2009 Web Conference with Dr. Arturo Alvarez-Buylla

- 00:00 Tell us about yourself and your career and how you started.

- 00:06 Well, I actually grew up in Mexico, where I did my undergraduate in a program called biomedical research, and I came to the U.S. to study my PhD at Rockefeller University, where I work with Fernando Nottebohm, studying neurogenesis in canaries and songbirds. And there is where I became very interested in the mechanism of adult neurogenesis, how can cells really get going in a complex environment like the adult brain. And there's a lot of different processes going on from the birth to the migration to then the integration within circuits that are already working, because it's not like the brain has been built. The brain is really put together at a different time, and here the brain is already functional. And circuits that are functional, while not losing function, have to integrate any neurons, and that fascinated me. So that's how we started. Then, I was working off that, I was offered a position at Rockefeller very soon after finishing my PhD at Rockefeller, and I had my first graduate student that came, and he actually told me that he couldn't care less about birds because they were so irrelevant for medicine. He wanted to do something relevant to medicine. And this actually is a very very brilliant student that then went on to do the pioneer work on the migration to the olfactory bulb. He actually, by the way, he's now professor, he has his own lab at MIT, and he's actually now working in birds. See how things go around? I got into stem cells. When we started, the field of stem cells really did not exist. In 1986, while I was still a graduate student at Rockefeller, there was a meeting in Waldorf Astoria where there it was proposed that the fact that this process were going on in the adult brain could open up a whole new field of progenitor studies, and I think that was a little bit one of the places where the stem cell field, at least for the brain, began. But there was very little insight into what it was going to become and how huge. It's more like a fashion now than sometimes real science. So that's a little synopsis of where I came from. And in 2000, I moved from Rockefeller to here, the other side of the continent, to California. And I've been here now nine years at UCSF. 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- 02:35 Out of curiosity, how big is your lab now?

- We're 11 people.

- What?

- Postdocs, graduate students, undergraduates. Do you have a full array?

- 02:49 Yes, it's right now, this year, three graduate students have graduated. There's actually one graduate student left in the lab, and two that are doing or planning rotations. So the rest of the lab is technicians and postdocs. This is a very interactive institution, so my lab is embedded within the stem cell program. In our ﬂoor is David Rowitch, Arnold Kriegstein, and Miguel Ramalho-Santos. We share lab space, we share a lot of equipment, and there's a lot of, also, studies that are in collaboration with a different lab, where postdocs and students are participating in projects that are interdigitated with different programs and different labs.

- Okay, so moving on to our questions. Our ﬁrst one's gonna come from Alexandra.

- 03:39 So, could you please summarize for us the structural characteristics of the adult neural stem cell niche and how its organization helps to foster stem cell renewal and neurogenic and gliogenic properties?

- 03:55 So how many hours do you have? That's a very deep question. As I mentioned before, when we were working in birds and canaries, we were already excited of how can new neurons be incorporated into a brain that is fully functional? And one of the questions, where these cells are coming from. And even back there, now it's almost 20 years ago, we made the, at the time, relevant observation for mammals because people thought it is was something special for birds that a glial cell was a stem cell. And years later, through work of another graduate student at Fiona Doetsch's lab, we found that also glial cell was acting as primary progenitor in the adult cells that were going to the olfactory bulb, that actually I had mentioned to 2009 Web Conference with Dr. Arturo Alvarez-Buylla

you Carlos observed. And that got us very interested in the whole lineage of going from brain ganglion to astrocytes. To the composition-- I guess the ﬁrst part of your question is how is this niche for neurogenesis organized, and our initial view from the works of Human Touch was that these glial cells that people had identiﬁed as astrocytes and we still call them astrocytes, we call them B cells in superventricular zone were actually right under the ependyma and that's why this region is called subventricular zone. Because something happened while they removed from the ventricle. And that D cells divided to generate another cell then, we discovered in the lab, that we call C cell, that's the transient amplifying, does everyone know what a transient amplifying cell is in the lineage?

- Yeah.

- 05:30 Just let me know if I'm talking in terms that are not understandable. So this transient amplifying cell then turns into the young neurons that go to the olfactory bulb. So if I can draw it here. So, the idea was that there was an ependyma and these cells are very interesting and we can spend time talking about them because they are really fascinating cells and I can tell you more about them. And then right underneath it there was these very complex cell that had processes in between other cells, that we call B cells, and next to them, there were clusters of more rounded cells that are these C cells. So it turned out that D cells that were not simple at all, give rise to the C cells, and then these C cells differentiated into young neurons and these young neurons from chains that migrate to the olfactory bulb. But this four-fold migration was another thing that Carlos Lois discovered in the lab. Chain migration is a way by which cells slip along by each other and migrate very quickly to the olfactory bulb. So the niche has to coordinate and organize three processes, birth, ampliﬁcation, and actually migration, because migration already happens within the niche. And so you can see that it is quite complex and this was our initial view of how it was organized. The recent work of one of the papers that you read actually suggests that these B cells are not this kind of mushy things that I drew, but actually very interesting cells. The 2009 Web Conference with Dr. Arturo Alvarez-Buylla

ependyma cells have little holes through which these B cells project, and then they have other process going back and then these cells have very long basal processes that actually end up in blood vessels. And some of your questions were related to this. So, as you can see, these B cells are much more epithelial than we had originally thought, and how both an apical where there's actually a prime receiver, another structure of great interest to us, and a basal structure and so, part of the clues to how this niche is organized has to do with these basal apical organization. And also this has another important clue that we had predicted before, that is that these cells are related to the early radial glia that are also epithelial cells, and the radial glia in turn, are also derived from another epithelial cell. So it goes from neuroepithelium to radial glia, to a cell that has actually something morphology in the adult, but it's actually retaining epithelial characteristics. So that's in terms of the progenitors. In terms of the C cells, we know very little. But we think that there is, we always see them closely interacting with C cells with the B cells, so they form clusters right next to the C cells, so we believe that there might be some interactions here that are important factors, that D cells are feeding these C cells. This remains a big question, because we do not know how many times these cells divide, how specified they are, how much program there is in the division of these cells, so a lot of this is unknown. And most likely, within these cells is that programs to start building neurons start to be created. And then from these cells generate the A cells, and then we know a little bit more. So this niche integrates, as I said before, birth, amplification, and migration. And one of the interesting questions is that these cells have migrate actually very far, go very very long distances. So how come they don't get lost in the brain? And what we have seen is that the organization of these epithelium, of ependymal cells with their cilia, plus chemo-repellents as Slit one and two actually there are secreting cerebrospinal fluid creating a directional gradient that actually moves these cells forward in the right directions to get to the olfactory bulb. So you can see the niche integrates both signals to regulate the behavior of the cells, signals that are still largely unknown to regulate the amplification of these cells, and this is very much related to cancer, because if these cells stop 2009 Web Conference with Dr. Arturo Alvarez-Buylla

amplifying, they go on to generate a tumor, and then signals to organize the migration, the correct migration of young neurons once they're in this other compartment. So there's a lot here to understand and study and we're just getting the initial hints of how it might be working. Is that clear?

- Yeah.

- Were you able to see my drawings at all?

- Yeah.

- That's fantastic, actually.

- 10:09 So is this structure is it unique to the lateral ventricle, or is it in other locations in the central nervous system, and in the body?

- 10:22 So, you see this little connection with the ventricle? When you look at in sections it looks like these, but as you saw from the paper from this student, Saman Usaday, When you turn this around and look it from the face, from within the ventricles and look at that face, a really big interesting surprise emerged. And perhaps I should tell you the background of this just to see that you get a little sense of how discoveries are done in the lab. I told you already that the orientation of these epithelium somehow controls the direction of migration of neurons one layer behind. So one of the questions that this student Saman Usaday, became interested saying well really, people are very focused on molecules molecules, molecules. This molecule controls this part of this mechanism and so on, but in this case, the mechanism is a two-fold problem. One is the molecule itself is being secreted into the spinal ﬂuid, but the other is the orientation of these epithelia. And that's a fascinating problem that many labs are working at now. How epithelium organized in this plane, that's called planar polarity, so is the organization of the epithelium within the plane of the epithelium? So there's two kinds of epithelial polarity, the apical basal polarity that I told you about is present also in stem cells, and the other one is planar 2009 Web Conference with Dr. Arturo Alvarez-Buylla

polarity. So one way to look at this epithelium is to open up the ventricles and look inside the ventricles. And this is what you see. So this is how a lot of ventricles in a mouse looks, this is the ventricles inside the forebrain. There's a little canal here that communicates the lateral ventricles with the third ventricle. And then goes through the fourth ventricle, and then to the spinal cord. And so cerebrospinal fluid flows from here down to third ventricle and then to fourth ventricle and so on. So, if you put this into context of the brain the cortex would be here, the olfactory bulb would be here, and the migratory stream goes one layer underneath with the cells migrating like this towards the olfactory bulb. So if you look, one way of looking at the polarization of these cells is that this is an epithelium made up of millions of these ependymal cells that I showed you before. So here these cilia are sticking towards you and each one of these cells has cilia, and each one of these cilia is moving very actively, and one way to see this polarization is, for example, to put a little India ink here in live, open ventricle that you just dissected out from a mouse brain, and what you see in real time is the India ink move incredibly fast like this. and then from back here it moves also like that. So that's one way of looking at the polarization, but that's complicated, because first you're not looking at the cellular level, you're just looking at groups of cells together, and it requires you know killing and looking at one hemisphere every time, which is very inefficient and difficult to do. So Saman, really, the way he got into this is he wanted an easy way to look at to see how this epithelium was polarized. So he took little fragments of this wall, and he kept the orientation of that fragment like it was, and he said I know, for example, in this part of the ventricle, the flow is like that, and now you take that and amplify it greatly, then I'm going to look for markers within the surface of those cells that will allow me to put an arrow in the direction of how the cerebral spinal fluid is flowing. And he first looked at the stains for just cilia, and it's very complicated because it's just like a field of grass. It's covered with cilia. But then he looked at the basal bodies of cilia, and he found that the basal bodies are actually all towards one side of the cell. Everyone knows what a basal body of the cilia is? 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Yeah.

- Yes, okay. So all the cilia are polarized to one side of the cell, and this allow him not to have a readout for polarity. You know, and again, this is an interesting question, because now he could study how this polarity comes about, and it's one of the things that actually he's now trying to do in the lab. At the same time he's actually trying to finish medical school, so it's a complicated thing. But this was his thesis, right? But when he was doing this, he was careful enough to notice something that was quite amazing and remarkable. That there was a pattern to all of these. So he looked at ependymal cells, and you all have seen the real pictures of the paper in these, and even though they were all polarized to one side, they were forming pinwheels like this, and this pinwheels had inside, in the center, very small apical endings. And each one of these endings have a single basal body. Sometimes they have two basal-- I believe that these have, that cells might have been G2, and these basal bodies were isolated to one primary cilia. to one primary cilium. So it turned out that these are the B cells. So you see, there is a pinwheel organization that you see throughout the wall, throughout these walls, and it correlates the hotspots where these pinwheels are actually places where we know a lot of new neurons are born. So your question was, is the patterns specific of these wall to these areas that are neurogenic, and the answer is absolutely yes. Because if you look throughout this system, in the third ventricle you do not see pinwheels, obviously this wall has another wall on the opposite side. The medial wall looking at the septum, and you there do not see these pinwheels. You only see them where neurogenesis happens, and you see them in areas where-- I'm sorry, you see them more frequently in areas where we know more neurons are born. So yeah, there's a specific organization to the neurogenic niches, and those areas that have neurogenesis maintain this structure, and those that do not don't have it. Does that answer your question?

- Yes. 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Please interrupt me if I say anything that you want me to clarify.

- 16:39 How does the pinwheel formation compare to the other stem cell niches in the body?

- How does the pinwheel organization compare to--

- Other niches...

- Of other

- 16:58 Yeah, I don't know, we haven't looked ourselves, but I mean, the description of the pinwheels is fairly recent. And we have seen other epithelium Saman was showing me the other day a skin stained, an embryonic skin stained with certain markers and there might be some evidence there of similar organization but I wouldn't be able to answer that question because we haven't looked at that and I don't know that there has been enough people still that have looked at the organization to see if this is a common feature of all your neurogenic, derminal niches. It's an interesting question but I don't think the information is there yet.

- Thank you.

- Next question is going to come from Elise.

- 17:40 Hi, my question comes from the discussion of your stem cell journal paper. Could you please elaborate on how the contacts that are made between the B one cells and the neural vasculature might inﬂuence their development, their differentiation and or their proliferation from adult neural stem cells and what factors, speciﬁcally how does it play?

- 18:05 Yeah, that's a very, very interesting question and again, it's one one that just looking more at the future. So, we speculate that these interactions with blood vessels must be very important, but that's based on work that other people have done. Work that 2009 Web Conference with Dr. Arturo Alvarez-Buylla

Sally Temple actually has done not very far from where you are, has shown that when you cold culture these stem cells with endothelium, and they don't need to be touching each other, but even if just the there's conditioned medium endothelium together with the stem cells, these cells are much better at making neurons as what they normally do within the So one possibility is that there's some kind of signal coming through here that makes these cells become neurogenic. Now, I think one of the things that comes out from the anatomy from looking at the way the cells are organized, is that most likely the cells are integrating information from this part of the cell, together with information of the optical end. So we know for example, that noggin, which is a protein that inhibits empty signaling is very important also to make the cells behave as neurogenic. So these cells might be computing information that arises at blood vessels and information that arises at the ependymal level and even information possibly that arises at the level ventricle. The primary cilia is now well known is like a little antenna, its for example where the sonic hedgehog receptor are that these cells integrate all that information to make a decisions to start dividing. So, again, a speculation. Fiona Doetsch, now in her own lab, she was a student in the lab that initially did the description that I told you before, has found that C cells also make contact with blood vessels. So one possibility is that this connection is dynamic and then once the cell becomes neurogenic, it can actually remove this contact with the blood vessel, and now, groups of C cells get in contact with that niche that's created by an open blood vessel that has no covering of epithelial cells. That was one idea. The other is that PDGF signaling, platelet-derived growth factor is known to be very abundant around these endings. And from works and another proposal in the lab we know that these cells are coated. With one of the receptors for PDGF. And actually, if you put PDGF into this niche, you can deregulate the proliferation of these cells and these cells go on to form tumor-like masses like gliomas, right next to this area. So one idea is that PDGF might be important at this ending. The other signal in time that is possibly important is AGF. And AGF2 has to be shown to be rich around blood vessels and that's another signal that might be interacting with the ending so this exercise says to make them act. But 2009 Web Conference with Dr. Arturo Alvarez-Buylla

all of these are hypotheses as our basis of data, but there are all hypothesis. Your third hypothesis is that blood vessels have associated to them, a very complex network of extracellular proteins, and some of those extracellular proteins have been shown to interact specifically with B cells, and that interaction actually either fosters the binding of growth factors to send them the right way, or directly might be necessary for these cells to act as the stem cells. And the unique thing is that even their blood vessels all around the brain, some components from these extracellular matrix are only present within this region. So again, suggesting extracellular matrix might be providing these signal that are important for the behavior of these cells. One final idea that Sofia, is probably going to be very interesting to all of you. comes if you see this is very similar so I can turn this around. And now, we can alert drawing here. So forget about these for a second. And you all know about the ventricular zone during development, and the cells have with radial glia now are known also during development to be the stem cells, they end up at the surface of the brain, where there is a very important vascular niche there also. And as the brain grows bigger, these cells make little, the right radial glia make little side connections with blood vessels that penetrate the brain. So, both in the adult and in development, this is an interaction of the stem cell with blood vessels. There is a recent paper from this institution from someplace that has identified that retinoic acid secreted by the meninges, which is heavily vascularized, actually triggers these cells during development to go from proliferative state to become neurogenic. So another possible factor we could put here is retinoic acid. So that gives you some, I mean your questions are very interesting, but there's a lot of possible ways to be installing these. But basically one of the very interesting aspects of how vasculature is the interacting with these cells. One additional thing, in the hippocampus Theo Palmer has also found the hippocampus is another region where neurogenesis continues. These stem cells are also astrocyte-like cells but these are away from the ventricle. And there they're also interacting with blood vessels, and there is a specific niche of blood vessels there right next to the stem cells that is supposed to be important for 2009 Web Conference with Dr. Arturo Alvarez-Buylla

neurogenesis there. So it's not a direct answer because there's no question but different leads into where we're thinking about the problem. Does that help?

- Yeah. Thank you.

- Our next question comes from Heba.

- 23:54 Hi, in general, what do you see as the role of cilia and adult stem cell function?

- Where are you, I can, I can, oh, okay.

- Speciﬁcally, B2 cells are described as active phase with multi-solar morphology of B2 cells affects its location.

- 24:22 Well, you know initially we described the cells as I said in an original finding, in sections show these cells were kind of amorphous and multipolar like your question indicates. But I think new work clarifies that even though they have these appendages, they have a prominent article-based cell organization. So, going back to your first question. I mean, these cells have a primary cilia here, and primary cilia have become very interesting in many ways. Now, this was not the first time that we saw primary cilia. We had actually seen primary cilia back in the very early 90s when we discovered radial glia in canaries has been the stem cells. And there we noted that these primary cilia is such a unique location that even though these organelles have been very interesting. Ellis have not been very interested in them, but they might be doing something very interesting. And this is how a post- doc now in the lab jungle who actually got into, he saw one of these publications he was working sensory organelles in drosophila And he wrote to me and said, I think there are now the tools available to start asking the question, what were the cilia important in what they're doing. So, these cells, like good astrocytes express glial fibrillary acidic protein. So, this is an intermediate filament, that is very abundant in astrocytes, and these cells express it and that's why it was thought for a long time that they were just differentiated astrocytes. Now it is well known that 2009 Web Conference with Dr. Arturo Alvarez-Buylla

actually GFAP is in many progenitors even during development including radial glia. So he thought that if we could, there is an animal that expresses CreE recombinant under the GFAP promoter that that could drive a specific expression of genes or mutations in these cells, and we throw these animals with animals that carry as flocks, And Qi 3 is sub-unit of a kinase 2. Kinase 2 is a mortar that is required for building the cilia. And even though there has been just by removing other things from all the recent work that my lab and other labs have done, it looks pretty specific that this mortar is essential to make the cilia. And again, here we could go for a long time because it's actually quite interesting. Kinase is life's cargo from the cytoplasm into the cilia to build the cilia. And what you see is normal cilia look like these, with microtubes inside, nine plus two structure. In the mutants it looks like these. Just a little blob without microtubes inside but still the basal body which is essential is associated with the structure. So, we decided to do this mutant, expecting a big phenotype here, and we did get a big phenotype here. But unfortunately, this phenotype is very difficult to interpret. Because ependymal cells, which are right next to them also have these long cilia which are essential for some of the things I told you before. And we also eliminate the cilia in this mutant when we do this cross. So these animals have hydrocephalus, they have abnormal ependymal cells. So all of the niches are messed up so we cannot know whether the things that we observe are specific to the cilia or are specific to all of these mess that we have made. But then, Yang Wu was sufficiently astute to look in other parts of the brain and he saw that in the hippocampus that I just told you, there was a phenotype, where there's also a stem cells where also we knew that they have cilia and the cilia was also removing those cells like I show here. And what he saw there is that in the transition, of going from an epithelium to the adult empty gyrus is part of the hippocampus and the stem cells are here. The cilia was essential to make the transition from these stem cells to these stem cells. These are the stem cells that are left throughout life, this is an empty gyrus. Is this CA3, CA2 and CA1, the rest of the hippocampus, so these transitions establishment of post-natal stem cells, the cilia play an essential role. So, we have focused more on this system to study how the cilia is working and I could tell you more if, I don't 2009 Web Conference with Dr. Arturo Alvarez-Buylla

know if, about how this is working and through which signaling pathway. But I can also tell you about other things because we can spend a long, time telling you about the, the cilia and the gyrus.

- Okay, thank you. So, next question.

- Okay, so this is from your latest paper... so yeah and the

- And that's you Melissa?

- What, oh, Alyssa,

- Oh, Alyssa, oh, okay,

- 29:19 Um, so the human GFAP 3 Kif3a ﬂ/ﬂ mice, they suffered from a smaller sub-granular zone suggesting that a primary ciliate do in fact play a role in the proliferation of progenitor cells. But it was observed that a small population of the BrdU stain cells survived in the granular zone. Do these cells appear simply because they developed earlier, or is it because they're possibly a different sub-population of cells?

- 29:49 Well, that's an interesting question but there are many, many answers to it. I mean the short answer we don't know. One possibility is the recombination was not complete. Another one is just what you mentioned that perhaps some of these cells escaped recombination because it comes from an early earlier stage, but the other is that we did not characterize those cells very carefully and those just could be cells that did not belong to the same lineage in which we cilia was eliminated. And one fourth possibility that's equally possible, is that in stalling the development of the cerebellum and we were among the ﬁrst two see this, actually Matt Scott and Ariel Ruiz i Altaba were the ﬁrst. It's known that the Purkinje 2009 Web Conference with Dr. Arturo Alvarez-Buylla

neurons that are deeper produce sonic hedgehog that then moves to where the cerebellum layer is. And there it activates in the cilia patch, which releases Smoothened repression and then allows those cells to divide. But whether this is a known or orphan phenomenon is not clear. I mean, some cells could be able to go past and divide a little bit, without any of the signal without minimal signaling. Or there could be some minimal signaling going on in those cells that are worrying it will just skip, because those cells might have some basal level of transcription activity already there. So, there is many explanations of why there is so few cells, I mean, the point is that there's very, very few cells and the cerebellum in those animals are much, much smaller than in normal animals. I mean, they're one fraction of the size of normal animals.

- Thanks Arturo, can you ﬂip back to the video?

- Did you guys had a chance to uh, did you guys had a chance to read a recent paper also from John Wu on the cerebellum again on neuroblastoma on the cilia?

- We did not read that one, no. So, should I tell you in two seconds when we had just found, the most recent thing?

- Sure, yeah. Because the last question--

- Yeah.

- The reason why I'm telling you, because it's very, very intriguing. So, I hope I'm able to explain this carefully. So, can you put it in the paper.

- Yeah.

- So, this is the cilia, and the cilia has Smoothened, which is the receptor for, sorry, it was considered core receptor, its a signaling pathway required for sonic hedgehog signaling. So Smoothened acts on lead 2 to provide a transcriptional activation but 2009 Web Conference with Dr. Arturo Alvarez-Buylla

also represses another transcription, transcription for cold lead three, which normally acts as a repressor. So you see, these D represses these repressors so it produces full activation. Is that clear?

- Yep. The way Smoothened gets activated a step before this, there is the receptor for Sonic hedgehog's patch, patched by sonic hedgehog and by banning Sonic hedgehog it now goes out of the cilia and allows Smoothened to go in. And these movement of Smoothened into the cilia is what activates the pathway. So, when we remove the primary cilia, we've removed the ability of these cells to respond to Sonic hedgehog and therefore, there was no growth in the cerebellum and most granular cells are not good use. Now, one interesting question was if we activate Smoothened 2 independent of Sonic Hedgehog, and there's a mutation for that. An important mutation was isolated from a human skin cancer that is permanently active so this Smoothened is bombarding the system with Gli2 signaling and then, is preventing Gli3 from forming so there's no repression so this is fully active. It is like the having the faucet fully open or a switch all turned on. When you do this. This produces a tumor in the brain. So the most common tumor in children called a neuroblastoma. So one of the ﬁrst simple questions that Jun Wu wanted to ask is, well what happened now. if we remove the primary cilia? Smoothened is an oncogene here and now we might have a case where Smoothened is able to act independent of the cilia. So he crosses animals with activated Smoothened to the key three A's conditionals, and when he crosses these animals, there was no medulloblastomas.

- Huh!

- So even in the context of an oncogene like this, A cilia is required for it to produce tumors. So in this case, if there's tumors in humans which there are, which are activated through the system. If we had them though I don't think we were removing the 2009 Web Conference with Dr. Arturo Alvarez-Buylla

cilia, we would kill those tumors and prevent those tumors from forming. Now here comes interesting twist. You would say, well now, instead of using to produce the tumors, We use Gli2 which is the activator. And there's also an allele that is permanently active of these that's actually truncated. So, if we put these, we should be able to produce medulloblastoma independent of the cilia. So we need these, and we need animals with deactivated Gli2. And these animals do not develop medulloblastomas. However, when we cross these animals again to the key Qi3 A conditionals So we have an animal that has activated D2, and no primary tumors. Now these animals give rise to medulloblastomas. So, depending on the oncogenic event, either the cilia facilitates or prevents the formation of tumors. Isn't that interesting? Totally Opposite growths. In one case is an on switch in another cases in off switch. So now we have gone to human tumors, and St. Jude's Hospital had a large set of tumors they had already tested for what signaling pathways were abberant. And what Jun Wu found is a group about 30 tumors all of those tumors that were activated, this was Sonic hedgehog and actually wind. Sonic hedgehog and wind. All this have primary cilia. All of the rest of the tumors except one, and this have different kinds of mutations, makes different kinds of mutation that none of them have primary cilia. So actually primary cilia can be a very important diagnostic tool, but its suggesting that some tumors need to keep the primary cilia to do their harm and get to grow. Other tumors actually need to remove the primary cilia to get to grow. And the reason why I'm telling you this is that the question before was very general. What, how do we see primary cilia working. The way we see primary cilia working is totally as an on and off switch. When you want it on, you want it totally on, when you want it off, you want it totally off. And actually, since the primary cilia is associated to the basal body, and these basal bodies are in fact, the centrioles that are going to give rise to the central zone. The primary cilia is tightly coupled to cell division. So some people have called primary cilia like the key to cell division. It is the thing that's controlling whether the cell is going to go into cell division or not. So one way of seeing based on what I just showed you. is this on off idea. So you can actually know based on what I had told you. Conceptualize 2009 Web Conference with Dr. Arturo Alvarez-Buylla

how these might work. We go back here. When you want to have it on you activate the activator and repress the repressor. When you wanted to have it off, just to make sure that its off. Obviously there is no Gli1 activation, but just to make sure that is off. You keep P3 there as a constitutive repressor. So I don't know if any of you has ever played around with a switch in the wall of your of your house but if you open it, they have little springs. The switch either goes on or off. There's some switches that stay in between but those are more either malfunctioning or they're done to regulate the intensity of the light. But most switches are either on or off. And they do this through switches, er, through springs. And the springs are there to either keep it on, or to keep it off. And I think the idea of having a repressor all the time there is exactly the same thing. It is like a spring that is keeping this thing off when it's off. So this gives you a little bit more conceptualization of how we're thinking about this little organelle that has now become center stage in biology.

- Arturo, in the context of the Gli2 mutant that you have, and when you crossed it with the Kif3a what do you think Gli3 is doing in those two different mutants?

- So, in the case here, in the case of Gli2-- So, in the case of Gli2, so we have Gli2 activation. We get no tumors.

- Right.

- When we remove the cilia, now we get tumors. And when we're removing the activity in Gli3. So in these animals with cilia, they have Gli2 activator but they still have Gli3 repressor. Right?

- Yeah.

- 39:54 So the Gli3 repression is sufﬁcient to counteract effectively of Gli2, so at least that's what we think. So again, part of the off switches remains there, even though part of the springs turning it on are trying to turn it on. But these springs are strong enough to 2009 Web Conference with Dr. Arturo Alvarez-Buylla

keep this off. Now, one prediction of these is that in the context of medulloblastoma and possibly other tumors, then Gli3 is going to be a very important tumor suppressor gene. And that's something that we're doing now. We have know from Alice Joyner, actually Mauser, has conditional Gli3s in it. So we would like to remove Gli3. Does that answer your question?

- Yeah, totally.

- So, moving on, our next question is from Beverly.

- 40:42 Hi, professor, it seems like these cells are derived from an Astra Sonic lineage and maybe due to the transition of late glial cells into astrocytes, especially since they expressed the actual glial markers, GFAP and GLAST. is there any way to further discern B cell populations from the actual, glial lineage, perhaps the down regulation of real, glial speciﬁc markers when D cells become present?

- 41:09 Well, that's a really interesting question that people have had almost for 10 years since we ﬁrst isolated these astrocytes as stem cells. And we have tried, you know, gene array. Other labs have tried and there's still no marker out there that people could point to, to say, Hey, this is a speciﬁc marker of those astrocytes that are of stem cells and not of of other astrocytes. There is some hints of some proteins that might be expressing one versus the others but there is no clear evidence of that. And to me what this is suggesting is that the cells are very closely interrelated and during development. So, it's well known that you know have radial glia. Can you switch the uh--

- You betcha.

- You have radial glia, and even before they were known as the stem cells, it was well known that the cells start separating from the wall of the ventricle. They start really retracting in this process, and then they start moving into the wall of the ventricle and become astrocytes. So this lineage was very well established and since the time of 2009 Web Conference with Dr. Arturo Alvarez-Buylla

Ramon y Cajal and even before that. And now, what we're saying is that there is a super group of cells of these that becomes sort of astrocytes they remain close to the ventricle and the insuperable group of them then go to the dentate gyrus. And these cell selectively can work like stem cells. So, this has stimulated other people to say, Well, can these cells behave as stem cells, and to our, in our lab, the answer is no. Yeah, there's a lab in Germany that has succeeded in making these cells divide and making them ternary neurons. These are the Frankie Milan astrocytes. Many labs now have seen that both these types of cells can work as neuro precursors. But structurally and functionally B cells might be closely related and those pronouns that are unique to this lineage, but differentiate from these astrocytes might be those are actually associated with them being stem cells. So I think it's still worth going after more markers that might differentiate B cells from D cells. I mean, you mentioned one of them, GLAST, and you know GLAST was characterized as a specific marker of astrocytes. And then people said, hey we have found something that really identifies the inter-astrocyte system that know your B cells, we looked at it and they also found it to have GLAST. One possibility is nesting. B cells are very rich in nesting and very few of Ds have nesting and yet there's some cells around blood vessels some astrocytes that burn both blood vessels that have nested. There is another one that Ben Barres' lab at Stanford had identified, dehydrogenase Ldh, and he has a large micro array analysis of different astrocytes and is probably one of the person of the world that knows the most about the signature. So, he also told me, Hey now I have found something that really identifies the true astrocytes I know your little cells that you are calling astrocyte, but are radial glia, modified radial glia. So he gave us the markers lo and behold, dehydrogenase does not identify radial glia early on in the sub=population, but it does identify the adult population. So it'd be probably very, very resistant to find these specific markers that just identify the stem cells.

- Did you want to follow-up.

- Oh sure, I'll follow up. Also, between the close similarities between-- 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Who's asking?

- A quick follow-up.

- Ah ha.

- 44:49 Because of the close similarities between mature astrocytes and neuro stem cells have differentiated astrocytes even transgenes to proliferate and generate other cell types of sort of natural re-programming?

- 45:01 Yeah, that's a really interesting question I alluded a little bit to this group in Germany that has claimed that they can use these cells to go on and divide. We had tried a little bit of that with so-called reactive astrocytes. So when you go into the brain and make a lesion, around the lesion site, there's all these astrocytes that become crazy and start expressing a lot of GFAP. And it's not clear whether they start dividing, making most of the field agree that they don't start dividing they just become hypertrophic and make a lot of processes to try to patch that lesion as much as possible. So, yeah I think it's a fascinating question on something that should be followed in more detail. Now I must say that astrocytes are very diverse and a lot of cell types, and I'm not talking also about the stem cells that look like astrocytes and other astrocytes, even where you were away from the regions where neurogenesis happens. Astrocytes are very diverse there is the classical differentiation between protoplasmic and fibrous astrocytes, fiber tracks and gray matter. But there are more diverse than that. There's a group of cells that are stained with a marker called the same with S100, beta, which is a calcium binding protein, and it was originally thought to be a very good marker for astrocytes and now people are finding that it is actually in a group of cells that look like astrocytes, but seem to be in the oligodendroglial lineage. So cells that give rise to the myelinating cells. So, yeah, is complicated. And astrocytes are very, very diverse and whether some of those cells can still be induced to behave like stem cells is a very, very interesting question. Especially now in this era of induced 2009 Web Conference with Dr. Arturo Alvarez-Buylla

pluripotency through transcription factors. I mean, there's perhaps cells all over our brains that could have very important pluripotent applications that with a little bit of manipulation, could be made to behave like very, very useful cells as progenitors.

- Okay, well next on track, Ivanka.

- 47:06 Hello, what are the rates of neurogenesis as organisms reach adulthood, compared to embryonic and postnatal neurogenesis? And also, why do you think lifelong neurogenesis has been conserved in the dentate gyrus of the human hippocampus while almost other all other areas of the brain have no adult neurogenesis and how does this arrangement speciﬁcally support continuous learning and memory?

- So, where are you?

- Here.

- This is very unfair because I look very big in a big screen here and very tiny--

- Yeah, that would be great.

- Now they're going to put me into smaller screen and you in a bigger screen. Ah, that's much better. Okay, so your questions. Your last part of the question was related to why neurogenesis occurs selectively in one part of the brain and not others?

- That's right.

- And what was the ﬁrst part?

- What are the rates of neurogenesis in organisms when they reach adulthood compared to the embryonic and postnatal stages? 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- 48:18 Okay, so the ﬁrst question is easy to answer because it's hugely different. So, in an adult organism, the, you know you, for example, the migration to the olfactory bulb, which is the most, the biggest germinal area in the adult brain, at least in a rodent It actually comprises about 10,000 cells per day going there. And 10,000 cells are produced in just in a few minutes through an embryonic development within the ventricular cell. So there's a huge difference. The embryo is really good and really fast at producing tons of neurons and a huge diversity of neurons. Which raises another question, now's my turn to ask questions. Can you put the paper? So I told you about the wall of the ventricle and the migration. And this distance in a mouse, of about 5 millimeters. You know, an embryonic brain is just a fraction of that. This is a late fetal brain it is even smaller than this. And this is producing millions of cells per day. While this whole structure is producing about 10,000 cells per day. Much bigger, much more expansive. Obviously it's doing many other things this brain but in terms of neurogenesis it's very small. And all of these cells need to be collecting to the olfactory bulb that's another three millimeters farther away. So that answers, more or less, your question in terms of numbers and sizes. But now it raises, before I answer your second question, let me ask you a question based on, on what you have read. Why do you think that the brain would keep such a huge germinal area, just to feed, generate such a small number of cells, and also to have them go so far away? Seems like totally counter- intuitive. If I was God and was deciding organisms. I would put just a fraction of what you have in the embryo, put it right here. Even inside the olfactory bulb, to generate the neurons the olfactory bulb requires, and that would do the trick. Here you have a huge structure many times larger than what the embryo needs, producing a smaller amount of neurons that go very far away.

- Anyone have an idea?

- Migration is good for your progenitors?

- Makes a 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Excuse me?

- Gets them to exercise?

- Maybe they have to mature and there are certain cell signals there that are necessary for them to become major neurons in the olfactory bulb?

- That's an interesting idea but that's not the answer.

- Anyone else?

- I think the answer is in one of the papers that you actually read. Or that you might not have read I don't know if-- but it's certain the answer was discussed in the paper with the pinwheels. Now I see how carefully you read my paper.

- Does it have to do with timing?

- No.

- 51:54 It has to do with another fascinating observation that another graduate student did, that cells born here are different from cells born here, and are different from cells born here. Almost like if this whole area, even though these are stem cells and here comes, you know, you have to learn that stem cells are able to generate the- neural stem cells are able to generate neurons, astrocytes, and oligodendrocytes the main cell types in the brain. And that's true for all of these regions. But when you're look in vivo, for example, these cells are very good at generating superﬁcial granular neurons in the olfactory bulb these cells are very good at doing deep granular cells in the olfactory bulb. These cells are very good at generating periglomerular cells that express speciﬁcally, TH upon the neuragic. And these cells are very good at producing cells that are in humans positive. Not only that, but we have now found that it's not only this wall, the wall that's actually here next to the cortex that in the adult does not just does not generate inter-neurons, 2009 Web Conference with Dr. Arturo Alvarez-Buylla

also generate cells for the olfactory bulb. And part of the medial wall, the other wall facing the septum generate cells that go to the olfactory bulb. So conceptually, why this is important is that even though all these cells are stem cells because they are able to do this when it comes to the production of neurons the ependyma decide this is gonna produce an N1, N2, N3, N4. So it's a space, I mean, timing. Michael's idea of timing might having a role is also important, there is a role for timing. But here the main answer is a space. That during the development, these cells become specialized dependent on their location to produce different types of neurons. And that once the brain has developed these niches are stuck with those progenitors in those domains. So the only solution to get the cells to where they're needed is to have very complex migratory phenomenas is to get them to the right place. So migration is a fundamental phenomena to be able to build the brain because the cells have to go work to go to make them dis-involve, they're like little umbrellas they know what to become. Once in the olfactory bulb, boom, they pull out their dendrites and axons, but their stuck, their dendrites and axons they have from where they were born. You know millimeters away. So, again, it's not like you make an neuron, it goes to right place and then sort of makes an axon, dendrite and the depend on the location where the signal cells is to become. It's not like that at all. The cell is already pre- speciﬁed and these are little packets of transformers that are already ready to make certain types of cells once they get to that area. And then the brain knows how to use them to make circuits that that make sense. Isn't it fascinating? Is that clear?

- Very clear.

- Next one's from Beth.

- 54:58 Hi, um, I guess now that we're on the subject of migration, what's the purpose of interkinetic nuclear migration during radial glia development, and what mechanisms control this behavior? 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- 55:09 So we had done very little work on interkinetic migration. I've been attracted by the same question that you are for many years since I was a graduate student. I don't think its really understood, all we have done with interkinetic migrations is we know that in these stages when cells are going from when cells are going from- Can you see this, or am I?

- Yeah

- Okay, from the radial glia to the adult stem cells, at this stage, cells are still undergoing interkinetic migration. One thing, in this stage, we think that they're is still attempting to do intricate migration naturally. Most of the cells we find in the G2N phase are stuck closer in between the ependymal cells than when they are not. So I think we still have a little dance going on there. Again, there's work from another colleague here at UCSF, Who has suggested that there is differential Notch signaling as the cell goes to the different parts of the basal apical movement, and that as the cell gets closer to the apical surface the Notch signaling gets much weaker, and then that allows the cells to release it, to make new neurons and or to divide and make progeny. So a grading of Notch signaling has been proposed and this is based on the zebrafish studies and breaking studies in zebrafish looking at telekinetic nuclear migration and looking where Notch signaling is happening within the movement of the cells. Yeah, there's nobody knows why this dance happens. Now, one thing that's important to understand, it's not unique to the brain. Interkinetic nuclear migration also happens in other epithelium as the cell undergoes division. So you might be something very primitive associated to epithelial development and division.

- 56:57 Okay, so the next question, actually mine, Is sonic, sonic hedgehog signaling Is sonic hedgehog also signaling a over the stem cell niche and are the cilia in a particularly appropriate position to receive these hedgehog signals? 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- 57:18 Yeah, that's an excellent question and I wish I had an answer and I can tell you some of us were attempting to answer that. So, in the case of the hippocampus, I know unfortunately, I have to tell you now more about the hippocampus. So in the hippocampus the cells that working as stem cells are these radial astrocytes. And they have- so the dentate gyrus is a structure that is full of granular cells and this is compact neurons that are very densely packed in this area and it's called the dentate gyrus because it looks like a tooth, and in Latin, dentate is if any of you speak Spanish is tooth, so it looks like a tooth. So that's why we call it dentate gyrus. But along the surface right here in this what's called the sub-granular zone there is these cells, that were called also astrocytes and were thought to be astrocytes. And these cells divide to generate intermediate cells. These are also transit amplifying cells that are unlike the ones I told you in the sub-ventricular zone, these cells only divide once to generate progeny, and then these cells differentiate directly into new granular cells. And they don't migrate very far and actually we believe it's most likely that they're staying within the same radial units where they are produced. So, one of the daughter cells actually, that produced just till it dies. So for each division of radial glia we believe you get one of new neurons here, and radial astrocytes, sorry. So, these are the cells that we found, they come also from the nerve epithelium. And somehow, somewhere between here and here, these fails. And the animals without primary cilia, end up with a very very tiny one fraction of the size and it doesn't have any of these regular cells. It has all the astrocytes all over the hippocampus, but it doesn't have these radial astrocytes. So, one of the very interesting questions here is who is providing Sonic hedgehog for this system? And, whether, you know, you said earlier development is produced in a graded manner and here it is not. And we don't know the answers we don't know. We have no trained insight to every gestational we see a little bit of Sonic hedgehog signaling here. But what is more, more interesting, perhaps, is that in the adult brain, it might be coming from neurons. There's a groups of neurons in the septum, which is another part of the brain that projects actually into the hippocampus, that has cells that express Sonic hedgehog and these cells may be transporting Sonic hedgehog and releasing Sonic 2009 Web Conference with Dr. Arturo Alvarez-Buylla

hedgehog here. So there might be mass regulators here within the brain that keeps these morph Agent like you just said, and keep it as as a factor to regulate these very important process. These might give us some hints of how the whole thing is regulated. So that's one thing that we're currently investigating in the lab. Now, you might ask, well what about the

- 1:00:20 Is there evidence in other systems showing that neurons are speciﬁcally releasing Sonic hedgehog like you're like you're describing?

- 1:00:29 No, he has been postulated, it has been observed and published that Sonic hedgehog contains neurons in the adult. And there has been proposals out there of people saying that yeah, what I've-- that Sonic hedgehog might be released by neurons. But still, nobody has characterized this process.

- Okay.

- 1:00:47 There is actually another of our neighbors a post-doc in Arnold Kriegstein's lab has found that earlier development within this region, there is a multi-cells that actually project an axon to be different in the B cells where the cells have projections that are also Sonic hedgehog positive. Interestingly the cells lose Sonic during post-natal development. So they cannot be cells that keep this going but they could also be a transition cell that maintain some kind of Sonic leveling going at the right time. But what I want to mention to you is some of the works that another post-doc in the lab is currently doing. So now you all know the answer to my question of, of why such a complex and long system. Because it cells are different in different parts of the brain. So one experiment that we have attempted is do we grab the cells dorsally to these now cells acquire these potential, or is it endogenous to the cells? And the answer is endogenous to the cells. So when we move these cells from ventral to dorsal maintain a ventral identity. So there seems to be something ventrally that is inﬂuencing the speciﬁcation of the cells. And you I already told you about Gli2 which is one of 2009 Web Conference with Dr. Arturo Alvarez-Buylla

the transcription factors involved in Sonic signaling. So when we look at Gli2, it is only ventral and not dorsal, suggesting that in post-natal brain Gli2 is speciﬁed in area just like in Morph agent development of ventral cells. We have now a very beautiful result I'm really excited about that if we are deﬁcient in Gli2 expression dorsally, now we can make these N2 cells behave like N1 cells.

- Really?

- So, is Sonic hedgehog still acting there as a permanent cell type? What we don't know again, it's where Sonic hedgehog is coming, and whether there are gradients. What you asked is also very interesting, whether there are gradients that are determining the behavior of the cells. For all we know, the cells that should be receiving the signal are through the primary celium. That primary Celia is through to the ventricle which is a liquid, just like water. Would be very difﬁcult to think how gradients would be created there. We also haven't seen that Sonic is expressing the choroid plexus which is the structure that secretes it, the cerebrospinal ﬂuid. So there's a lot of questions in place in what you just asked, both related hippocampus and to this ventricular zone.

- Thank you, next question from Elise.

- 1:03:21 Hiya, um, in your nature neuroscience paper you mentioned that the loss of Kif3A function in adult stem cell proliferation can rescue some of the effects of a loss of Smoothened expression because Kif3a has a role in hedgehog signaling. So why does the loss of these two required elements both Kif3a and Smoothened actually helps to reduce the phenotypic severity instead of making it worse because I have two elements in the same?

- Excellent question. but you have the answer already. If you understood what I explained before you should be able to answer that.

- Okay. 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Yeah, we do.

- Do you?

- Yeah, we got it from the ﬁrst screen--

- Don't, don't, you don't say it, Michael, what?

- We got it in terms of how your earlier, your earlier explanation of the experiments, dealing with the medulloblastoma formation and lack thereof.

- Right, ﬁne. But I wanted to make sure that students got it. So can someone explain it to me?

- Does it have to do with the fact that you talked about the on off switch and the double, the two things that are activated and the two things that are oppressed?

- That's right.

- 1:04:40 So it makes up for it because you have repression of the repressor so the pathway can still go on?

- 1:04:47 That's right, so when you remove your repressor when you remove the cilia, you remove the reppressor, so now you release a little bit of that effect of having this cilia so you get an improved phenotype than if you just had the Smoothened because Smoothened would still have Gli3 there. Is that clear?

- Yes.

- If you make the diagrams, if you draw to yourself I think you understand it.

- Okay. It's explained a little bit in the paper, but I think these kinds of sophistication is what you're going to be dealing with as, biologists in the future because we all thought 2009 Web Conference with Dr. Arturo Alvarez-Buylla

when I was a student at USC State, that everything was going to be simple just one more peeling one more, one more peeling and then the answer to everything was going to be evident. And biology was going to just be in our hands and it's obviously not like that. Every time we peel a little bit more more complexity emerges. And one of these is, you know, this ability to think that systems are having both repression and activation at the same time. And this is thinking molecularly obviously that one signal in the pathway when you put all these signaling towers together concurrently, incredibly interesting and complicated. And that's I think where you guys are headed.

- Thank you, so our last question comes from Wendy.

- Hi Dr. Alvarez.

- Yes.

- 1:06:11Your sonic hedgehog paper, would you expect that the human GFA Qi creates Kif3afl/fl mice to exhibit mental retardation targeted cognitive deficit, or a shorter life expectancy had they not been sacrificed? With these mice be a good model for severe cognitive deficits in humans?

- I have trouble hearing the ﬁrst part of what you said.

- Sure, would you expect that the human GFAP CreE Kif3afl/fl mice to exhibit mental retardation, cognitive deficits or shorter life expectancy had they not been sacrificed?

- 1:06:47 Yeah, well, these animals as I mentioned before, are very abnormal because they have hydrocephalus. Hydrocephalus is really a very important, even in humans, a very important malformation because it increases internal ventricular pressures and it compresses the brain so makes, produces a decrease in the amount of tissue that you have of real brain white matter. So, these animals have bloated brains, with 2009 Web Conference with Dr. Arturo Alvarez-Buylla

cerebrospinal fluid have very thin cortices, so I'm sure that they're very deficient in many, many ways and actually they're very ataxic. So, yes, it could be a model for what you're saying. But, in fact, there are so many things wrong with them in terms of the normal development of the brain that I think will be very difficult to isolate specific effects on the kind of cognitive phenotypes that you're interested in. It might be possible to induce the, in fact, possible, to induce the mutation more selectively in other groups of cells and then, more specifically study the effect of removing the cilia in groups of neurons, for example. And this has already been done by a group in the Midwest. I forgot the specific location but it's very interesting when they removed the cilia in-- By the way, cilia are not only present in progenitors like I told you, but are present in many many cells, including the migrating cells and including neurons themselves. And in neurons, it has been seen that the cilia concentrate in core receptors like serotonin receptors. So a group has removed the primary cilia in groups of neuronal hypothalamus and these neurons become obese. And it's still not clearly understood why they become obese, but clearly the primary cilia there is acting as some kind of receptor, is concentrated as some kind of receptor, and it's leptin, which is one of the hormones that's relating to energy metabolism and weight. It's something else. So, again, the role of primary cilia is going to go way beyond progenitors and it's going to probably going to have a very poor role in other cells. In neurons, these neurons in the hypothalamus that have these primary cilia had a very long primary cilias, about five microns long So the primary cilia I've been telling you about is one micron long, so about five times longer than the one I just told you. Another example of this ciliated cells is your olfactory mucosa the olfactory receptors themselves are ciliated, they have a long cilia also where they accumulate the olfactory receptors. So there's all kinds of interesting things that cilia are doing. In fact, there was a fascinating paper in like two or three issues back in Science, showing that your ciliated cells in your-- airways, I'm just recovering from a cold here and coughing, because probably all those cells are all messed up. How's the situation in the East Coast for the flu? Out here its pretty bad. Even though we have better weather than you have, but there's a lot of people that are sick. And 2009 Web Conference with Dr. Arturo Alvarez-Buylla

what happens is that these ciliated cells in your bronchi and in your trachea, get decimated and they need to regenerate and, by the way, they are not regenerated from other ciliated cells, they're regenerated from progenitors that are one layer underneath. But the fascinating observations that this group made is that these cells in your airways have taste receptors and is the only other place in the body except your tongue where taste receptors have been found. And, interestingly, they changed the beating, the movement of the cilia according to the taste. So if you put bitter substances on this epithelia they've began beating faster. So, here you have an example of another ciliated cell that has taken advantage of these appendages to put receptors, in this case from the tongue, for, probably, you know the nauseous signaling that makes them beat faster to get rid of whatever is noxious there. So, yeah, the primary cilia are going to be involved in many things. These are not primary cilia, these are mood cilia cell like ependymal cells. We suspect that ependymal cells also have some receptors in their cilia. And so yeah, in neurons, it's going to be very interesting the role, in circuit formation and development and doing it selectively in groups of neurons. I think would be the way to go.

- She's asking a follow-up.

- 1:11:17 What types of human diseases are the direct result of defects in neural stem cells and their niche, and are current therapies being designed based on our understanding of the stem cell niche?

- 1:11:30 Yeah, no, there's a lot of work done in these areas. Now, in terms of your ﬁrst question. I don't know. There are many genetic mutations that have been associated with malformations during development of the cerebral cortex, lissencephaly and heterotopias. But I didn't know any that has speciﬁcally associated with malformation for example disease of the ventricular zone. Now that's because we just don't know. We know very little about the humans ventricular zone. And in fact there is a big controversy between my lab and a lab in Sweden. Where the lab 2009 Web Conference with Dr. Arturo Alvarez-Buylla

in Sweden suggests there is migration to the olfactory bulb in humans, and where we don't find migration to the olfactory bulb, so this group in Sweden for example, claims that people with Alzheimers disease, one of the first things that they lose is olfaction and that that might be due to the fact that there is a defect with migration of cells to the olfactory bulb. But, in fact, even in children less than one year of age, we still do not see a large number of cells going to go back to olfactory bulbs in humans and we think that in humans most of these processes are prenatal. Now there is a sub-ventricular zone in humans. and it has been associated with, for example, multiple sclerosis and it becomes activated multiple sclerosis. And it seems that the oligodendrocytes might be ramped up. The production of oligodendrocytes might be ramped up in these patients. So that's as far, as you know, the sub-ventricular zone. In terms of the hippocampus, there's many, many studies suggesting diseases associated with neurogenesis or problems with neurogenesis from simple things to stress, depression, and this is all correlative work where they find that people that is depressed have less dividing cells in the superior zone of the hippocampus and work in mice and rats suggest that you can, that anti-depressant drugs actually increase the number of new neurons formed in the hippocampus. So there's a lot of work being done there in terms of trying to relate disease to this specific niche of neurogenesis. And then, your second part of the question was what, I forgot.

- 1:13:44 Are there current therapies being designed based on our understanding of the stem cell niche?

- 1:13:49 Yeah, that's also true for example, in the hippocampus people are thinking that anti- depressants might be modulating having part of at least part their action with changes in neurogenesis in the hippocampus. And there's people also playing around with an idea, for example for activating oligodendrocyte regenesis in patients with multiple sclerosis to have more legolin produced to those areas that have been the deprived of oligos. But it's all in very early stages of experimentation. Also in newborn children the sub-ventricular associate is playing 2009 Web Conference with Dr. Arturo Alvarez-Buylla

a very, very important role because it's the side that bleeds very easily. The blood vessels somehow there can bleed and these early hemorrhages in children that are either premature or have had some kind of of toxic events. late in development or during birth where there's some anoxia going on, are thought to be largely done to bleeding around the sub-ventricular zone. So I think that there is large groups of people trying to understand what's going on within the niche there that makes this area so prone to bleeding and whether there's a way to prevent or even detect this early on to prevent you know, from killing off a lot of especially the oligodendrocytes and white matter tracks that ended up creating like cerebral palsy and problems of that sort. So, yeah, I think that there is a lot of people working along those lines. No, we're not doing work along those lines. In fact, my impression is that, the sub-ventricular zone, in terms of brain repair is going to be not that useful. It might be useful for oligogenesis but my impression is that just based on what I have been telling you is that it has a very important biological meaning, in terms of how it's integrated into animals, especially with very strong olfaction. So based on that, we're now trying to do what the sub-ventricular zone does for the olfactory bulb to try to ﬁnd the right cell that will do the same kind of thing for cortex. And we're actually working collaboration with other groups here in using plasticity and cortex. The true transplantation of speciﬁc neuronal progenitors because I don't think that these cells in the sub-ventricular zone are meant to do that. Even though there's people that claim so, I don't think that that's true.

- Thank you.

- 1:16:07 So, in closing we'd like to know, in your opinion, what are the most pressing questions in the ﬁeld of adult stem cell research, and their use in human disease therapy, and what steps is your lab taking to address those questions?

- That sounds like a question from a reporter. But you said you are in the neuroscience program, in neurobiology? 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Yes.

- Someone was in some other thing that had nothing to do with neurobiology, no, when you were introducing yourselves.

- No, that was me.

- So, I mean, I guess you want me to tell you what you should do?

- That'd be helpful.

- Basically, what are you, when you think of the ﬁeld of just understanding the neural stem cell niche and what your lab does and focuses on, what are the most pressing questions in the next year or next ﬁve years? And what's your lab doing to address those questions?

- I guess whatever things looks exciting and interesting and new.

- What do you most excited, before you come in.

- What?

- What makes you the most excited to get you in the lab, right now, as it stands right now?

- 1:17:31 Well, I just told you is this ability to switch phenotypes within the speciﬁcation because that might gave us a hint of what's going on in terms of the speciﬁcation. But I must tell you that I'm so confused by so many things that we even have found that I'm not sure exactly. I think we need a new generation of thinkers to think about clearly, we'll keep poking at things that look exciting and interesting. I guess that there's two style of scientists. I mean there's scientists that are have a very clear plan of an idea of something that they want to do, and that's not me, unfortunately. And there's people like me that just go after and things that are 2009 Web Conference with Dr. Arturo Alvarez-Buylla

really exciting and interesting and I just get excited by the biology and the possible ramifications that it could have, possible ways it intersects with other things that we're doing. So that's one of the difficult things when you write a grant and you sort of want to tell them what you're excited about and what do you think is going to be important within the next years. And then when you turn our your report at the end of the five years you figure out that you have done absolutely or very little of what you have to post, But doing so you have found a lot of other interesting things and I have seen a lot of other very boring scientists that do exactly what they have proposed to do, and have actually missed a lot of opportunities to do the things on the sides. Unfortunately I cannot tell this to NIH because they won't give us any more money but, I think science should come from looking at something and becoming really intrigued about it. And from there, going on to make the great discoveries. Not from very careful planning, I'm going to discover a to b to c because I don't think we're that smart. I think we have good intuitions and that's discoveries come can but I think our ability to integrate especially now that there's so much information around to make meaningful hypotheses. There are cases where it is possible, but most of the cases I think is a little bit of trial and error. Now, I'm sure that many scientists if they were hearing this would kill me because they would want to make science look much more professional and directed. But I just don't think that it is like that. i think it's a matter of being excited about something and it might be something very trivial, I mean there were people interested about the orientation of hairs in the wings of flies. And people said oh, you're wasting your time looking at the wings of flies and the orientation, and they end up doing incredibly basic discoveries. People like my advisor, Fernando Nottebohm, as a kid was incredibly intrigued by sunbursts and the little songs that he was hearing the woods while he was growing up in Argentina and then he was able to guide his his career to answering that question He discovered the whole whole song control pathway in birds and adult neurogenesis in birds and all these other things that have been important, you know, and so just do what you like and go for the things that's that smell and taste good and you enjoy. 2009 Web Conference with Dr. Arturo Alvarez-Buylla

- Thank you very much.

- Sorry, sorry about my -- thanks for calling my cold.

- It's my pleasure. Thank you so much for your time, Arturo. Okay, thank you guys. Good luck to everybody.