So what makes the olfactory system so appropriately sculpted for that? 00:03:04.371 --> 00:03:06.791 Well, not all decisions made by scientists are rational. 00:03:07.571 --> 00:03:13.331 There's also something called gut feeling. And back then I felt this was a wonderful new field. 00:03:13.451 --> 00:03:15.931 Also, it was new, right? It was a new approach. 00:03:16.731 --> 00:03:20.531 And I haven't regretted since then. There may be more interesting systems for 00:03:20.531 --> 00:03:25.611 other questions. But the questions I started to get interested in about 20 years 00:03:25.611 --> 00:03:28.491 ago are still the questions I'm 00:03:28.491 --> 00:03:31.171 interested in now that I think about every morning when I take a shower. 00:03:31.311 --> 00:03:34.131 And as long as that's the case, I think I will continue to work on it. 00:03:35.371 --> 00:03:40.691 So now, what's the – can you give – so this morning, you spent quite some time 00:03:40.691 --> 00:03:45.711 to sketch out for us the basic structure of this mouse olfactory system. 00:03:45.951 --> 00:03:49.471 So before we start looking at the details of your discoveries, 00:03:49.711 --> 00:03:54.351 what are the key elements of this olfactory system that we should keep in mind? 00:03:55.451 --> 00:04:01.651 So anatomically, a molecular, there is a division in different chemosensory 00:04:01.651 --> 00:04:03.911 structures in the nasal cavity, in the nose. 00:04:04.231 --> 00:04:10.191 You have the main olfactory epithelium, which contains several million olfactory sensory neurons. 00:04:10.311 --> 00:04:12.971 We don't really know the number, by the way, but it's several million, 00:04:13.131 --> 00:04:19.791 each expressing expressing one of these 1,200 auto-receptor genes that Bocanaxo described. 00:04:20.231 --> 00:04:26.131 Then you have the septal organ, a specialized structure, which contains approximately 10,000, 00:04:26.891 --> 00:04:30.291 olfactory neurons, and half of these express the same receptor called SR1, 00:04:30.531 --> 00:04:37.551 which is a rule breaker as these neurons are activated by a very wide variety of chemicals. 00:04:37.551 --> 00:04:43.231 We have the vomeronasal system, abbreviated VNO, vomeronasal organ, 00:04:43.431 --> 00:04:45.951 which doesn't express these auto-receptor 00:04:45.951 --> 00:04:49.851 genes, but two other families of G-protein-coupled receptor genes, 00:04:50.171 --> 00:04:55.211 V1R genes and V2R genes, V-vomeronasal, approximately 300 in total. 00:04:55.631 --> 00:05:00.591 And then finally, a very small chemosensory structure at the tip of the nose 00:05:00.591 --> 00:05:05.491 called the The Grüneberg ganglion, abbreviated GG, maybe 500,000 cells, 00:05:05.711 --> 00:05:10.271 that's perhaps mostly active in neonatal newborn mice. 00:05:11.671 --> 00:05:15.671 So there's already quite some complexity of chemosensory anatomical structures 00:05:15.671 --> 00:05:20.011 in the nose and the corresponding repertoires of chemosensory receptor genes. 00:05:20.971 --> 00:05:27.151 Not surprisingly, as mice are highly chemosensory organisms, 00:05:27.531 --> 00:05:31.531 they rely less on their sense of vision as we do, for instance. 00:05:33.410 --> 00:05:38.630 These neurons are all projecting one axon, one nerve ending, 00:05:38.810 --> 00:05:43.670 to the brain, to the main olfactory bulb or the accessory olfactory bulb. 00:05:44.130 --> 00:05:52.510 And in the bulb, each axon terminates only in one of the so-called glomeli, 00:05:52.750 --> 00:05:55.990 of which are about 3,600 in the mouse. 00:05:56.170 --> 00:06:00.030 So for the main olfactory system, which detects what we call garden-variety 00:06:00.030 --> 00:06:05.710 type of odorants, The picture is as follows. We have 1,200 genes. 00:06:06.650 --> 00:06:11.630 Most of these are expressed in olfactory neurons, but only one gene per cell, per neuron. 00:06:12.310 --> 00:06:17.530 Each neuron has one axon, and it enters one glomulus in the bulb, 00:06:17.590 --> 00:06:21.110 where it then synapses with second-order neurons. 00:06:21.570 --> 00:06:26.250 Okay. So now we have this structure, which is fairly layered. 00:06:26.250 --> 00:06:32.570 And so it seems to have it must have some tight control over how it is this 00:06:32.570 --> 00:06:37.730 is wired up and I think an added complexity of the system is that actually there's 00:06:37.730 --> 00:06:43.190 a continuous turnover of these receptor neurons so now the real problem becomes okay, 00:06:44.070 --> 00:06:48.790 this layer where it hits the olfactory bulb these nerve endings form then these 00:06:48.790 --> 00:06:56.070 glomeruli and how is the specificity of the organization of this glomeruli now assured. 00:06:59.110 --> 00:07:02.210 So during development, and quite quickly during development, 00:07:02.390 --> 00:07:07.090 I would say the first few days after birth, this glomerular array matures, 00:07:08.510 --> 00:07:14.250 finally forming these approximately 3,600 glomeruli, and the neurons that express 00:07:14.250 --> 00:07:19.110 the same receptor project their axons to the same glomeruli. 00:07:19.210 --> 00:07:22.010 In fact, they form these glomeruli. It's not that the glomeruli are there and 00:07:22.010 --> 00:07:23.830 they just have to project their accents to them. 00:07:25.230 --> 00:07:31.350 And this glomalor array, or glomalor map if you wish, develops quite reproducibly, 00:07:31.390 --> 00:07:34.750 left and right bollop in the same individual and from mouse to mouse. 00:07:35.610 --> 00:07:40.370 It's not so reproducible that it's stereotyped, we cannot draw a map with coordinates 00:07:40.370 --> 00:07:43.150 on the bollop and say this is this glomalus for this receptor, 00:07:43.310 --> 00:07:46.970 but it's very reproducible, or recognizable. 00:07:49.516 --> 00:07:55.176 What that means for the function of function for decoding is not clear, 00:07:55.296 --> 00:07:59.056 but there is clearly a spatial map that develops quite early, 00:07:59.176 --> 00:08:01.496 very precisely in the mouse. 00:08:02.636 --> 00:08:07.996 And also you then went to describe how this kind of labeling could occur. 00:08:09.116 --> 00:08:12.896 So there was this idea of zones, right? Maybe there are zones that sort of define 00:08:12.896 --> 00:08:19.496 a sort of a rough kind of chemotopic map in which you can then place your glomeruli. In the olfactory bulb. 00:08:19.596 --> 00:08:22.156 In the olfactory bulb, yes. Do you think that's still a plausible scheme? 00:08:22.556 --> 00:08:26.616 So there have been several attempts and several papers over the years in different 00:08:26.616 --> 00:08:33.416 model systems to ask with various electrophysiological or other methods, 00:08:34.256 --> 00:08:39.796 are there particular types of chemicals that certain regions of the bulb are 00:08:39.796 --> 00:08:41.496 more sensitive to than others? 00:08:42.496 --> 00:08:46.456 That's called chemotopy. entropy so there would be a chemical structure somehow 00:08:46.456 --> 00:08:50.796 to the glomerular array we don't know of course a priori what are the valid 00:08:50.796 --> 00:08:54.776 features that would be extracted is it chain length. 00:08:56.676 --> 00:08:59.576 Saturated unsaturated bonds molecular weight and so on we don't really know 00:08:59.576 --> 00:09:03.816 this in advance but have been attempts and there is some evidence that finds 00:09:03.816 --> 00:09:07.416 that and some other evidence that doesn't find it to some extent one can find 00:09:07.416 --> 00:09:12.016 what one wants to find right and no but wait But there's an important thing you said earlier. 00:09:12.096 --> 00:09:17.756 You said, okay, across individual mice, you find a rough chemotopic map. 00:09:18.456 --> 00:09:22.916 There's just some jitter with this glomerular, how it's placed in that, if you want, matrix. 00:09:23.256 --> 00:09:28.836 Yeah. But that would suggest that there is some sort of zoning, 00:09:28.896 --> 00:09:32.256 if you want, of this bulb in development. 00:09:33.336 --> 00:09:37.116 So the chemical logic of the glomerular array is not overwhelming. 00:09:39.416 --> 00:09:43.336 It's not the case that each part of the bulb is equally sensitive to all odorants, 00:09:43.416 --> 00:09:44.636 right? That would be the other extreme. 00:09:44.776 --> 00:09:48.716 Yes, there is some kind of regionalization, but it's not overwhelming and we 00:09:48.716 --> 00:09:51.676 don't really also know in the end what that means functionally, 00:09:51.716 --> 00:09:55.116 you know, if it's a byproduct of it doesn't make sense. 00:09:55.496 --> 00:09:58.916 No, let's do a thought experiment. I mean, if we take a mouse, 00:09:58.956 --> 00:10:01.376 we present it with lemon. 00:10:01.376 --> 00:10:05.836 Lemon, now we see some glomerulus light up in the olfactory bulb, 00:10:06.056 --> 00:10:11.036 and I ask you, go show, go find that same glomerulus now in another mouse. 00:10:11.596 --> 00:10:16.356 Then you would take the location of this identified glomerulus to start your search. 00:10:17.776 --> 00:10:22.916 What's the probability distribution around that point in space to find the glomerulus 00:10:22.916 --> 00:10:26.676 that will also respond to this lemon odor? 00:10:29.297 --> 00:10:32.877 There have been a few studies looking at that anatomically, and the level of 00:10:32.877 --> 00:10:37.557 uncertainty, if you wish, of variability is maybe in the order of 1.52%. 00:10:38.137 --> 00:10:41.877 So even between the left and the right ballop, that's probably a better way of looking at it. 00:10:41.957 --> 00:10:44.937 If you know the positions of glomeruli on the left ballop, you can't really, 00:10:45.097 --> 00:10:50.257 to the level that we would like to, find the positions of the glomeruli on the 00:10:50.257 --> 00:10:52.257 other ballop. We cannot make an atlas, in other words. 00:10:52.717 --> 00:10:57.477 Unlike, for instance, in the honeybee or the drosophila, where people have been 00:10:57.477 --> 00:11:01.657 able to make an atlas and say precisely this glomulus and this position with 00:11:01.657 --> 00:11:05.857 this shape, volume, and so on, that is that glomulus, and they give it a name. 00:11:05.977 --> 00:11:08.517 That we cannot do with the mouse or the rat thus far. 00:11:10.657 --> 00:11:15.017 But implicitly, I have the feeling you're saying that you believe that there's 00:11:15.017 --> 00:11:16.057 not a strong structuring. 00:11:16.777 --> 00:11:19.937 But in some sense, if we would try to do this more quantitatively and say, 00:11:20.017 --> 00:11:24.997 okay, if I have X, Y, Z of an identified glomerulus, now I need X, 00:11:25.017 --> 00:11:30.457 Y, Z plus some delta to find in another animal with probability 90%, let's say. 00:11:30.697 --> 00:11:33.717 Yeah, there's certainly a… How big is that delta? Yeah, there's certainly a probability. 00:11:33.917 --> 00:11:38.177 So I think the best way of visualizing this variability. 00:11:39.217 --> 00:11:44.417 Is to look at two glomeruli at the same time, for instance, of two strains of 00:11:44.417 --> 00:11:47.937 mice with a different marker, maybe one like Z, the other one GFP, 00:11:48.157 --> 00:11:54.497 cross them together, And then glomalus A and B would be on the left side, for instance, 00:11:54.737 --> 00:11:59.997 A more lateral than B, and on the right side, it may be the other way around. 00:12:00.617 --> 00:12:05.017 And this is more than whole distortions of the map. 00:12:05.097 --> 00:12:08.377 You could think perhaps that the bulb would be a little bit squeezed and things 00:12:08.377 --> 00:12:09.917 would be a little bit more anterior, posterior. 00:12:09.917 --> 00:12:14.657 Here, but if you have internal rearrangements, medial-lateral relative positioning 00:12:14.657 --> 00:12:18.317 that is inverted, then that clearly shows there is an underlying variability, 00:12:18.677 --> 00:12:22.557 which we have to acknowledge, unfortunately, to the point that we cannot make, 00:12:22.697 --> 00:12:27.557 even in inbred mice, a real atlas. 00:12:27.717 --> 00:12:31.297 And one actually may then also wonder if there is really a glomelon map. 00:12:31.457 --> 00:12:33.157 That word gets used a little bit loosely. 00:12:34.177 --> 00:12:38.217 Depends what kind of resolution and detail you want, but one can actually wonder, 00:12:38.277 --> 00:12:40.557 is Is that really a glomerular map for a species? 00:12:40.977 --> 00:12:46.397 But now if I would take my two mice again and we have our identified glomerulus, 00:12:47.397 --> 00:12:52.537 how big is the probability that I would find the matching glomerulus in another 00:12:52.537 --> 00:12:55.777 animal in exactly the opposite position? Yeah. 00:12:57.015 --> 00:13:01.795 Yeah, that I don't know. Maybe we have only emphasized the cases where there is a mismatch. Okay. 00:13:01.975 --> 00:13:06.815 But if you look more like the circle or the area, I would say about 20 or 30 00:13:06.815 --> 00:13:09.355 glomeruli for the two or three studies where this has been done. 00:13:09.755 --> 00:13:13.975 So 20 or 30 glomeruli out of 3,600 is quite small, actually, 00:13:14.075 --> 00:13:14.955 if you wish. That's what I'm saying. 00:13:15.115 --> 00:13:20.515 Yeah. So you could imagine that you have a rough patterning with a resolution of 10 to 20 glomeruli. 00:13:20.775 --> 00:13:24.755 And that's still, what, 10% of your total structure? 00:13:25.875 --> 00:13:31.355 1%. Oh, 1%, exactly 1%. And then within that, okay, you have some jitter. 00:13:31.475 --> 00:13:37.655 But this is actually pretty precise, I would say, as sort of a non-expert in this domain. 00:13:37.775 --> 00:13:40.855 Well, from a practical point of view, it's not precise enough for us to make 00:13:40.855 --> 00:13:42.755 some kind of an atlas, right? 00:13:42.875 --> 00:13:47.735 Which we thought a few years ago we could do. We could make a probabilistic atlas. Exactly right. 00:13:48.015 --> 00:13:55.915 But maybe we, in Drosophila again, although the number of glomeli and receptors is much lower, 00:13:56.275 --> 00:14:00.215 there you can there it's really been possible to do so yeah but the interesting 00:14:00.215 --> 00:14:04.235 thing is of course you apparently were expecting to find some precision in this 00:14:04.235 --> 00:14:08.655 atlas that was not satisfied no i i wasn't but but others were and they and 00:14:08.655 --> 00:14:11.115 they use terms even now still in print of, 00:14:11.715 --> 00:14:16.855 stereotyped which from the old french type i think the stereotype where you 00:14:16.855 --> 00:14:19.675 really make a photocopy right you have some kind of a template and you print 00:14:19.675 --> 00:14:23.715 an exact copy with maybe some little bit of dirt or so but it's basically an 00:14:23.715 --> 00:14:26.775 exact copy and that's not really the case and i wouldn't use that term either. 00:14:26.995 --> 00:14:30.135 What we like to say after thinking about this for quite a while, 00:14:30.915 --> 00:14:33.095 recognizable or reproducible, something like that. 00:14:33.175 --> 00:14:37.255 It's not stereotyped. You cannot even from the position of the glomalin, 00:14:37.355 --> 00:14:39.015 the left ballop predicts. 00:14:39.875 --> 00:14:42.775 With high degree of certainty, those in the right bulb. Okay. 00:14:43.035 --> 00:14:47.775 But then would you still go along with the idea that it's a rough patterning 00:14:47.775 --> 00:14:53.075 and then through some self-organizing process, this gets filled in and out of 00:14:53.075 --> 00:14:55.035 result of the self-organization of the variability? 00:14:55.535 --> 00:14:59.415 Or would you say at this stage, look, it's better not to commit ourselves too 00:14:59.415 --> 00:15:03.355 strongly to the idea of a map because it's too ambiguous at this stage? 00:15:03.355 --> 00:15:09.895 Yeah, and it can also be misleading to think in terms of mechanisms, right? 00:15:09.975 --> 00:15:14.155 If you really believe, I think some people still believe that this map is extremely 00:15:14.155 --> 00:15:21.855 precise and is in variable positions, then the mechanisms that enable to produce 00:15:21.855 --> 00:15:24.455 that must be extremely complicated, right? 00:15:24.455 --> 00:15:28.835 But if you acknowledge that there is indeed a bit of jitter or variability or 00:15:28.835 --> 00:15:34.175 uncertainty, then it relaxes a little bit the demands you would put on these mechanisms. 00:15:34.195 --> 00:15:38.995 So it's more than just being a little bit too strict or philosophical. 00:15:39.115 --> 00:15:44.655 We really want to know what kind of precision do we expect from the mechanisms 00:15:44.655 --> 00:15:50.115 that put that into place, which are largely unknown in my view today. Right. 00:15:50.595 --> 00:15:54.455 But now on top of that, it's not a case that we can think about this glomeruli 00:15:54.455 --> 00:16:00.255 as being really clean sphere type structures that are just neatly stacked together. 00:16:00.575 --> 00:16:03.795 You also have quite some variability in the shape of this glomeruli, 00:16:03.935 --> 00:16:06.355 so this cluster of nerve endings themselves. 00:16:06.855 --> 00:16:12.575 So what kind of variability do you see there? So yeah, glomalus is acellular. 00:16:12.655 --> 00:16:14.615 It doesn't have any cells in it, no nuclei. 00:16:14.795 --> 00:16:17.515 It's a few. 00:16:18.846 --> 00:16:22.586 Hundreds of incoming axons that terminally arborize and make synaptic contact 00:16:22.586 --> 00:16:25.266 with the dendrites of the second-order neurons. 00:16:25.406 --> 00:16:30.466 So this ball of synapses, that is a glomelus, which is a discrete structure 00:16:30.466 --> 00:16:34.466 surrounded by some glia and pyroglomelar neurons and so on. 00:16:35.246 --> 00:16:39.966 We would like them to be, of course, all perfect spheres, but that's indeed 00:16:39.966 --> 00:16:42.846 not the case. Some are a bit more oblongated. 00:16:43.886 --> 00:16:47.706 Some are in the form of a haltier with a central stalk and two balls hanging 00:16:47.706 --> 00:16:51.126 on it, which makes it a bit difficult sometimes to say is this one or two glomeruli. 00:16:52.666 --> 00:16:56.586 The average diameter would be about 55 micron in the Mars, but also there, 00:16:56.606 --> 00:16:58.866 there is quite some difference. 00:16:59.566 --> 00:17:04.406 So it's true, it's not 3,600 perfect spheres that are aligned. 00:17:04.586 --> 00:17:10.286 Moreover, there are different layers in the dorsal part of the bollop. 00:17:10.366 --> 00:17:13.886 It's typically only one glomerulus, but 00:17:13.886 --> 00:17:17.546 more ventrally in the ball up there you have multiple glomeruli on top of each 00:17:17.546 --> 00:17:22.046 other and deeper into the tissue and so on so there is an another another dimension 00:17:22.046 --> 00:17:29.486 there right so then in but whatever the the structuring that takes place to 00:17:29.486 --> 00:17:31.206 get the patterning of the glomeruli, 00:17:31.826 --> 00:17:37.286 there is a belief that that the the single genes expressed by the receptor neurons 00:17:37.286 --> 00:17:38.726 that in the end would also give 00:17:38.726 --> 00:17:42.866 identity to their exons, would in some way play a role in this process. 00:17:43.926 --> 00:17:48.606 Yes, that's been known for quite a while from genetic data. You can replace 00:17:48.606 --> 00:17:51.826 the coding region of a receptor by that of another one. 00:17:52.406 --> 00:17:56.646 In fact, we have even done in one case with an other G-protein copper receptor, 00:17:56.726 --> 00:18:00.706 the beta-2-anergic receptor, and you get a glomalus. 00:18:00.886 --> 00:18:03.406 Again, a reproducible, recognizable place. 00:18:04.066 --> 00:18:08.426 In some cases, it looks like it's a completely new glomalus that wasn't there 00:18:08.426 --> 00:18:11.986 before, but there is of course space if you have 3600 for a few more. 00:18:13.346 --> 00:18:19.346 You can also make smaller mutations down to point mutations in other receptors 00:18:19.346 --> 00:18:25.746 using gene targeting and you can create novel glomelar identity, 00:18:26.026 --> 00:18:28.206 so glomelar that probably are not there before. 00:18:28.946 --> 00:18:34.206 The receptor protein is also there along the axons all the way to the end and early in development. 00:18:36.320 --> 00:18:40.640 Suggesting that it has something to do mechanistically with fasciculation, 00:18:40.880 --> 00:18:41.920 coalescence, convergence. 00:18:42.660 --> 00:18:47.040 Exactly how that works at the molecular level is, I don't think, 00:18:47.040 --> 00:18:51.460 clear, or at least there is no consensus of how an old receptor could do that 00:18:51.460 --> 00:18:52.520 with such precision again. 00:18:53.400 --> 00:19:01.180 So then you spent quite some time then to try to show or identify the location 00:19:01.180 --> 00:19:06.540 of these different genes that the receptor neurons express in the genome. 00:19:07.640 --> 00:19:09.800 So what kind of pattern do you find there? 00:19:11.420 --> 00:19:15.220 The pattern I like to, the term I like to use is actually haphazard, 00:19:16.260 --> 00:19:21.380 which means maybe there is no clear pattern, but maybe it's not excluded that there is a pattern. 00:19:21.700 --> 00:19:26.480 There are in the mouse approximately 40 loci sites in the genome where you can 00:19:26.480 --> 00:19:27.580 find old receptor genes. 00:19:28.960 --> 00:19:34.880 Eight of them are solitary genes, which means megabases up and downstream, 00:19:35.080 --> 00:19:37.760 there is no other receptor gene in the genome. 00:19:38.200 --> 00:19:41.080 These are the exceptions. Most genes come in clusters. 00:19:41.480 --> 00:19:44.660 The largest cluster is approximately 300 auto-receptor genes, 00:19:44.860 --> 00:19:46.540 all stacked one next to the other. 00:19:46.760 --> 00:19:51.740 And the typical cluster has no other genes in them, non-auto-receptor genes, 00:19:51.880 --> 00:19:53.880 although of course there are also exceptions. 00:19:54.500 --> 00:20:00.520 So this pattern, this distribution is, I would like to say, haphazard. 00:20:00.520 --> 00:20:02.100 That there is no clear logic to it. 00:20:02.200 --> 00:20:06.300 It's not that every chromosome has one cluster or there are three huge ones 00:20:06.300 --> 00:20:10.440 or each cluster of genes is expressed in the same zone in the epithelium or 00:20:10.440 --> 00:20:13.500 encodes genes of the same family. Even that is not the case. 00:20:14.780 --> 00:20:19.060 For lack of a better term, and I welcome new terms, I'd like to call it haphazard. 00:20:19.720 --> 00:20:21.480 But how many genes do we have in the mouse? 00:20:23.069 --> 00:20:27.049 Genes with an intact open reading frame, approximately 1,200. 00:20:27.589 --> 00:20:31.869 In fact, the mouse genome is not completely sequenced. We check our favorite 00:20:31.869 --> 00:20:35.089 clusters once in a while, and the number of genes decreases, 00:20:35.089 --> 00:20:36.669 actually, from month to month. 00:20:36.749 --> 00:20:39.249 There are genes disappearing that are probably mistakes of assembly. 00:20:39.709 --> 00:20:44.289 That's a recent experience. So the jury is still out, but the number seems to 00:20:44.289 --> 00:20:46.589 converge to 1,200, maybe 1,100. 00:20:47.029 --> 00:20:48.129 But the total mouse genome? 00:20:49.349 --> 00:20:54.009 Probably 20,000, 25,000, so quite a significant fraction, yeah. Right, exactly. 00:20:54.949 --> 00:21:02.109 So here we have these 20,000 genes, but now of those 1,200 that relate to the 00:21:02.109 --> 00:21:06.089 receptor neurons, how much is the variability in base pairs of these genes? 00:21:07.429 --> 00:21:18.169 If you take two random odontoreceptor genes, the average is a nucleotide amino acid. 00:21:18.349 --> 00:21:22.669 I think amino acid homology was 33% or something like that. It's not very high. 00:21:23.609 --> 00:21:27.129 There are some motifs in the amino acid sequence. 00:21:27.949 --> 00:21:32.489 One of them is May-Dry-Vac. Another one is Fast-Cash. Easy to remember. 00:21:32.689 --> 00:21:36.749 Those are kind of typical for autumn receptors. 00:21:36.829 --> 00:21:40.889 If you see those two in a sequence, you have a very, very high likelihood that 00:21:40.889 --> 00:21:41.909 these are autumn receptors. 00:21:43.329 --> 00:21:46.829 So they come in families. You can define a family however you want. 00:21:47.269 --> 00:21:50.329 There have been, of course, debates about that. If it's 80% cut-off, 00:21:50.389 --> 00:21:55.589 60% cut-off. a nomenclature developed in 2002 by Stuart Firestone was based 00:21:55.589 --> 00:22:01.469 on these families and he was in the 200-something families of these 1,200 genes. Mm-hmm. 00:22:02.958 --> 00:22:07.658 So, but how long is the longest one you would find of all these 1,200 genes? 00:22:08.018 --> 00:22:13.818 They're all very similar in length. Approximately 1,000 nucleotides of about 330 amino acids. 00:22:14.038 --> 00:22:17.698 They have quite a short N-terminus and quite a short C-terminus. 00:22:17.798 --> 00:22:22.818 So, a big part of the protein is presumably in the membrane. Okay, good. 00:22:23.138 --> 00:22:28.838 So, as a family, as a total family, they're fairly uniform. So now we have this 00:22:28.838 --> 00:22:33.398 strange phenomenon that some seem tightly clustered on the genome and others 00:22:33.398 --> 00:22:37.718 are sort of singular, sitting out there somewhere in isolation. 00:22:38.958 --> 00:22:43.318 So what could be the consequence of grouping all these genes together? 00:22:43.478 --> 00:22:50.458 What could be a possible advantage in transcription or reproduction of a genome? 00:22:50.638 --> 00:22:55.078 Is there anything you can say about that? Well, typically gene families are clustered. 00:22:55.918 --> 00:23:01.538 That's the case in general genes that are related for which then multiple copies 00:23:01.538 --> 00:23:07.038 typically come in a cluster they could have evolved by unequal crossing over 00:23:07.038 --> 00:23:12.678 so by mistakes actually during meiosis we have an extra copy of the gene duplicated 00:23:12.678 --> 00:23:15.698 created which then can then mutate away, 00:23:17.078 --> 00:23:21.878 whether or not that creates an advantage is another issue but obviously having 00:23:21.878 --> 00:23:24.898 genes with the same function clustered. 00:23:26.818 --> 00:23:31.938 Opens the opportunity for local control of the cluster of regulatory elements that. 00:23:32.878 --> 00:23:39.638 Decide or help decide within that cluster which of the genes is turned on right but now so in, 00:23:40.338 --> 00:23:43.918 terms of the control this might already make a difference right so for olfaction 00:23:43.918 --> 00:23:48.178 do you have in mind that there is such a variability and also these control 00:23:48.178 --> 00:23:50.138 signals that for instance you want 00:23:50.158 --> 00:23:55.918 to have one control signal to switch on a whole group of receptor genes while 00:23:55.918 --> 00:23:58.138 you want to have a tighter control over another one. 00:23:58.238 --> 00:24:04.578 This might be also, let's say, differential for, let's say, the different subsystems 00:24:04.578 --> 00:24:06.598 that you would find in the epithelium, right? 00:24:06.638 --> 00:24:10.918 Where maybe one, you might have very coarse control over the gene expression 00:24:10.918 --> 00:24:13.598 and other regions, you want a very tight control over gene expression. 00:24:13.938 --> 00:24:17.278 So do you mean in terms of numbers of cells that express it? For instance, yeah. 00:24:17.418 --> 00:24:21.798 Yeah. Yeah, that's something that is, I think, underappreciated and also not 00:24:21.798 --> 00:24:28.018 so easy to quantify is that you find often in papers also that each receptor 00:24:28.018 --> 00:24:31.498 gene is expressed in one out of a thousand cells. Actually, it should be one in 1,200. 00:24:31.858 --> 00:24:36.038 Even that is not true. There is two orders of magnitude of difference. 00:24:36.138 --> 00:24:38.038 The champion is more 28. Right. 00:24:38.976 --> 00:24:42.216 For which, as far as I know, still no ligand has been found, 00:24:42.296 --> 00:24:46.216 and that's expressed in about 100,000 cells in a mouse. It's truly, again, a rule breaker. 00:24:46.996 --> 00:24:50.196 There are other genes that are expressed in just a few hundred cells. 00:24:50.636 --> 00:24:54.296 So the probability of expression, which we mean operationally, 00:24:54.296 --> 00:24:57.916 the frequency or the number of cells in a given mouse that expresses a given 00:24:57.916 --> 00:25:00.056 gene varies over two orders of magnitude. 00:25:01.636 --> 00:25:06.016 Is that evolutionarily determined? Are the genes that are expressed in many 00:25:06.016 --> 00:25:10.076 cells, are they more important? Therefore, there need to be more cells of them. I don't know. 00:25:10.756 --> 00:25:14.516 But it's, of course, interesting for us in the long run to look at it experimentally. 00:25:14.776 --> 00:25:21.896 What in the promoter, just upstream of the coding region, affects this probability. Right, exactly. 00:25:22.516 --> 00:25:25.876 Because another angle on this could also be because you're saying, 00:25:25.936 --> 00:25:29.736 well, from the perspective of olfaction, it looks haphazard, right? Like arbitrary. 00:25:30.536 --> 00:25:35.376 But you could also argue, well, look, but it's not impossible that some of these 00:25:35.376 --> 00:25:39.956 genes are expressed in other parts of the body of playing a different role. 00:25:40.496 --> 00:25:44.216 And that's for that you want to have certain kinds of control sequences at work. 00:25:45.016 --> 00:25:51.196 So that's an emerging story. There has been, ever since the beginning of the 00:25:51.196 --> 00:25:52.316 auto receptor gene saga, 00:25:52.556 --> 00:25:56.316 there's been papers about who are genes expressed in the testis, 00:25:56.356 --> 00:26:00.156 particularly in sperm, but many genes are expressed there perhaps less interesting. 00:26:00.316 --> 00:26:05.936 There has been some evidence that they may be involved in perhaps chemotaxis 00:26:05.936 --> 00:26:10.216 of sperm, swimming towards the egg and so on, but that's all quite limited. 00:26:10.916 --> 00:26:15.256 So apart from that, there are isolated cases of genes, odom receptor genes, 00:26:15.416 --> 00:26:16.936 that are expressed outside the nose. 00:26:17.076 --> 00:26:19.316 If they are also expressed in the nose and olfactory neurons, 00:26:19.476 --> 00:26:23.176 they would qualify as an odom receptor, not with the O from odorant or olfaction. 00:26:24.312 --> 00:26:26.812 If they're not expressed in the nose and only outside the nose, 00:26:27.012 --> 00:26:31.452 then you cannot even call them a nodal receptor. They have just been hijacked 00:26:31.452 --> 00:26:34.232 or used in evolution for something else. 00:26:35.332 --> 00:26:39.252 Now, the one that comes to mind is this Moritine-2. 00:26:39.392 --> 00:26:45.792 It has several other names, which is really a strange gene. It is expressed clearly in the nose. 00:26:46.772 --> 00:26:49.152 There's ligands for it and so on and so on. They have glomeli. 00:26:49.752 --> 00:26:52.312 It's also expressed in a small number of cells in kidneys. 00:26:52.312 --> 00:26:57.132 Kidneys and mice with a knockout in that gene have a problem with regulating 00:26:57.132 --> 00:27:02.552 their blood pressure, which is something you would never, never have thought to even look for. 00:27:03.452 --> 00:27:07.552 So it's something that needs to be looked at further. 00:27:07.712 --> 00:27:12.572 And indeed, in those cells in the kidney, the mechanisms that control the expression 00:27:12.572 --> 00:27:16.312 are probably different from those in the nose. They're not random. 00:27:16.612 --> 00:27:21.572 They're probably much more regulated and so on. So perhaps they have a different promoter. 00:27:21.712 --> 00:27:25.432 The same coding region can have two promoters, a few kilobases apart, 00:27:25.532 --> 00:27:28.552 and one promoter is used in the nose and the other one is used outside the nose. 00:27:28.652 --> 00:27:30.212 That would be one simple solution. 00:27:30.572 --> 00:27:34.892 Would you be willing to reconsider the notion of haphazard? 00:27:35.012 --> 00:27:37.992 That maybe it looks haphazard because not all the constraints have been taken 00:27:37.992 --> 00:27:42.812 into account, but if you would take on board the idea that these same receptors 00:27:42.812 --> 00:27:46.572 are also expressed in other parts of the body, at other points during development, 00:27:46.892 --> 00:27:52.152 for other functions possibly, Possibly that this requires other levels of control 00:27:52.152 --> 00:27:56.492 that then require this kind of structuring of the genome. Would you find that 00:27:56.492 --> 00:27:57.732 a reasonable alternative interpretation? 00:27:58.292 --> 00:28:02.452 So I referred with haphazard to the organization in the genome, 00:28:02.492 --> 00:28:04.032 not the expression per se. Okay. 00:28:04.372 --> 00:28:08.932 I don't think there is rampant expression of auto-receptor genes outside the 00:28:08.932 --> 00:28:10.972 nose, even in certain parts of development. 00:28:11.152 --> 00:28:13.772 Probably would have been found by now. 00:28:14.832 --> 00:28:19.012 So it's not the case that every odontoreceptor gene is at some point expressed somewhere else. 00:28:20.392 --> 00:28:25.532 On the other hand, many senior libraries, many express sequence stacks, 00:28:25.672 --> 00:28:30.332 ESTs, many microarrays that keep popping up odontoreceptors all the time to 00:28:30.332 --> 00:28:33.392 the annoyance often of these investigators because they don't know what to do with it. 00:28:33.632 --> 00:28:40.292 So I think it's been ignored a bit or underestimated too long in our field, 00:28:40.352 --> 00:28:41.872 this non-olfactory expression. 00:28:43.372 --> 00:28:46.932 But on the other hand, there is no rampant expression of OR genes outside the 00:28:46.932 --> 00:28:48.552 nose, I think, any time in development. 00:28:49.632 --> 00:28:53.912 Even though there is plenty of chemical sensing going on in other parts in the body. 00:28:54.252 --> 00:28:59.992 Yeah, so these O-receptors in the end don't have a very unique receptor structure. 00:29:00.172 --> 00:29:02.732 They're G-protein copper receptors, seven transmembrane proteins, 00:29:02.892 --> 00:29:06.612 so they're amino acid sequence snakes seven times through the membrane. 00:29:06.612 --> 00:29:12.572 Mean there's many other gpcrs in fact many of the drugs you buy in a pharmacy 00:29:12.572 --> 00:29:18.112 with a prescription are designed against gpcrs agonist or antagonist and what 00:29:18.112 --> 00:29:23.512 have you so from that point of view it's not surprising that some of these so-called 00:29:23.512 --> 00:29:26.552 or genes so because they have an or like sequence, 00:29:27.072 --> 00:29:34.152 are simply used by other cells to detect small molecules as their colleagues the non-or gpcrs, 00:29:34.692 --> 00:29:36.612 non-olfactory GPCRs as they do, right? 00:29:36.752 --> 00:29:41.672 Exactly right. That's indeed how pharmacologists talk now about their GPCR genes. 00:29:41.752 --> 00:29:43.932 They call them non-olfactory GPCRs, by the way. 00:29:44.032 --> 00:29:48.192 That's their GPCRs minus the 1200 that we work on. 00:29:48.572 --> 00:29:52.932 But it might then also imply that the direction the field is taking is also, 00:29:53.072 --> 00:29:57.432 well, maybe what we used to call an olfactory receptor gene, 00:29:58.212 --> 00:30:03.912 is actually part of a larger family of, let's say, ligand-bound receptors Receptors. 00:30:04.912 --> 00:30:08.292 And by accident, a subfamily of these have a specialization that they express 00:30:08.292 --> 00:30:12.092 in the epithelium. Is that not where things are going now? 00:30:13.092 --> 00:30:16.452 Well, I mean, this was known from the beginning. There are G-protein coupled 00:30:16.452 --> 00:30:21.472 receptors, and that's an ancient motif for many receptor types. 00:30:21.792 --> 00:30:28.052 So in that vein, 10 years ago, we reported this rather surprising finding. 00:30:28.132 --> 00:30:29.772 We did what we call the receptor swap. 00:30:29.952 --> 00:30:33.832 We replaced the coding region of a northern receptor, M71, for which we had ligands. 00:30:35.392 --> 00:30:38.032 Acetophenolbenzaldehyde, we replace it with the beta-2 adrenergic receptor, 00:30:38.212 --> 00:30:43.392 and that's the most, the best characterized GPCR, also I think the first one 00:30:43.392 --> 00:30:44.732 to be cloned 20 years ago. 00:30:45.572 --> 00:30:50.072 And lo and behold, the neuron that expressed now the beta-2 adrenergic receptor 00:30:50.072 --> 00:30:58.632 from the M71 locus now form a new glomalus at a location quite far from the M71 glomali. 00:30:58.632 --> 00:31:03.632 We also know that these neurons respond to beta-2AR agonists, 00:31:03.772 --> 00:31:08.052 isoproterol, in a dose-dependent fashion. 00:31:08.612 --> 00:31:13.752 So if you didn't know that in these mice the beta-2AR was expressed from this 00:31:13.752 --> 00:31:18.512 M71 locus, and I would show you all these images, you would not be able to tell the difference. 00:31:18.772 --> 00:31:24.212 So that's another, of course, artificial experimental evidence that perhaps 00:31:24.212 --> 00:31:27.132 there is nothing so special about organ receptors in these regards. 00:31:28.632 --> 00:31:31.932 Now, there are a lot of GPCRs expressed in the nose, in olfactory neurons. 00:31:32.012 --> 00:31:37.632 That's another thing that's been neglected, perhaps a bit on purpose by our field. 00:31:38.172 --> 00:31:43.412 Dozens and dozens of GPCRs are expressed. It's come up in several screens already, in several papers. 00:31:43.772 --> 00:31:47.192 Some are expressed in olfactory neurons, like the dopamine type 2 receptor, 00:31:47.712 --> 00:31:51.932 very frequent used for drugs against schizophrenia. 00:31:52.752 --> 00:31:57.772 All olfactory neurons, all mature neurons express the dopamine type 2 receptor, which is also a GPCR. 00:31:57.772 --> 00:32:04.152 And so why is the OR, can it instruct axons to form glomeruli and not other 00:32:04.152 --> 00:32:08.852 GPCRs that are expressed there anyway is another issue for further research. 00:32:08.912 --> 00:32:12.512 Perhaps it's a question of the timing of expression, the level, 00:32:12.632 --> 00:32:17.712 or are they expressed at extremely high levels or something else we are missing. Right, exactly. 00:32:19.552 --> 00:32:26.012 So now we have a bit of an idea of this sorting problem you've seen in the olfactory bulb. 00:32:26.012 --> 00:32:32.352 Bulb, that means you have a huge population, a million plus receptor neurons 00:32:32.352 --> 00:32:36.652 sitting there, sending their projections into the olfactory bulb, 00:32:36.832 --> 00:32:40.892 initially in a rather disorganized way. 00:32:41.332 --> 00:32:46.512 And then if you're by some sort of magic, it comes out sorted at the other end 00:32:46.512 --> 00:32:50.232 and all these processes terminate in their preferred glomerulus. 00:32:54.236 --> 00:32:57.776 And in some sense, we looked at the different aspects of that sorting story 00:32:57.776 --> 00:33:01.136 that could play a role. And we see actually all of them are rather problematic. 00:33:01.776 --> 00:33:04.516 Like the idea of zoning is problematic. 00:33:06.336 --> 00:33:10.236 So what are the alternative interpretations of this? What would be an alternative 00:33:10.236 --> 00:33:12.596 view of how this sorting process could work? 00:33:13.896 --> 00:33:17.236 So a model that we proposed a while ago, and we talked about this this morning, 00:33:17.256 --> 00:33:21.036 is that of homotypic and homophilic interactions. 00:33:21.036 --> 00:33:25.116 Interactions, that the auto-receptor protein, which we know is expressed very 00:33:25.116 --> 00:33:27.576 highly along the axons all the way to the end, 00:33:27.756 --> 00:33:32.476 that auto-receptor proteins of the same type, so homotypic, interact with each 00:33:32.476 --> 00:33:39.736 other, homophilic, and that would either by itself create some kind of a specific adhesion or perhaps, 00:33:39.896 --> 00:33:44.016 combined with a signal transduction event in the axons, that would cause these 00:33:44.016 --> 00:33:46.876 axons to fasciculate, to form fascicles, to form bundles, 00:33:47.036 --> 00:33:50.656 which is probably a step that leads towards the formation of a glomerulus, 00:33:50.776 --> 00:33:56.616 axons that stick together and at some point perhaps even stop growing and form 00:33:56.616 --> 00:33:59.356 their glomerulus. That is one model that we favor. 00:33:59.456 --> 00:34:03.476 It would be a parsimonious model evolutionarily because if a new-ordered receptor 00:34:03.476 --> 00:34:08.596 is created in evolution with an amino acid sequence that's sufficiently different. 00:34:08.816 --> 00:34:13.236 Then these homophilic interactions are different now, and now you would have 00:34:13.236 --> 00:34:17.056 a new identity and a new glomerulus created without anything else. 00:34:17.056 --> 00:34:21.916 But wait, I'm not sure if that's the whole story then, because you would still 00:34:21.916 --> 00:34:27.456 might have to generate an overabundance of these processes to have this sorting 00:34:27.456 --> 00:34:30.036 process, self-organizing sorting to work out, 00:34:30.196 --> 00:34:33.236 because you lack specificity now. You mean neurons and axons? 00:34:33.356 --> 00:34:36.456 That's right. I mean, these receptor neurons that throw out these processes, 00:34:36.676 --> 00:34:41.896 if they have these attractive and repellent interactions, you might want to 00:34:41.896 --> 00:34:46.536 throw out quite a large number of them in the hope that a small subset will 00:34:46.536 --> 00:34:47.856 actually reach the target or not. 00:34:47.856 --> 00:34:53.536 So that's the issue of selection and, again, something that is, I think, ignored or…. 00:34:54.974 --> 00:34:59.254 Overlooked in the field is how many, what's the success rate of the whole process? 00:35:00.014 --> 00:35:06.194 To put it in a simple way, of every hundred neurons in the epithelium that are born and project an axon, 00:35:06.734 --> 00:35:13.014 that gets to the bulb, how many of those send their axon to the correct place 00:35:13.014 --> 00:35:16.474 in the sense that it innervates a correct glomalus and it survives, right? 00:35:16.594 --> 00:35:19.614 That's a very simple question. What is the success rate? And we don't know that. 00:35:20.374 --> 00:35:24.454 We intuitively think that it could be very high, Perhaps as close as 100%, 00:35:24.454 --> 00:35:25.934 but there's no such thing in biology. 00:35:26.334 --> 00:35:30.794 If it's quite low, though, let's say it's less than 50%, then there is an opportunity 00:35:30.794 --> 00:35:35.454 for selection, for negative selection, if you wish, for weeding out processes, 00:35:35.694 --> 00:35:39.414 as you call them, axons that are completely off the wall and project to the 00:35:39.414 --> 00:35:41.394 wrong part of the bulb and are hopelessly lost. 00:35:44.154 --> 00:35:48.874 Or axons that enter a glomalus that is very close, but not exactly of the same 00:35:48.874 --> 00:35:53.354 type, right? And they could, over a period of hours of days, being eliminated. 00:35:53.734 --> 00:35:56.534 So there is no method to look at the success rate. 00:35:57.654 --> 00:36:02.434 And also when people look at these images, I don't think they think about that, 00:36:02.554 --> 00:36:05.494 that some axons could not make it. 00:36:05.534 --> 00:36:08.854 It's just not, in that sense, these pictures are somewhat misleading perhaps. 00:36:09.034 --> 00:36:13.114 They give you the end product, the final results, but it doesn't tell you about 00:36:13.114 --> 00:36:14.014 the mechanism necessarily. 00:36:15.463 --> 00:36:20.603 Possibly in different stages of development, you might see signatures of that process at work. 00:36:20.943 --> 00:36:25.003 So in general, the brain has an exuberance, as it's often called. 00:36:25.463 --> 00:36:30.043 More neurons produced than necessary, more axonal processes produced than necessary. 00:36:30.763 --> 00:36:34.723 That's not the case, by the way, with olfactory axons. They never have multiple 00:36:34.723 --> 00:36:39.543 processes, just one per, at least that's a dogma, one per neuron. 00:36:40.443 --> 00:36:45.503 But again, I would not be surprised at all if someone finds out that the success rate is very small, 00:36:45.823 --> 00:36:49.463 very low, that there is an exuberance of neurons being produced, 00:36:49.563 --> 00:36:55.143 that many of them never make it, and they get removed so quickly from the system 00:36:55.143 --> 00:36:58.963 within hours of days, and everything is asynchronously developing anyway, 00:36:59.123 --> 00:37:00.363 that we would just be missing them. 00:37:00.363 --> 00:37:04.423 But if you would look specifically for them, and really with very specific methods, 00:37:04.523 --> 00:37:08.543 perhaps you could find them. There is a lot of cell death going on in the olfactory epithelium. 00:37:08.703 --> 00:37:13.163 You can use any marker you wish for apoptosis, even in adult mice. 00:37:13.283 --> 00:37:16.563 Plenty and plenty of cells dying, as it decays in every epithelium. 00:37:17.003 --> 00:37:23.203 So at least there is a basis there for cell death playing a role in scalloping, 00:37:23.323 --> 00:37:25.403 if you wish, these projections. 00:37:26.003 --> 00:37:33.063 Right. So now, another step, because there's a lot of work behind these observations 00:37:33.063 --> 00:37:34.823 that you're sharing now with me. 00:37:36.143 --> 00:37:41.783 But then as one step in that whole process, you also decided to clone a mouse. 00:37:42.503 --> 00:37:45.703 So how did that help you? How did that help you in understanding this system? 00:37:45.983 --> 00:37:51.323 So that was for the gene regulation issue. issue, a very popular idea back from 00:37:51.323 --> 00:37:52.403 the early days from 1991, 00:37:52.643 --> 00:37:59.163 the Bercouzel paper, was that the way an olfactory neuron expresses one gene 00:37:59.163 --> 00:38:06.523 stably and irreversibly at high levels is by some kind of genetic alteration in the genome. 00:38:07.203 --> 00:38:08.743 That's the case with lymphocytes. 00:38:09.443 --> 00:38:14.223 B cells make one antibody with a heavy and a light chain, and T-cell receptors, 00:38:14.303 --> 00:38:17.803 the Let's go for beta t's as if one alpha and one beta chain. 00:38:19.047 --> 00:38:22.967 And they do so by irreversibly changing their genetic material. 00:38:23.147 --> 00:38:26.987 There are pieces of DNA that are lost, obviously irreversible, 00:38:27.087 --> 00:38:33.527 and in some cases inverted, that underlie this. 00:38:34.267 --> 00:38:38.487 But surely the problem of cell faith is broader than that, right? 00:38:38.547 --> 00:38:42.547 It's not only for the lymphocytes. I mean, any cell at some point in development 00:38:42.547 --> 00:38:49.787 is committed to become of a certain identity, like a skin cell or a liver cell or a heart cell, etc. 00:38:50.067 --> 00:38:52.907 So why do you highlight the lymphocytes in this case? 00:38:53.647 --> 00:38:56.147 Because they have so many similarities with olfactory neurons. 00:38:56.407 --> 00:39:02.647 They have a large number of genes at their disposal, and each neuron or each 00:39:02.647 --> 00:39:06.147 lymphocyte expresses only one of them. I mean, it's not only me who makes this analogy. 00:39:06.527 --> 00:39:11.367 So this was pervasive in our thinking. I must say that many of the people working 00:39:11.367 --> 00:39:13.967 in olfaction, at least in the early days, were actually ex-immunologists, 00:39:14.247 --> 00:39:18.067 had a PhD or a postdoc in immunology, so maybe they were thinking along these lines. 00:39:19.567 --> 00:39:24.427 But don't you agree that today, we might as well say, well, the analog might 00:39:24.427 --> 00:39:25.587 as well have been a skin cell? 00:39:26.207 --> 00:39:30.367 Well, but skin cells don't have to acknowledge a large number of genes of the 00:39:30.367 --> 00:39:33.727 same family at the disposal of which they have to pick one for expression. 00:39:33.907 --> 00:39:35.007 That's just not the case. 00:39:35.227 --> 00:39:38.687 But in the end, they have to pick a subset of all possible genes to express. 00:39:38.987 --> 00:39:42.927 So, to go back even longer in history, 00:39:43.127 --> 00:39:48.247 when these gene rearrangements in B lymphocytes were discovered in the 70s by 00:39:48.247 --> 00:39:53.547 Susumu Tonagawa and others, there was the idea that other aspects of differentiation 00:39:53.547 --> 00:39:57.207 and development would also be regulated by irreversible gene rearrangements. 00:39:57.927 --> 00:40:01.287 But the cloning experiments of John Gurdon and others were a strong argument 00:40:01.287 --> 00:40:07.267 against that because you could produce normal or fairly normal animals from differentiated cells. 00:40:07.447 --> 00:40:11.447 And if these cells had shattered pieces of DNA irreversibly, 00:40:11.587 --> 00:40:13.447 then they would not have been able to do that. 00:40:13.727 --> 00:40:18.407 But indeed, there was a period of time, 30, 40 years ago, where it was thought 00:40:18.407 --> 00:40:24.087 that many differentiation processes would be…. 00:40:25.105 --> 00:40:29.025 Governed by irreversible changes. And we don't think that's the case anymore. 00:40:29.025 --> 00:40:32.305 So it's basically like, look, your faith is set by some switch. 00:40:32.785 --> 00:40:36.505 And now basically, in some sense, you irreversibly wipe out a bit of your genome. 00:40:36.705 --> 00:40:38.225 So from then on, that's it. 00:40:38.565 --> 00:40:43.225 And also the discovery of the possibility of making induced pluripotent stem 00:40:43.225 --> 00:40:45.445 cells, the iPS cells, was another argument against that. 00:40:45.465 --> 00:40:50.925 You can take a differentiated cell and sort of reconvert it into an embryonic 00:40:50.925 --> 00:40:53.845 stem cell or like cell that can go any way. 00:40:53.845 --> 00:40:56.925 Way that's also an argument against that irreversible changes 00:40:56.925 --> 00:40:59.765 of any type actually not necessarily genetic but then 00:40:59.765 --> 00:41:02.905 how did this this cloned mouse that that you build 00:41:02.905 --> 00:41:05.805 was quite a tour in itself to to build it 00:41:05.805 --> 00:41:13.125 help you to understand this this determination and stabilization of of the cell 00:41:13.125 --> 00:41:16.945 faith in the olfactory system so if if neurons that express a given receptor 00:41:16.945 --> 00:41:20.525 let's take our favorite receptor again m71 if neurons expressing that receptor 00:41:20.525 --> 00:41:25.245 have made an irreversible genetic alteration to do so, 00:41:25.345 --> 00:41:31.345 then a mouse cloned from the nucleus of such a neuron would be monoclonal, essentially. 00:41:31.585 --> 00:41:34.965 All the neurons, or the majority of them, would express M71. 00:41:35.845 --> 00:41:39.805 That was the prediction. If these changes are irreversible, if they're somehow 00:41:39.805 --> 00:41:42.845 reversible during the cloning procedure, which not many people have thought 00:41:42.845 --> 00:41:45.825 about, but I sometimes think about this, if they would have been reversible 00:41:45.825 --> 00:41:48.825 during the cloning procedure, then we draw the wrong conclusion. 00:41:49.785 --> 00:41:53.845 So it was essentially a negative result. We got, let's say, a normal mouse out 00:41:53.845 --> 00:41:56.245 of the nucleus of a neuron expressing M71. 00:41:56.385 --> 00:41:59.845 These neurons expressed not necessarily M71, but other own receptor genes. 00:42:00.225 --> 00:42:05.705 Hence, there were no irreversible changes. If you clone a mouse with a lymphocyte, 00:42:05.705 --> 00:42:09.145 Rudy Yenish has done that around the same time, then you really get a monoclonal 00:42:09.145 --> 00:42:11.285 mouse. These are spectacular phenotypes. 00:42:11.505 --> 00:42:14.525 All the B lymphocytes in that mouse make just the same antibody. 00:42:14.685 --> 00:42:17.985 Right. But how come that cloned mouse is even viable? 00:42:18.285 --> 00:42:21.705 It might have been possible. It would not even have been a viable cell. Thank you for watching. 00:42:22.900 --> 00:42:26.980 Yeah, I think our paper and that of Rudy Inej and Richard Axel was the first 00:42:26.980 --> 00:42:29.860 to clone with post-mitotic mature neurons. 00:42:29.960 --> 00:42:33.460 That was not necessarily given. 00:42:33.640 --> 00:42:36.880 The success rate of cloning by nuclear transfer is quite low, 00:42:36.960 --> 00:42:39.280 I must say. It's in the single-digit percentages. 00:42:39.880 --> 00:42:45.040 Some of the data are a bit massaged in these tables, but it's never more than 10%. 00:42:46.140 --> 00:42:51.060 And so it could still be that the other 90% die for fundamental biological reasons. 00:42:51.060 --> 00:42:55.260 We will only know when we're able to get the frequency higher. 00:42:55.720 --> 00:43:00.680 Right. Okay, but so now that you know that there might still be an irreversible 00:43:00.680 --> 00:43:07.540 change, but it doesn't translate if you clone from that cell a whole new mouse, right? 00:43:07.920 --> 00:43:11.180 So what now is your alternative interpretation? 00:43:12.640 --> 00:43:17.900 Well, if it's not genetic, it must be epigenetic. That's the more modern, 00:43:17.940 --> 00:43:19.520 perhaps fashionable way of looking at that. 00:43:19.520 --> 00:43:25.300 But wait, that could also be, imagine I have another cell that's expressing 00:43:25.300 --> 00:43:30.540 other genes that is controlling the expression of the genes in this receptor neuron. 00:43:31.320 --> 00:43:35.640 So now you clone only from this receptor neuron, but this controlling unit is gone. 00:43:36.360 --> 00:43:41.040 Now the control signal is gone. So this receptor neuron again is expressing its full genome. 00:43:43.569 --> 00:43:47.929 Yes, but at least that gene or receptor gene that was expressed in the original 00:43:47.929 --> 00:43:50.269 cell, that gene was not irreversibly altered. 00:43:50.749 --> 00:43:53.549 That's right, exactly. That's what we said, nothing more. Okay, 00:43:53.609 --> 00:43:58.589 so not irreversibly changed just within that one cell. 00:43:59.249 --> 00:44:03.809 So you do consider the possibility that within the organism there might be additional 00:44:03.809 --> 00:44:05.429 control signals that regulate that. 00:44:06.109 --> 00:44:10.409 Well, what I was worried then and still a bit now is that experimentally during 00:44:10.409 --> 00:44:13.469 the process of nuclear transfer, which is a complex procedure, 00:44:13.589 --> 00:44:19.909 which we don't understand, that change that was present in the M71 locus was somehow reverted. 00:44:20.089 --> 00:44:25.389 If you have a thing that flips back and forth, it just flips back, right? 00:44:25.509 --> 00:44:28.149 And then we don't see it anymore, but that doesn't mean there was no change 00:44:28.149 --> 00:44:30.289 before that. Sure, absolutely. 00:44:30.329 --> 00:44:43.889 Because maybe the whole confirmation of the DNA was such that it was put into 00:44:43.889 --> 00:44:47.509 a metastable state that prevented further transcription. 00:44:48.829 --> 00:44:52.729 But that by, let's say, perturbing the system again in the cloning procedure, 00:44:53.049 --> 00:44:55.809 it could sort of flip back into a state that could express itself. 00:44:56.129 --> 00:45:01.129 I can see that. But so now if you say epigenetic, that's of course a little 00:45:01.129 --> 00:45:05.109 bit tricky because at this point, epigenetic just means, well, 00:45:05.209 --> 00:45:08.389 some factor that's not genetic, right? 00:45:08.469 --> 00:45:13.969 But that basically could be from the whole of the universe to the diet to other 00:45:13.969 --> 00:45:16.689 cells, developmental trajectories, whatever. 00:45:16.809 --> 00:45:19.609 So if you say epigenetic, what would you have exactly in mind? 00:45:20.009 --> 00:45:23.449 Well, that's not what I say, but that's what other people say. 00:45:23.529 --> 00:45:28.109 And indeed, it's very broad. I mean, in some ways it doesn't say very much because 00:45:28.109 --> 00:45:29.489 it only says it's not genetic. 00:45:29.649 --> 00:45:34.169 That's exactly right. That we sort of knew there is no genetic change. The DNA isn't changed. 00:45:34.509 --> 00:45:39.429 We think it's not changed in the organ receptor gene locus. But I would…, 00:45:40.716 --> 00:45:44.256 I think that at this point in time, we really don't have a good view of how, 00:45:44.456 --> 00:45:49.656 I mean, I'm the first to admit that, how a neuron expresses one allele of one 00:45:49.656 --> 00:45:51.896 gene at high levels. We just don't understand that. 00:45:52.116 --> 00:45:55.276 It sort of have clues of why it could be a small number of genes, 00:45:55.416 --> 00:46:02.736 but why it's just one, that is very difficult for anyone to think on or show experimentally. 00:46:03.016 --> 00:46:05.816 Okay, but then what's the next step there to solve this? 00:46:07.616 --> 00:46:12.276 Probably development of new technologies miniaturization we have to work with 00:46:12.276 --> 00:46:16.436 single cells uh one can sort cells expressing the same receptor using a cell 00:46:16.436 --> 00:46:20.736 sorter with gfp but even then you look at the population of cells so we have 00:46:20.736 --> 00:46:25.416 to look at single cell and that's all coming uh coming along now there's rna 00:46:25.416 --> 00:46:27.136 seek is now being developed for single cells, 00:46:28.336 --> 00:46:33.696 um so i think it's a as usual new new technologies will um give us new ways 00:46:33.696 --> 00:46:34.536 of looking at all problems. 00:46:36.656 --> 00:46:41.436 But without a clear hypothesis, the technology might also lead you astray. 00:46:43.916 --> 00:46:48.176 Yeah. Or hypotheses, multiple hypotheses. 00:46:48.316 --> 00:46:51.636 You want to ask, basically, neurons that express receptor A, 00:46:52.376 --> 00:46:54.856 how are they different from neurons expressing receptor B? 00:46:54.956 --> 00:46:59.376 And let's say that A and B are two similar genes in the same cluster, as close as you can get. 00:46:59.496 --> 00:47:04.856 What is different between those cells? That would be a very simple approach approach, right? 00:47:05.316 --> 00:47:08.836 And you would take anything you find differentially expressed or differentially 00:47:08.836 --> 00:47:12.276 methylated or hypomethylated because one of them could be causative. 00:47:12.616 --> 00:47:16.856 Others could be the consequence of neurons expressing receptor A versus B, 00:47:16.956 --> 00:47:21.936 but other changes or differences could be explained or help explain why it's 00:47:21.936 --> 00:47:24.536 receptor A in one versus the other. 00:47:24.596 --> 00:47:29.196 But I think in the long run, we have to look at single cells, but it's possible now. 00:47:29.276 --> 00:47:32.716 There is really a big, big progress in single cell studies. 00:47:33.136 --> 00:47:36.456 But you think to do that effectively, you do need new technologies. 00:47:38.402 --> 00:47:42.542 Yeah, but that's always been the case, no? Well, it depends. 00:47:42.842 --> 00:47:47.282 I mean, given the question you posed, you might have to develop a specific technology, 00:47:47.502 --> 00:47:49.982 but you don't have to wait for things to show up at the horizon. 00:47:50.882 --> 00:47:55.962 But I mean, in your research, you're very technology heavy in your approach, right? 00:47:55.982 --> 00:48:02.142 So one thing you described is this nanostring system that you have been using 00:48:02.142 --> 00:48:06.782 to identify, and also other aspects of this whole regulatory system 00:48:07.022 --> 00:48:12.822 within the cell for gene expression around this notion of the P element. 00:48:13.922 --> 00:48:18.642 So what has been the insight there with respect to this regulation question? 00:48:19.142 --> 00:48:23.542 So we have adopted nanostring because there was probably still is no good method 00:48:23.542 --> 00:48:27.442 to look at all these organ receptor genes at the same time in the same sample. 00:48:28.742 --> 00:48:33.162 Typical way of looking at it is by qPCR, quantitative PCR, but that's really 00:48:33.162 --> 00:48:37.422 asking a lot doing that for hundreds of genes from one sample, a lot of pipetting. 00:48:37.962 --> 00:48:44.062 There's microarrays. I think their specificity is somewhat questionable, to say it nicely. 00:48:45.042 --> 00:48:48.842 Often these probes are from the 3-prime non-translated region, 00:48:48.982 --> 00:48:52.802 which is computationally determined, so not experimentally validated, 00:48:53.102 --> 00:48:54.762 because it's difficult to do so. 00:48:55.162 --> 00:49:00.262 So we have chosen for this nanostring, with which, since we chose only to make 00:49:00.262 --> 00:49:04.422 probes against all receptor coding regions, we could only look at at half the genes. 00:49:04.762 --> 00:49:09.702 So it's for us an assay. We can really look at close to 600 genes have the repertoire 00:49:09.702 --> 00:49:14.202 in one sample of RNA of about a microgram without any kind of amplification. 00:49:14.882 --> 00:49:18.562 It's an assay. I mean, by itself it doesn't show anything. And with that assay, 00:49:18.682 --> 00:49:25.042 we showed that mice that lacked at a mutation in this 317 base pair element, 00:49:25.122 --> 00:49:26.102 which we call the P element. 00:49:27.702 --> 00:49:32.202 That there are 10 genes differentially expressed, nine are downregulated, 00:49:32.282 --> 00:49:36.722 there is less RNA in the mutant mouse versus the wild type, and one is slightly upregulated. 00:49:36.862 --> 00:49:40.882 And they're all within about 200 kilobase from the so-called P elements. 00:49:41.162 --> 00:49:48.362 So that shows that that element somehow regulates the expression of OR genes 00:49:48.362 --> 00:49:50.462 in the cluster at the organism level. 00:49:51.382 --> 00:49:57.342 And you believe that this might be a key step in determining the faith of these receptor neurons? 00:49:57.702 --> 00:50:03.522 Yeah, one of them. It's not the only one. One, there is, we also said that clearly in our paper, 00:50:04.622 --> 00:50:08.962 that it's of course not by itself explaining how an olfactory neuron expresses 00:50:08.962 --> 00:50:12.962 one allele of one gene, but it's one of the several layers, 00:50:14.808 --> 00:50:19.868 Possibly hierarchical layers of control mechanisms that altogether ensure that 00:50:19.868 --> 00:50:23.448 the large majority, if not all, mature neurons expressing one allele of one gene. 00:50:23.568 --> 00:50:27.028 But by itself, it cannot be responsible for that. 00:50:27.328 --> 00:50:30.388 So how complex do you think is this control hierarchy? Yeah. 00:50:31.348 --> 00:50:34.668 Again, we will answer that when we know the whole hierarchy. 00:50:36.648 --> 00:50:41.428 It's not going to be solved immediately. I still have many years to go before my pension. 00:50:43.988 --> 00:50:48.248 And it has multiple aspects to it, multiple levels. There is also cellular selection. 00:50:48.468 --> 00:50:52.788 I believe that occasionally neurons are produced that make two receptors or maybe even more. 00:50:53.268 --> 00:50:57.668 And perhaps they are lost somehow, lost in the system by negative selection 00:50:57.668 --> 00:50:59.568 and that you can look for that too. 00:50:59.728 --> 00:51:03.908 It would be another control mechanism, right? You produce, you eliminate the cells you don't want. 00:51:04.948 --> 00:51:08.048 So it's not going to be solved anytime soon. The journals, of course, 00:51:08.068 --> 00:51:11.988 like us to claim in our papers in top journals in the title and so on that this 00:51:11.988 --> 00:51:13.348 is now the final breakthrough. 00:51:15.348 --> 00:51:20.888 But how many layers of control do you expect? Is it like single digits? 17. 00:51:23.348 --> 00:51:27.148 No, multiple. And at a different genomic level, 00:51:27.788 --> 00:51:33.968 positioning of clusters for expression, there's some evidence for that recently 00:51:33.968 --> 00:51:40.448 with nuclear aggregation that the gene that's expressed is in a different position 00:51:40.448 --> 00:51:42.048 of the nucleus than the other genes. 00:51:42.128 --> 00:51:46.368 That could be one of the mechanisms, but it cannot explain why it's only one, right? Right. 00:51:46.868 --> 00:51:48.768 That's always the problem. Why is it just one? 00:51:51.428 --> 00:51:56.668 But it's not, I mean, I think a satisfying level of insight, 00:51:56.868 --> 00:52:00.628 the question is how much, when are you satisfied with saying, I've understood this. 00:52:00.648 --> 00:52:06.908 A satisfying level of insight is feasible, conceivable, the next decade or two, but not sooner. 00:52:08.268 --> 00:52:15.028 And we hope, of course, that it has some general relevance, that we have not 00:52:15.028 --> 00:52:19.108 just explained how this particular weird family of genes is controlled, 00:52:19.288 --> 00:52:22.268 but that it has some insights that are more generally relevant to biology. 00:52:22.268 --> 00:52:27.368 Exactly right, because we started the conversation with how you use the olfactory 00:52:27.368 --> 00:52:29.228 system as a model to study development. 00:52:29.528 --> 00:52:33.328 Yeah. Right? But now, in some sense, we ended up dealing with a question that 00:52:33.328 --> 00:52:37.128 seems rather specific for the olfactory system, which is, how do I get such 00:52:37.128 --> 00:52:40.528 a precise expression of a single gene in a single neuron? 00:52:40.688 --> 00:52:44.308 So, what's this telling us in the end about development? 00:52:46.721 --> 00:52:50.641 Well, again, we will say this at the end, whether it was worth it and whether 00:52:50.641 --> 00:52:52.401 it was. I want to know it now. 00:52:52.821 --> 00:52:58.561 I was once a few years ago with Michael Brown in Beijing, coincidentally at 00:52:58.561 --> 00:53:01.241 the same time at an institute, and the graduate students all asked him, 00:53:01.261 --> 00:53:04.361 you know, how do I find an interesting biological problem, right, 00:53:04.441 --> 00:53:06.461 that gives me a Nobel Prize like he got? 00:53:07.321 --> 00:53:10.161 And he had a very good answer. They said you can pick almost any problem to 00:53:10.161 --> 00:53:12.541 start with, and you have to dig deeper and deeper and deeper. 00:53:12.541 --> 00:53:16.961 And when you hit very deeply, you will come to fundamental principles and mechanisms. 00:53:17.801 --> 00:53:24.041 So the angle or the approach point at which you start to study biological phenomenon 00:53:24.041 --> 00:53:26.801 or question perhaps matters less. It's how deep you go. 00:53:27.421 --> 00:53:33.901 But I constantly try to remind myself that we are not working on just the sense of smell of a mouse. 00:53:33.901 --> 00:53:39.581 You could just say that very honestly, that we hope that what we find and what 00:53:39.581 --> 00:53:44.581 we uncover, that is more important than just olfactory system of the mouse. 00:53:44.761 --> 00:53:47.361 That is a conscious leitmotif. 00:53:47.941 --> 00:53:53.861 If you wish, it's a hope and a plan. 00:53:54.241 --> 00:53:58.841 Right. So then to get to the finish line, two things. 00:53:58.841 --> 00:54:04.041 So, actually, you told me that it's exactly 20 years ago that you got initiated 00:54:04.041 --> 00:54:08.601 in this experimental study of olfaction, when you entered the lab of Axel as 00:54:08.601 --> 00:54:10.641 a postdoc. Yeah. That's correct, yeah? 00:54:12.341 --> 00:54:16.681 And so, now, given your experience in this field and also your many accomplishments, 00:54:16.961 --> 00:54:22.081 what would be Peter's law that we should adhere to in trying to understand how the brain works? 00:54:24.021 --> 00:54:27.421 Well, I don't like this question to begin with, how the brain works. 00:54:27.421 --> 00:54:32.481 That is so overly ambitious and probably even nonsensical, you can only hope 00:54:32.481 --> 00:54:37.821 at least with the present technologies to understand a bit of it, an aspect of it. 00:54:38.081 --> 00:54:43.101 Perhaps one circuit, one small step, it's some kind of Flemish modesty perhaps, 00:54:44.261 --> 00:54:49.481 that we, you know, perhaps with time and when we understand more smaller bits 00:54:49.481 --> 00:54:54.181 of it, more deeper and fundamental, we can… But I definitely never said that 00:54:54.181 --> 00:54:56.941 or never will claim that we want to figure out how the brain works. 00:54:56.941 --> 00:54:59.041 That is such a big, big question so far away. 00:54:59.401 --> 00:55:01.621 One has to maybe say this in grant proposals. 00:55:02.421 --> 00:55:04.061 So Peter's law is to be modest. 00:55:05.601 --> 00:55:08.241 And try to answer a problem that 00:55:08.241 --> 00:55:11.901 is answerable at that time with the technologies available at that time. 00:55:12.181 --> 00:55:14.781 But I think other people have said that too and realized that too. 00:55:15.461 --> 00:55:19.141 It's solving the solvable or Peter Medawar said that, right? Okay. 00:55:19.381 --> 00:55:25.361 But then the other hand is, so five years from now, I'm going to go to Frankfurt. 00:55:25.361 --> 00:55:28.641 For it and I'm going to confront you with your predictions of today. 00:55:29.921 --> 00:55:35.921 So what's the prediction that you're most passionate about today that you really 00:55:35.921 --> 00:55:39.601 spent most of your time on that you really want to have tested five years from 00:55:39.601 --> 00:55:42.961 now that I can confront you with then? Well the. 00:55:44.336 --> 00:55:46.576 But that's, again, technologically is the single-cell studies, 00:55:46.676 --> 00:55:50.176 I think. But I'm not the only one who's thinking that. But I want a prediction, Peter. 00:55:50.896 --> 00:55:53.876 But that is coming so clearly there, and there's not much published, 00:55:53.936 --> 00:55:55.656 and it will give us really new insights. 00:55:56.676 --> 00:56:00.536 Because we typically, when we look at the biological phenomenon, 00:56:00.656 --> 00:56:02.856 we look at the population of cells, and we are missing a lot. 00:56:02.996 --> 00:56:08.076 For instance, to come back to our p-element, the p-element shows a reduction 00:56:08.076 --> 00:56:11.456 in expression at the population level. 00:56:11.556 --> 00:56:14.796 We all hope that we have a nice Gaussian curve, right, which is shifted. 00:56:15.216 --> 00:56:21.016 But if the Gaussian curve is split into a bimodal curve, you would get exactly the same results. 00:56:21.116 --> 00:56:24.116 And it's actually erroneous results because we're now misled. 00:56:24.796 --> 00:56:28.616 So I really believe for our system and the things we are interested in, 00:56:28.696 --> 00:56:31.156 we have to look at single cells. It is doable. 00:56:31.336 --> 00:56:33.636 We are doing and other people will do it. And in five years, 00:56:33.676 --> 00:56:36.376 I think there will be nice insights coming from that. 00:56:36.536 --> 00:56:39.176 But that's a very modest prediction, right? So you're saying five years from 00:56:39.176 --> 00:56:43.636 now, I'll be able to look into single cells in the olfactory system. 00:56:44.016 --> 00:56:46.616 And see their genetic expression dynamics. 00:56:47.236 --> 00:56:50.596 It's already possible, yes, with RNA-seq. Yeah, but if you can already do it, 00:56:50.616 --> 00:56:52.696 it's not a very exciting prediction for five years from now. 00:56:52.716 --> 00:56:55.076 Well, but also, do we get something interesting out of that? 00:56:55.176 --> 00:57:00.096 Or will we be lost in details or in variability and so on? 00:57:00.176 --> 00:57:03.336 I hope and I believe that there will be new insights coming out of it, 00:57:03.376 --> 00:57:04.556 which we didn't even imagine. 00:57:04.776 --> 00:57:08.996 But would you say five years from now, you have nailed this problem of the cell 00:57:08.996 --> 00:57:10.676 phase stabilization? No, not at all. 00:57:10.856 --> 00:57:13.856 No? No, I don't want to put any year on that. 00:57:14.936 --> 00:57:19.276 That's really a longer project five year plans for the communists I know that, 00:57:19.276 --> 00:57:25.236 that's why I'm asking so Peter Wombart and for grant organizations so Peter 00:57:25.236 --> 00:57:28.656 Wombart, thank you very much for this conversation thanks Paul. 00:57:28.880 --> 00:57:34.800 Music. 00:57:34.736 --> 00:57:40.376 The CSN podcast was produced by the Convergent Science Network of Biometrics 00:57:40.376 --> 00:57:47.176 and Biohybrid Systems A project funded by the European Sevens Research Framework Programme. 00:57:48.216 --> 00:57:53.636 For more interviews, recorded lectures or upcoming conferences in the field 00:57:53.636 --> 00:57:59.996 of biometrics and biohybrid systems, go to csnnetwork.eu. 00:58:00.196 --> 00:58:01.136 Thank you. 00:58:01.040 --> 00:58:08.400 Music.