 A couple of disclosures. I have no financial conflicts of interest. In the US, we always have to disclose that. Secondly, I have bad hearing. I can only hear in my left ear, and I have a hearing aid here, which doesn't work very well. Third, I have terrible back problems. I'll be happy to show you my x-rays and MRIs afterwards. So it means that I have to lean on something all the time, and we'll probably have to sit down. When I teach, I have occasionally gotten, so in the US, when we teach, we get all this feedback from students, and we're evaluated on that. And I have occasionally gotten feedback that I was so bored with my teaching that I was sitting in my lectures. So it wasn't that I was bored, it was just that I was in pain. So I have a question to begin with. I know that this is a very famous institute for mathematics, and I was told by one of the chairwomen of this session that the mathematicians, some of the audience are actually famous mathematicians, or young mathematicians who were very bored with what they were hearing about biology. How many people, maybe I could get a show of hands, are mathematicians or non-biologists? You're so bored. So there are no mathematicians or physicists or anyone like that? Can I go see if they're in the lobby? I teach an applied math course. Sorry? I teach an applied math course. Are you a mathematician? No. Not a pure one. But applied math is fine. I don't care. I'll take that. OK, so I'll give you a little bit of personal math background. So I'm not a very good mathematician at all. But I'm sorry? You're a mathematician, sir? OK, so I'll give you a little bit of another mathematician. They brought in some ringers. We don't have more. More, more, more. Do we have a minion? More mathematicians and a mathematical dog? Good, good, good. So as I was saying, I was told the math, I know, I was saying before people came in, ladies and gentlemen, I know this is a very famous mathematics institute. I know about famous, I've had a lot of mathematician friends. I know about Borbaki. So I myself am not a good mathematician, but I have. So my father, my late father was a fairly good mathematician. He was a physicist. He got his PhD in physics from Columbia in 1951. He's been long dead. But I can tell you that he grew up in New York City. And in 1939, he was captain of his high school math team that won the New York City Mathematics Championship for high school. And so he got his PhD in physics from Columbia in 1951 and was a postdoc at the Courant Institute. So I imagine you know what that is. And so I have three children. Our two older ones are not at all interested in science, and they weren't good mathematicians. But our 14-year-old is very good, much better than I ever was. So next year, I know he'll be taking calculus, which is pretty good for a 14-year-old, I think. Much better than I was. I didn't take calculus till I was 16. So anyway, and so I know something about mathematical talent that I don't have it, but some of my relatives did. So my name is Mostov. There's a George Mostov, the Mostov-Virgidity, who's probably related to me. He passed away. I'm Spen. Pardon? I think you mean Judge Mostov is W. Yes, but they're all translated from the Cyrillic. And back in those days, it was usually translated with a W or an OFF when they came over the immigration in Ellis Island. It doesn't matter. They're all shortened. It was a Ukrainian name, Mostovoy. And it was all shortened from that. It's a Ukrainian-Jewish name. So it doesn't really matter how it's transliterated. I have letters in the original Cyrillic, Ukrainian, or Russian. And I studied Russian in school, so I know how it's transliterated. It doesn't matter how it's spelt in English. OK, so I know about the, I don't really know the details of the Mostov, but Virgidity. He passed away two years ago, but we're probably related. I'm told that all Mostovoys are related. So who's it for? Sorry? Time's up. OK. Pardon? OK, well, that's the most interesting result I have to say. OK, now for the rest of you, my talk only takes 35 minutes. And I was, and I started, I got up here at 306. For the rest of you, many of you in the audience know me as a cell biologist. I grew up, I was a student of Günter Blöbel, who passed away three weeks ago, and know me in the membrane traffic field. And I'm not going to talk about membrane traffic at all. I'm going to talk about classic straight up developmental biology, which most many of you are probably not very familiar with. So I have a lot of background, including some bioengineering and geometry. So I'm going to actually talk about geometry today. Classic Euclidean geometry. So this work was all done by Yang Yang, a postdoc. In my lab, in collaboration with Jeremy Reiter's lab at UCSF. And the overview, this is all work that's known. Most internal organs in metazoa are made of epithelial tubes. Tubes have a characteristic length in diameter. The control of length of the small intestine, which exists in chordates, vertebrates and related, have a characteristic length. And it's a fundamental and almost completely unsolved problem. Most people aren't even aware of it as a problem. And the control of epithelial tube length, in general, in vertebrates is a largely unsolved problem. And there's a clinical correlate, a short bowel syndrome, a severe disease of intestinal length, which is an unsolved problem clinically. Now, the new data to summarize is that, surprisingly, primary cilia, and I will explain everything in here, that this talk, an earlier version of this talk a year ago, was designed for UCSF faculty lunch, which includes mathematicians for this chemist. So I'll explain every term in here. Surprisingly, primary cilia controls small intestine length through the hedgehog signaling pathway. The loss of function of intestinal cell kinase, which is a poorly understood kinase, and it's a cilia gene causes short intestine. Other cilia genes also controls intestinal length. And hedgehog signaling, they work through mechanical forces, involving smooth muscle acting and YAP, which is abbreviation for YES-associated protein in mesenchymal cells. I'll explain every word in here, so don't worry. So we're going to start with one of the simplest metazoa. Metazoa means simply multicellular animals, as opposed to plants, or single cell organisms. So one of the simplest, one of my favorite organisms, is Hydra, and, excuse me, related things like jellyfish. So basically, can we have the lights down a little bit? So Hydra are one of the simplest types of metazoa. They contain two tissue layers. They have an endosperm, which lines the interior cavity. They basically have an interior cavity. And they contain an ectoderm, which lines the external external. These are both layers of epithelia. That is, a single layer of cells that form like a layer of bricks. And they have some loose cells in between neurons. This is from a standard textbook. And by Bruce Albert, one of the original great textbooks of cell biology in 1983. I'd just like to mention, in 1983, I was still in grad school. And completely unrelated work of mine was in this book. So it's always very gratifying that when you're still in grad school, your own work is in the book. But it's not related to this story. So most internal organs in metazoa are made of epithelial tubes. And so just to give some examples, this is a cast of the branching airways in a million lung. This is a diagram of the branching tubes that make up of them in a million kidney. This is a diagram of the branching tubes that make up the circulation of a human being. And this is a diagram of the airways that make up what's called the trachea of the embryo of a fruit fly from Marc Krasner's lab. This is the only one where we understand how it's formed. But it's formed in a completely different manner than all the ones in mammals. For instance, it's not involved. It's formed without any cell division. So it's a completely different way of forming things. Now we're going to focus today on one model system, which is the human digestive system, which those of us who ate lunch a few hours ago, that lovely buffet. We started by chewing our food in our mouth, then it went down our esophagus and into our stomach, and then our small intestine and our large intestine and out the anus and the rectum. And that is really one continuous tube. I went to medical school once upon a time for the one simple reason that I was a good Jewish boy. And in the United States, in a certain period, every good Jewish boy went to medical school. And those of you like Dave Drubin here who failed to do that probably disappointed their mothers. So I wasn't smart enough to be a physicist like my father. I should add, when I was in graduate school, I was a government-funded MD-PhD program that the US government likes to fund. When I was in grad school, when I was around 23 or so, I managed to sort of explain to my father, the physicist, what I was working on. And he had one comment to me, which was, if you have to do experiments, you must not be very smart. And he told me how he did his PhD, basically his whole thesis work, in about six weeks. And the first week was doing the work, and the next five weeks was writing it down. So then he got my mother, who was engaged to him and knew how to come over to his apartment and type it up. And she said that was the hardest thing she ever did because there were subscripts to the subscripts and superscripts to the superscripts. And his whole PhD thesis was about, I don't know, 30 or 40 pages. So yeah. So anyway, let's continue. Now we're going to talk about mechanical engineering because I got this figure out of a handbook of mechanical engineering, which is that every tube has a diameter, a wall thickness, and a length. And that applies to the vertebrate gastrointestinal tract. We showed you that. But a schematic of that is we have an esophagus, a stomach, small intestine with different segments and a large intestine. This happens to be a diagram of a mature chicken, which is what I could find the diagram of in a textbook. But you'll notice each part here has a length that's very characteristic and reproducible from one individual to the next. And a characteristic diameter and length. And what controls that? Why are they so reproducible from one individual to the next unless you're mutant? How reproducible is our good data on how reproducible it is? I don't know that I have statistics that I could find. But I think if you have genetically homogeneous individuals like in mice, humans are sort of outbred, although they're not really, in the grand scheme of things, they're not really outbred. I mean, if you have a 4 foot 6 person and a 6 foot 10 person, I'm sorry, I can't translate that into meters right now. But it's going to vary. But if you take inbred mouse strains, I think they're probably, well, I'll show you why it's not measured in a minute. So I'll tell you why we don't know exactly. But I think in an inbred mouse strain, they're probably pretty good. I don't know the exact answer. And I'll show you in a minute why we don't know. Because if you have 10% change in intestinal length, you know you have inflammation if it gets shorter. Okay, thank you. So 10% change is a big change. Okay, well, I'll tell you why we don't, why nobody measures intestinal length in a mouse in a minute. But I'll tell you the adult small intestine in both a mouse and a person, we know exactly how it's organized and exactly how we make it through the work of a lot of people, but especially in the last 15 years, the lab of Hans Klebers who's in Amsterdam. So this goes back really 50 years or more, but in molecular terms, the intestine, small intestine is organized in what's called a crypt, in crypts, which are imaginations below the surface and that in villi, and the whole point of villi is to increase the surface area because the role of the small intestine is to absorb nutrients out of the lumen. And you want a maximum amount of surface area. And we know though that how these cells are produced is that there are stem cells here. This is the best understood and stem cell system in the adult or one of the best understood stem cells in the adult, the other being in the blood. The stem cells turn over slowly and then their daughters go through what are called rapidly transient amplifying cells and they move up like an escalator up here through the villi and they get sloughed off here. And this whole process takes three to five days and they're shed here at the villus tips and they go through differentiation process. They form four or actually five different differentiated types of cells as they go up here. That's a few, one type goes actually backwards down and one of the remarkable things, the whole process, the control of this is very well known in detail, is that a single stem cell, if you kill off one of these systems and you transplant a single stem cell, a single stem cell can regenerate this whole cryptvillus unit. So we know about how this regenerates. So regeneration of adult organs is a major field of medicine. We all want to be able to regenerate missing organs. And this is one example where you can do that very efficiently. Now development of this in the mouse is, or in the human, it's very well understood. It's much easier to study in the mouse because you can manipulate it genetically and you can go in and kill the mouse, study the mouse embryo it will. One doesn't like to do this in humans normally. And so in humans you have to depend on abortion or spontaneous abortion and so forth. But the mouse takes 20 days to develop from conception and you start to see around what's called embryonic, oops, embryonic day 13 and a half. You always deal with half days because you assume the mouse mates in the middle of the night and then you come in around noon, that's a half a day. This is just the convention. So at about embryonic 13 and a half days you have a single, simple layer of epithelium and around 15 and a half you have villi, the no crypts, the crypts in the mouse and the human don't really start to form until after birth. So as an adult you have these crypt phyllis units with different cell types. So going back to this, excuse me, I need a glass of water. Only in France can you get this nice fizzy water without having to go to some fancy system. Remember we talked about you have a characteristic diameter like the stomach is very wide and you have a characteristic length. Now the control of the length is a fundamental and almost completely unanswered question unlike diameter and why is diameter so hard to know? This is why I said to you, when someone asked this I think in the front row, we're talking about geometry here, although I suppose you would say this isn't really geometry because we don't have any Euclidean proofs. Why is it so hard to know? Well the problem is the intestinal epithelial tube is tightly coiled by the associated connective tissue. When you dissect it out, what you get is this tightly coiled bundle, okay? And there's that connective tissue that holds it in this tightly coiled bundle and for even a very skilled, practiced dissectionist person it takes 15 or 20 minutes to dissect the tube free from this and the connective tissue then collapses and that takes about 15, 20 minutes of work to do this and no one ever bothers which is why, you know... Oh yes Tommy. I'm just gonna name my rhyme. Pardon? I'm gonna name my rhyme. Well you can do this now on people or on babies. Or on mice. I don't know that anyone has forgotten this working on mouse routinely. Maybe you have this now on mice but it's not a part of the routine characterization that I know. If you look through dozens and dozens of phenotypic reports of mice, of mouse mutants I've never seen anyone report on intestinal lines. It's just not done, okay? It's not part of the phenotypic report. I suppose with MRI maybe it could be done, okay? But, and maybe with their... I think it could be done. It isn't. I haven't seen it done. It will be. Pardon? It will be. It will be maybe. It will be maybe. I haven't seen it done. And so I haven't seen it done. So you can look through. It's not routinely done at this point, okay? And so there, when you get, someone characterizes a new mouse mutant that dies at some point either in utero or at birth or sooner or after. It's not a routine part of the procedure. So, okay, let me just say this. It's now, you can routinely knock out, there's about 20,000 genes in the mouse genome or the human genome and you can knock out, people are systematically, systematically, excuse me, knocking them all out. A lot of them have no phenotype under normal lab conditions which doesn't mean anything by itself because it may be that they have a phenotype under some kind of stressful conditions or a phenotype when they are combined with some other knockout or some other mutant, meaning a synthetic, what's called a synthetic phenotype. But, okay, so, and there's a whole list, standardly of say, once, if you have a mouse that say dies at birth or dies in late embryogenesis, there's a whole long list of things that are normally measured or normally examined. Intestinal length is just never examined on anything that I've ever seen, okay? So that's why we don't have data on intestinal length. Okay, okay, I think I have my slides out of order. Okay, oh, I'm sorry, going the wrong way. So there are some humans that are born with very short intestines and these are called short bowel syndrome, okay? And these people, babies generally, the small intestine is so short that they do not have adequate nutrient absorption. So as I mentioned, the goal, the main role of the small intestine is to absorb nutrients and if it's more than about 50 to 70% too short, shorter than normal, you don't have adequate nutrient absorption and the baby is basically malnourished and starved to death. This is a high morbidity and mortality. There are 9,000 cases per year in the US. The only treatment is IV nutrition and that works very poorly in the long term. They essentially die because you can't get real adequate nutrition. There are things that don't work in the long term and the only cure is an intestinal transplant and that generally doesn't work in the long term. So there have been mutations in a few genes that have been mapped. These are just the abbreviation names of these genes and but there's been little further investigation of these, okay? And one thing I'll mention here is that regeneration of the small intestine, which I'm not going to talk about, but regeneration of the length of the small intestine is a long term goal, although it's not what I'm directly talking about. Now radially, as I said, a single stem cell can regenerate an entire cryptvillus axis and it can actually make it bigger than normal. So you can make, if necessary, if you stress the intestine, the intestine can get wider than normal. But longitudinally, small intestine never regenerates in length. So if it's too small at birth, it doesn't get bigger. Then it grows, it normally grows a great deal. I mean, a baby's small intestine is much shorter than a full grown adult's small intestine. But if it's 50% too short at birth, it will be 50% too short in adult if it lives that long. So our goal is if we understand the control of the length of the small intestine and they ultimately enable us to have some kind of therapy to increase or generate the length of the small intestine in short bowel syndrome. A lot of cases of short bowel syndrome are because for some external reason, we have to remove part of the short bowel, like if it's damaged for some reason. Okay, and I won't go into all those things. Now, intestinal cell kinase, which I will talk about at some length to explain to everybody. And I'll tell you that both the biologists here and the mathematicians and everyone in between probably don't know about it, is provided to us with a serendipitous clue to intestinal length control. Intestinal cell kinase is a ubiquitous serine, three unique kinases, a class of kinases, for those of you who are mathematicians. Kinases are enzymes that add phosphate groups to proteins that are involved in signaling. It's pretty obscure. It hasn't been studied much at all. It belongs to a very obscure family of kinases. It's particularly abundant in intestine, but it's found in almost most cell types. Now, the way we got into it is it was a substrate of a cell cycle related kinase, which Ying Yang was studying as a PhD student in the lab of Tommy McKayla at the University of Helsinki before she came to my lab as a postdoc. And another little bit of serendipity. So she wanted to come to the Bay Area because her husband was a computer scientist. She came from someplace in China to do her PhD with Tommy McKayla because he had a fellowship available. And her husband came to graduate school in Helsinki to study computer scientists. And he worked some years ago for Nokia when Nokia sort of really existed as a cell phone company. And he helped develop this cell phone game called Angry Birds, which some of you probably heard of if you're old enough. And so Nokia sort of went broke. But Facebook was interested in cell phone games. And so they hired him. And so he moved first to the Bay Area to work at Facebook. I have a connection to Facebook, unfortunately. I never use Facebook or any social media. But the guy who founded Facebook was his name Zuckerberg. His wife was a medical student at UCSF. And so since I teach all the first year medical students, I give them one lecture on epithelial cells. And so I must have taught her some years ago for better or for worse, although I have no memory of this. My lecture now on epithelial is now on the web. So they don't even get to see me in person. They just get to watch me as a talking head with slides on the web. So, here we go. Now intestinal cell kinase, you don't have to concern yourself with any of these details. But there's a way of making a mutant mouse that lacks it. And you can just buy the sperm for this. As I said, all the genes, every gene in the mouse has been knocked out by some consortia. And you can just buy the mutant sperm and breed the mice. And she made an intestinal cell kinase knockout mouse. And while she was doing this, two other labs published the same mouse. But she was very thorough. And unlike these other two labs, she dissected out the small intestine. Because she didn't know better that no one bothers to dissect it out. And so she dissected out the small intestine. And lo and behold, it was shorter. So I'm going to use this pointer. So this is the small intestine, stomach, small intestine, the secum, which is a structure at the end of the small intestine dividing the small intestine from the colon. And mice, I think, don't have an appendix. Oh, yeah, they do. They do a little thing here. The appendix is what gets taken out by doctors in all the movies and things. And the small intestine was shorter in the mutants. And so it's much shorter. So this is actually a graph. Over time, 13 and 1 half days through 18 and 1 half days. And you can see by 18 and 1 half days, it's much shorter. This is the wild type. And this is the mutant. The large intestine is also shorter. But it's harder to measure it as reproducibly. So possible mechanisms why this happens. So one is that there could be decreased proliferation of the cells. She's done just one experiment to look at this. It's promising. So one way of measuring proliferation is you inject into the mice a nucleotide called which is an analog of thymidine, which gets incorporated into the DNA. You can detect it fluorescently. And so the blue is a mutant. And the black is the wild type. You can see there's a small, but statistically somewhat significant difference here. These are the epithelial cells. And these are the mesenchymal cells that surround it. I'll explain that more in a minute. She needs to reproduce this using different variations of this experiment. There are a few other possible mechanisms. There could be cell death. There could be more cell death in the mutant. We haven't detected that. There could be a change in orientation of cell division. That is, when they divide, you could have the cells instead of dividing next to each other in the longitudinal axis, they could divide next to each other in the radial axis, the circular axis, around the circle. That happens as the tube would become wider instead of longer. And that actually happens in long airway development, as shown by our colleagues in UCSF, Gail Martin, and Wallace Marshall's labs in airway development. The airways, as they branch, they change the diameter in their length. But the shortened testins that we see are unchanged in diameter, at least as far as we've measured it. So let's talk about intestinal cell kinase. So intestinal cell kinase is concentrated in primary cilia, which I'll explain, though also present in cells that lack cilia completely. So could cilia be involved in intestinal length control? So there's no previously reported link between cilia and intestinal length. And there's no previously reported link between cilia and any aspect of intestinal development. So what are cilia? So cilia comes from the Latin word for eyelash, like supercilius. I don't know if that's a word in French. It's a Latin word. It means high eyebrows. So is that a word in French? Front? Eyebrows? Cilia? I don't know. I'm just trying to explain this to the non-sobalogist. But so cilia are little projections, very skinny little projections, from the surface of the cell. This is what they look like by scanning EM. And this is what they look like if stained with appropriate marker. So primary cilia, most cells in the body have one cilia projecting from them that are non-motile. There are other kinds of cilia that are motile that whip back and forth. And they have a core of microtubules that are called the axonine. And then they have a membrane surrounding them. At the base, they have a centriole that's turned into a basal body. And cilia contain more than 500 proteins. The mutations in many of the genes that code for these proteins cause diseases called ciliopathies. So there are at least 125 known ciliopathies. And many of them affect many organs, affect the retina, skeleton, a liver, cardiac defects, brain, kidney. Kidney cysts aren't the most common. But notably, the small intestine is not known to be affected. So I need to sit down for a minute. My back is really getting to me. Don't think I'm really bored. As I said, I have terrible back problems. And I just can't stand. And I think some of the people who came in late missed my point about I'll be glad to show you my back x-rays if you want. Can we see it? Pardon? Can we see it? I don't have it in the talk, but I have some slides with them. I have scoliosis with a 72-degree curve. So this is a normal mouse that E16.5. And this is an ICK mutant mouse. It's got facial distortions, polydactyly, extra digits, gross edema, face problems. So it corresponds to the human disease, ecosyndrome, endocrine, cerebral oste dysplasia, which is perinatal lethal. The mice and probably the humans die of respiratory failure at birth because the lungs are messed up so they can't breathe when they're born. It's very rare in terms of cases reported. But I think that's probably because, first off, it resembles a number of other perinatal lethal syndromes. And the only way you can diagnose it is by sequencing. In places with advanced health care, if you do an ultrasound, you'll see it's a grossly distorted fetus that's incompatible with life and it would get aborted because it's going to die at birth. And in places, and those aborted fetuses, generally no one's going to pay for sequencing. And although I did tell some people that I had my own genome recently sequenced for $350 from a new company called Genose.co. And $350 is quite cheap. People used to talk about $1,000 genome, but if you can get it done to $350, but no one's going to pay for that usually. And if it's in a place that doesn't have advanced health care, it'll be born, it'll just be stillborn. Almost all the cases that have been reported were consanguinist families. And so generally tend to be in places that don't have advanced health care. So it's not easy to diagnose. You can only diagnose it by sequencing because it resembles a number of other severe sygneopathies. So the psyllium is assembled and maintained by intraphlegia transport, which was discovered by Joel Rosenbaum at Yale using chlamydomonus as a model system because you can see things most clearly. So there's anterograde transport and retrograde transport, which moves particles up and down. And depletion of ICK causes a bulge in the cilia tip. This is the ICK depleted. This was in cell culture. Although you actually do see this in cultured cells from an ICK fiberglass. So that is generally characteristic of a decrease in retrograde transport. So things that get to the tip and then accumulate there. And what Ying did is actually collaborate with Hero Ishikawa with Wallace Marshall's lab at UCSF who has the TERF microscope. And he could measure live imaging of IFC cargo in IMCD cells, which have long cilia in culture. And so he measured the rate of IFT speed, anterograde, which was unchanged, and retrograde, which was decreased. This is an actual movie of live imaging. So we can measure the velocities. So with a loss of ICK, there was a reduction in, oops, reduction in retrograde intervigelar transport and accumulation of cargo at the tip of the cilia. Now, so with Jeremy Ryder's lab at UCSF, he had three other severe ciliopathy mutants that all died at birth, around birth, and apparently lethal. And they all have human ciliopathy that associates them, which are also all perineatal lethal. INPP5E is a lipid phosphatase concentrated in cilia. B9D1 and TCTN3, that's tectonic 3, are required for cilia formation. And they're all expressed in the fetal small intestine. And he had made the mice for these. And all of them also had short intestine. Here, INPP5E, B9D1, and tectonic 3 had short small intestine. They were less severe, but that just could simply be a matter of penetrance or of timing, of when they actually get turned on in the small intestine. OK, so the summary part one, I know I'm covering a lot, the length of the small intestine, and epithelial tubes in general is an unsolved and fundamental problem. I keep on telling Ying, in terms of when she wants to get on the job market, that she's discovered a whole new problem that people just have been sort of ignoring. I mean, the only people who have worked on tube length are in Drosophila trachea, which is a different thing. It's post-myototic. Takes place without cell division. And there's a translational relevance of short bowel syndrome. And the small intestine never regenerates in length. So there's, and everybody talks about regenerative medicine. And this is a whole area that is wide open. And ICK, as well as three of the cilia genes, control intestinal length. OK, now to the second issue, we get into real developmental biology. And so I've got some very elementary slides here. So an epithelium, I've been studying epithelium my whole career since, my whole graduate school career since 1979, when I started grad school with Gunter Global, who just passed away three weeks ago at Rockefeller. And he had spent 51 years at Rockefeller, won the Nobel Prize. So an epithelium is a tightly packed layer of cells that divides one surface from another. It's a topological division. It's, in biology, you can think of it as a two-dimensional surface. Although it's really not exactly two-dimensional, but you think of it as a two-dimensional thing. And underneath, essentially, epithelium is a basal lamin of a thin layer of connective tissue, extracellular matrix. And underneath that is a layer of mesenchymal cells during development, which is loosely packed. Now, this is development, OK, loosely packed cells. Now, during development, there is bi-directional signaling between the epithelium and mesenchymal, mesacon, by several signaling pathways, which are the blue arrows. And one major pathway is the hedgehog pathway, which was developed, which was discovered, of course, in Drosophila, and named a hedgehog. Because in the great classic screening by Eric Wiesthaus and Yanni Nuslin-Walhard, who did this great classic screen at the European Electrobiology Lab, the EMBL, for which they won the Nobel Prize years ago, the flies looked like hedgehogs. Hedgehogs, of course, only grow in the old world. They're not indigenous to the new world, although I'm told and I've read that one species of hedgehog that come from Africa, one type of desert hedgehog, have become very popular as pets in the US and elsewhere. And we thought about getting them as pets for the lab, but they're illegal to have as pets in California, it turns out. So that ended that idea. So anyway, the hedgehog signaling pathway requires invertebrates cilia, although actually does not require cilia in flies, as it turns out. And it was Catherine Anderson who discovered that the hedgehog signaling pathway needs cilia for signaling in mammals, but not in flies. So the pathway involves a hedgehog ligand, which is secreted by one type of cell, generally epithelia, and binds to a receptor called patch on the receiving cell, and patch is on cilia. So the receiving cell requires, oops, sorry, requires intact cilia for hedgehog signaling to function. So when the pathway is off, patch is on the cell surface, but it is not in the cilia. This is a simple diagram of the cilia. Cilia here is rather not very long, just to fit it in here. But when hedgehog ligand comes along, it binds to patch, and that causes patch stays here, but it causes smoothened to move from inside the cell. Smoothened is here to move to the cilia membrane, and that causes glee. Glee stands for glioblastoma, actually, to cause glee to bind to the microtubules inside the cilia, and glee gets proteolytically processed and moves into the nucleus, and glee is processed to what's called a glee activator and binds to the DNA in the nucleus and causes transcription of hedgehog responsive genes. And the hedgehog, this whole hedgehog pathway, is involved in developmental events in many, many different tissues. And this is a major developmental pathway in many, many different tissues, including, we think, control of intestinal length. So now hedgehog ligand is generally, in many tissues, secreted by epicillial cells and binds to patch receptor on the cilia in mesenchymal cells. And one particular reason for thinking this is an intestine is that in a small intestine, the intestinal cells generally don't have cilia, except at very early times. They have cilia at embryonic day 13.5, but they lose them in a few days after that. And by embryonic day 15 or 16, the cilia are gone. But the mesenchymal cells have cilia all the time. So what we think is happening is that the epicillial cells are secreting hedgehog ligand and it's acting on cilia on the mesenchymal cells. Now one way to test for this is that we can delete ICK specifically in the mesenchymal. And this involves some engineering trickery that's been developed. Dermal 1 is a promoter that causes expression of genes in mesenchymal cells. And it expresses, oh, that's not me, is it? OK, it expresses Cree, which is a recombinase which acts on these flocc sites. This is some modern day genetic engineering which causes the excision or loss of ICK specifically in the mesenchym. So loss of ICK specifically in the mesenchym causes short small intestine. We can see that here, embryonic 15.5 and embryonic 17.5, the black versus the white. And so ICK is there for acting in the mesenchym. And I can just tell you, if we do the reverse experiment and lose ICK just in the epithelium, it has no effect at all. Not showing you that. Now, to just show you some old results published by Andy McMahon's lab in 2010. And John Halmau, who did this experiment when he was at Harvard, he's now directs an institute at the University of Southern California. And John Halmau, I think, is at the University of Massachusetts when he did these experiments. When these guys deleted the hedgehog ligands, and there are two of them in the intestine that are redundant, Sonic hedgehog and Indian hedgehog. And I'll tell you that the Sonic hedgehog is actually named after the Sonic hedgehog video game character. And when you deleted both of them because they're overlapping and redundant, this is the entire intestinal tract from the stomach all the way out in the small intestine and large intestine. The whole intestinal tract shrinks by 90%, not just like the 70% or something that we can get, but 90% because it's a much more efficient, complete shortening. OK, so this is their old result. Andy McMahon is one of the titans of the hedgehog field in mice. So this we know that the hedgehog can do this. The hedgehog is needed for this. But then we wanted to go on and find out, well, I talked to Andy two years ago or something about this. And he said, oh, it's just hedgehog. But then as a cell biologist, I said, well, how does hedgehog do this? Andy's content to just say it's hedgehog. But I want to know how does hedgehog do this? Well, first off, two factors that are downstream of hedgehog or need for hedgehog to work, glee, which is this transcriptional activator, and patch, which is a receptor, are decreased in the ICK knockout relative to the control. So that's glee here and patch. And you can see they're both decreased. The black bar is versus the white bars. And so how did the decrease in hedgehog signaling lead to short intestine? So we asked, could change in mechanical force be involved? Because everybody in cell allergies been working on mechanical force. And I have to say, we were partly inspired by Cliff Tabin, who is my classmate from my undergraduate days in Chicago, and his student, Amy Shire, who is now a fellow at Berkeley. His name is Dave Drew, and you can tell us. I think, you know Amy, don't you? She's one of these fellows at. Is she in Richard Harlins? Yeah, she's in Richard Harlins' lab, yeah. And so there's the epithelium, which is a single layer, and it's surrounded by mesenchymal cells. And some of those mesenchymal cells are precursors to the smooth muscle cells that surround the intestinal epithelium. So I showed you about mesenchymal cells. Now, some of them, our intestine has a single layer of epithelium, which absorbs the nutrients. And it's surrounded by, as I mentioned, I teach histology, or I used to teach histology, to medical students. When I was a medical student in 1977, I started, I had, am I running out of time? OK, then I'll stop telling stories. OK, so become smooth muscle cells. And these smooth muscle cells contain alpha smooth muscle actin, and that can produce mechanical force. So here in the control, we stain for smooth muscle actin in green, and blue are just the nuclei, the DNA. And in the intestinal cell kinase mutant, the smooth muscle cell actin are very disorganized. And we know from other people's work that these smooth muscle actin that are disorganized probably produce much less mechanical force when they're disorganized like that. Now, so then there's this protein called YAP, which everybody works on now. And YAP, which stands for Yes Associated Protein, is a key mechanosensitive transcriptional regulator. So both biochemical and mechanical cues control the movement of YAP between the cytoplasm and the nucleus. Cytoplasmic YAP is inactive, but when it moves into the nucleus, it becomes a major regulator of cell proliferation and organized control. And in YAP is here in red. And you can see this is the control. A lot of it's in the nucleus. But here, a lot of it's in the cytoplasm. It means it's diffused. You don't see it, especially it corresponds to co-localizers where there's a lot of the smooth muscle actin, which is sort of much more disorganized compared to the control. So the summary, again, just to repeat, the length of the intestine and tubes is an unsolved problem. There's a short bowel syndrome. Small intestine never regenerates in length. And the number of cilia genes control intestinal length. Cilia likely control intestinal length through hedgehog signaling in the mesenchymal. Hedgehog probably acts in mechanical force generated by smooth muscle actin, mesenchymal cells, surrounding epithelial cells. Mechanical force probably acts in YAP, mechanically sensitive transcriptional regulator. So Yang Yang did all this work with help with Jeremy Ryder's lab, Wallace Marshall, and Hiroki Shikawa. And Tommy McHale's lab, when she started this, with Pekka Pavinet, an undergrad who actually came to visit us one summer two years ago, Johann Piranen, who's in Tommy's lab, funding from the usual suspects. Thank you. Sorry to take up so much time about math, but you did make me start eight minutes late. Tommy has a question? So how do you know that? I can never answer his questions. Yes, go ahead. Try. Try. Yeah. How do you, in the last part of your talk, how do you establish that the relationship is causal and not causal? It's causal and not correlative. We haven't yet. But we are going to. And the way you presented it, you said it was... I said may. I always had a may or might. But we're doing experiments, like killing all these smooth muscle actin cells. Because what you can do is you can put, we're in the middle of doing this. Actually, we have some results that are looking indicative. You put this, you use a smooth muscle actin for motor hooked up to diphtheriotoxin. So that kills the smooth muscle actin expressing cells. And that restores the lengths of the intestine. So it's the opposite. What? It's the opposite, right? No. If you get rid of smooth muscle actin contractility. Wait, is this right? Yeah, it's the opposite. Try to remember this. If you have no contractility, well, it doesn't make it short or long. We're going to make it short, okay? But we're also manipulating YAP activity, okay? You manipulate YAP activity and you manipulate actin contractility, you manipulate those independent of the upstream things like acilia. We're manipulating those things independently, okay? And that can establish causality, or at least begin to establish causality, okay? That's the basic key. Keith, we have questions over here. In what cells do you see changes in the YAP? The epithelial or the mesenchymal? The mesenchymal. The YAP is mostly active in epithelial cells. Well, these cells are- That's where it controls cell survival and cell proliferation. Well, here we're seeing- I don't know that the YAP is important in mesenchymal cells. No, here we're seeing it in mesenchymal cells, okay? These cells are the ones that have mechanical force. It's strange. No, but YAP is in a lot of cells. Yeah, but the YAP phenotypes are usually epithelial. No, we're seeing it in mesenchymal cells. We will manipulate it. As I said to Tommy, our plans, I don't know if we have a result, our plans are to manipulate YAP and act in independently of the cilia and hedgehog, okay? So that will give us something. Yes. What is the ICK target? Our ICK is a kinase. I know, what is the target? Well, okay, so we are doing that using the Keven-Schokac techniques. Because with that, you can identify the Keven-Schokac using artificial substrate. You can identify the directory, but that's not the point. Hedgehog is phosphorylated by ICK. Pardon? Hedgehog. What? Hedgehog is phosphorylated by ICK. But, okay, I can't run this. I'm, Hedgehog is phosphorylated by ICK. Sorry? Is hedgehog phosphorylated by ICK? Oh, I don't know, but the issue is it doesn't matter because other cilia genes give the same phenotype, which are not kinases. So ICK, it's not ICK per se, it's cilia that are needed. Okay? Three other cilia genes give the same phenotype. So it's not limited to ICK. ICK was fortuitous. Yes, the question is why ICK is required for cilia. No, ICK is not required for cilia. ICK is required, is, ICK stimulates retrograde transport. But, ICK is not required for cilia. And three other cilia genes, at least three of the cilia genes give the same phenotype, which are not kinases. Okay, so. At each stage of embryo development, actually. I'm sorry, I can't hear you. At each stage of embryogenesis, this length being determined. A length of the. Of intestine being determined at each stage. Which stage? It's a continuous process. So it's not decided where anomaly happens, at which stage the development anomaly happens. It's a long process. Yes, and it continues postnatal. I mean, the intestine continues to grow until the organism reaches adult. I see, so anomaly happens if it attracts all development organ, it makes slow everywhere, what? So it becomes slow in every way, it stops, right, it becomes slower. Or something stops, some stage stops. All stages. Or everything becomes slower in some stage. I don't know that that's known. But I know that the intestine continues to grow in a human, the intestine is approximately three times body length. Okay, now it varies in species depending on the diet. So in general, carnivores have shorter intestines and herbivores have longer intestines. That's a species difference, and that's a very different question. No, no, no, my question is time, time dependence. Yeah, time dependence. I don't know that that's been studied in great detail, but it's growing throughout both embryogenesis and postnatally until you reach full adult length. But anomaly happens at which stage, when you see something develops slower. It's kept as early or later. Both, I mean it's, when we see it. What's the first stage to become a human? Different human. Perhaps we should continue this at the coffee break. Yeah. The two of you. So let's thank Keith while he's searching for the picture. And.