 Can you hear me now? Yes. Okay. So good afternoon, everybody. It's a really great pleasure to inaugurate our series of seminars with our external speakers with Alex Polen. This is you know also a special instance of our NeuroLoquia which exceptionally takes place on Fridays this week for Alex rather than on Thursdays but it's also more broadly the first HE seminar by an external speaker. So we are really very, very, very happy and honored to welcome on this occasion Alex Polen, a really not thought of a better guest to inaugurate our series. And also very grateful that Alex accommodated this visit in the aftermath of a number of COVID related pandemic changes of flights. And so again very grateful for accommodating that. So just very few words of introduction because Alex achievements don't need a lot of introduction but it's just a pleasure to do so. So Alex started off with a bachelor in Harvard and then moved to Oxford for his master. I think it's also a very nice combination of training that bridges environmental science first to neuroscience. And during the time he was also an awardee of the Rhodes Scholarship which I'm sure you will know is one of the most prestigious scholarships in the world. And he then moved to Stanford and then to San Francisco where he trained in the lab of Arnold Crickston. And during the time he contributed to really fundamental discoveries and insights about what makes us human including with the identification of outdoor radial glia. And he then went on to establish his own lab as an assistant professor of neurology at University of California San Francisco in the Mission Bay campus. And already as a PI he has yet again provided a similar contribution to the field the most recently as you have read in nature with a dissection of the evolution of inhibitory neurons in the primate setup. Throughout he has been at the leading edge of the development and applications of new technologies to inquire into the evolution of the human brain. And this is also one of the main reasons why his research is so relevant and aligns so nicely with a lot of the themes that we are pursuing in the Neurogenomics Research Center and more broadly at UNTECR. So without further ado I leave the podium to him thanking him again and it's really an enormous pleasure to welcome you. Thank you Alex. Thank you for the kind introduction and you know it's a special privilege for me to be visiting an institute and program dedicated to neurogenetics and these interests are really close to my heart. Today I want to talk to you about some contrasting developmental mechanisms that can underlie the evolution of entirely new cell types. And let's see if this will work. We've got the computer running up there to accommodate the zoom link. So dramatic changes in brain structure and function have evolved along the lineage leading to humans. Our brains are at over a thousand times larger than the rodent brain and have tripled in size in just the last few million years. As Giuseppe mentioned in my training I was really focused on genetic molecular and developmental mechanisms that could contribute to brain expansion and cortical expansion itself and using single cell RNA sequencing we were able to identify specific marker genes and signaling pathways that distinguish these outer radial glia neural stem cells or basal radial glia from those at the ventricle. But the genes and pathways that we found were shared across primates and to try to find human specific changes I generated polypotent stem cells and brain organ loads from human and chimpanzee and we were able to identify some human specific signaling pathway changes and quantitative increase in mTOR activation in human outer radial glia. As I started my own group I took a step back and we thought more about some of the special features of brain evolution. And one of the major features is that not all brain regions change equally. The, let's see, the laser pointer. So the gray matter of the neocortex, the underlying white matter and the striatum have expanded disproportionately compared with other regions like the olfactory bulb or ventral midbrain. And, oh, can you go back? Go back. Sorry, I'm trying to go back to the other side. So we, in my group, we've become really interested in how this process of unequal scaling combined with new functional requirements may drive cell type specific adaptations to the large brain failure ecology of human and other non-human primate brains. And so today I'm going to talk to you about how do new cell types evolve in the primate brain. And here's a picture of one of the cell types that Matthew, who's here in the audience, identified the cell type that lines the striatum and expresses some of the biosynthetic machinery for making dopamine. And so how is this cell type specified in primates and not rodents? Where did it come from developmentally? And ultimately we'd like to think about how do qualitatively new neurons integrate into existing circuits? And so I'll talk about three different scenarios of cell type evolution today. Next slide. And the first one would involve an entirely new initial class of neurons that is qualitatively distinct and generated early in development by progenitor cells with different gene expression networks. In the second scenario, I'll talk about a conserved initial class of neurons that looks similar to that, which is seen ancestrally, is moved to a new location where it may acquire new gene expression programs and new functions post-mythotically and later in development. And in the third scenario, I'll talk about what I think is the most common in recent human evolution sensor divergence from other apes. And to take a play on Sean Carroll's quote, this would involve teaching old cell type new tricks where changes in gene regulatory networks in otherwise conserved cell types could lead to specialized functions. Next slide. And today I'll mainly focus on inhibitory neurons. Over 100 years ago, Ramoni Kahal appreciated the increased morphological diversity of inhibitory neurons in primate in the human brain, even speculating that these were connected to the functional superiority of the human brain. Indeed, there's an increased proportion of inhibitory neurons in human and primate cortex compared with rodents and also in dorsal thalamus compared with rodents. And recently, Fennecrenin and Steve McCarol's lab discovered these primate-specific HAC-3 stridal interneurons that are inhibitory. And there've also been multiple reports of more neurons that expressed hyraxine hydroxylase, a rate-limiting enzyme in dopamine and catecholamine production in human white matter and striatum compared with other non-human primates and especially with rodents. In addition, these inhibitory neurons migrate long distances to their final destinations, so they might be particularly impacted by the increased duration and increased overall size of the brain during development. It's a little tough. There's some animations here, but I don't have control over the slide. So the neurons from the MGE in blue are the major contributor to the cortex, and then neurons from the CGE are also contributors to the cortex. The neurons from the LGE are mainly associated with making the projection neurons of the striatum as well as multiple types of olfactory bulb inhibitory neurons. And with Matthew, we wanted to ask, are these initial classes of inhibitory neurons for specified early in development shared between primates and rodents, or can we already see distinctions at these early stages of development? And so there's Matthew here and also in the audience and, you know, as one of the first grad students, it's been great having Matthew as a real eye for patterns in the computational and histological data. So with Matthew, we surveyed the, let's see if I can get this. I'm not sure. Okay, well, we surveyed the germinal zone origins of these inhibitory neurons as well as their destinations using single cell RNA sequencing. And we did this across the span of cortical neurogenesis. Here's a projection of about 100,000 inhibitory neurons colored by the region from where they were sampled, but organized based on transcriptional similarity. And you can see most of the germinal zone neurons are over here. And next slide, please. And if we cluster these by transcriptional similarity, we can see that these are mainly the progenitors which cluster by cell cycle progression. But next slide, if we look at the post mitotic neurons, Matthew could identify these discrete initial classes of post mitotic neurons. And we're calling these initial classes because they're later partitioned into one or many terminal classes by a process of post mitotic fate refinement. But at these early stages of development, we don't yet see a partition related to the terminal classes that they will generate. Instead, the transcriptional variation is mainly related to maturation. For example, that, okay, well, we can go next. Next slide. And so we wanted to ask this simple question, to what extent are these initial classes of inhibitory neurons conserved between primates and rodents? And we did this a few different ways at the level of individual cells as well as at the level of clusters. For simplicity, I'll show a comparison at the level of clusters. You can already see that the mouse inhibitory neurons form a very similar manifold. So how correlated are these clusters? If we look across marker genes, we in general see one-to-one homology. But there was one case that really stood out where there's two populations of MGE neurons that extract crab P1 in primates, but only a single population in rodents. And one of these was also defined by the expression of TAC3 and other markers from this recently discovered adult population of TAC3 stridal interneurons that kind of green and showed represents about 30% of the stridal interneurons in primates, but is not found in rodents or carnivores and thus likely evolved within the last 90 million years. And so our developmental data gave us the chance to look at how early in development our evolution early novel cell types specified. And in the adult data, this terminal class was very similar transcriptionally to the TH and PV positive stridal interneurons. And indeed in the developmental data, does anyone have a laser pointer? I usually use the cursor of the mouse, but okay. Thank you. So the math class, so these TAC3 neurons are very transcriptionally similar to the class that ultimately generates the TH and PV cells, but they already express distinct neuropeptides and neurotransmitter receptors. And then next slide, they also distinctly express transcription factors like LHX8 at the MGE septum boundary that are associated with the production of cholinergic neurons, but transcriptionally they look quite distinct from the initial class that makes cholinergic neurons. Next slide. So how early are they specified? Is it post-metautically? Could we already see a gene co-expression signature in the progenitors? So look at this, Matthew looked at the co-expression of markers of the TAC3 class as well as the math class and shared markers in progenitor cells. Next slide. At G1 and G2M, and these edges represent significant co-expression patterns. So we could already see co-expression of some of the cell type specific markers for TAC3 or math with the shared markers at these early stages of development. And then we went back to the tissue to test whether we could see these co-expression signatures. Next slide. And we looked along the MGE both at the ventricle and in the subventricular zone. And in the subventricular zone we could see co-expression of TAC3, this neuropeptide that marks the cell type with several other markers and crab P1, suggesting that indeed this gene co-expression network already is present in the progenitor cells. And then we wondered is there a spatial bias or even a zone that produces the TAC3 distinct from the math? We had seen some co-expression of transcription factors signatures with the cholinergic neurons. And what Matthew found is that the progenitor cells that expressed, next slide, that expressed these markers and the newborn neurons were, there was not a discrete location producing math versus TAC3, but there was a bias where the TAC3 newborn neurons were more likely to appear caudally across the MGE. As these neurons continue to differentiate, these co-expression signatures strengthened and more genes are included. And we could use these as markers of the dispersion of these sister classes in the striatum and evaluate the extent to which their partitions there are already mixed. Next slide. And we could see, in a single plane, the yellow cells represent co-expression of multiple TAC3 markers and the green cells represent the math class. We could see these evenly dispersed in the striatum. Next slide. As well, along a rostocautal axis with no spatial bias. So these are specified very early. And to look, next slide, at the gene regulatory networks that specify these, we did analysis called a regular analysis where we look at the co-expression between transcription factors and their putative downstream targets based on transcription factor motifs. And unsurprisingly, the math class was distinguished by networks related to the math transcription factor, as well as some other transcription factories like MF2C involved in neuronal maturation and in the cortex PV specification. But interestingly, the TAC3 class was distinguished by multiple regulons involving early response genes, suggesting differences in calcium activation or potentially neuronal activity at these early stages. So we have this model where an ancestral crab P1 expressing MGE progenitor is partitioned by distinct transcription factors to express different neuropeptides and functionally distinct, next slide, functionally distinct neurotransmitter receptors, suggesting that this cell type is talking to and listening to different neurons in a circuit. And to underscore how early this occurs, the math class goes on to specify well-conserved PV and TH stridal interneurons, but we don't see any signatures of those classes at this stage when we can already see the TAC3 neurons. So this for us would represent an entirely new initial class specified early in development. How common is this across all the inhibitory neuron classes? This stood out to us. So Matthew constructed a taxonomy of inhibitory neurons organized by the predicted birthplace, transcription factor signatures, birth date, and the TAC3 class was really an exceptional case. The only example that was qualitatively distinct between primates and rodents. And if that's the case, then how do neurons diversify in the primate brain or what might explain this morphological diversity? Next slide. So here there was a surprising cell type to find in the neocortex. I mentioned that the vast majority of cortical inhibitory neurons come from MGE and CGE, but in our sampling we found multiple classes that appear to come from the dorsal part of the LGE, lateral ganglionic eminence. Next slide. In addition, when we looked at the data, so here's the CGE and MGE cells relatively homogenously distributed across the rostocatol axis of the cortex. Next slide. But the dorsal LGE derived interneurons were enriched in the prefrontal cortex. And so we wanted to track this down further, what's going on. And when we looked back at this heat map of correlations with rodents, one of these classes, the Mystupex 6 class, showed its strongest correlation actually with adult born granule cells of the olfactory bulb. Next slide. And so what are olfactory bulb-like neurons doing in the cortex and how did they get there? Normally these olfactory bulb neurons are generated in the dorsal LGE and then the adult SVZ and migrate in parenchymal chains along the rostromigatory stream to the olfactory bulb. And we presumed their, you can just advance a few, we presumed their dorsal LGE origin by the expression of markers, low expression of CGE markers, high expression of serrated CGE and LGE markers, the expression of some LGE markers, but low expression of the medium spiny neuron marker Fox P1. And first I'll focus on this Fox P2 TSHC1 class, which is found in both our cortical sampling, but also in our subcortical and stridal sampling, and which correlated with the cell type found in mouse ventral tilencephalon. Next slide. And so here's a picture of the anatomy. I'll use some of those marker genes and their co-expression or co-immunoreactivity along with this anatomy to follow the origin and migration of these populations. So you can see that these classes of inhibitory neurons are, the markers are expressed along the dorsal LGE here. And if this is more of a kind of dorsal lateral portion of it, if we go to the next slide. First focusing on this TSHC 1 class, when you look at the UMAP, and you're not supposed to do this, but it looks like this TSHC 1 class is almost converging with the direct and indirect medium spiny projection neurons, and which are generated in the ventral LGE. And we wanted a way to ask this more quantitatively, is this class born in a distinct place, but then converging on a similar transcriptional program? Next slide. And to do that, this is a heat map of the overlap in activating genes in the upper right and inactivating genes in the lower right along the trajectory of differentiation. And what we can see is these direct and indirect medium spiny neurons, we can distinguish them even in progenitors. We can see different co-expression signatures, but they follow very parallel tracks in the UMAP, and that is more quantitatively reflected. They activate very similar genes, and they inactivate very similar genes as they differentiate. This TSHC 1 class, on the other hand, it also overlaps quite strongly in the gene that activates with the direct and indirect, but it inactivates a different set of genes, and up here to diverge from the mice 2 pack 6 class. And you can see that here, the overlap of activating genes is quite strong between the TSHC 1 class and the medium spiny projection neurons, and quite a bit lower for the mice 2 pack 6 class. And so what are the genes that are activated and inactivated? This class is inactivating the signatures of its dorsal LGE origin. So when it gets to the striatum, its dorsal LGE origin may almost be camouflaged in a sense, and at the same time, it's up regulating these projection neuron programs. And some of these markers corresponded to a recently discovered cell type, these eccentric spiny neurons that were thought to be a deviation of direct medium spiny neurons, but our data led us to hypothesize they're actually specified in a distinct place and converge along this trajectory. And so to look further at that, we zoomed in at the dorsal LGE here and asked, can we already see these medium spiny neuron genes turning on? And next slide. And we can see many examples of TSHC 1 overlapping with CASC 1, and then OPRM 1 turns on later in differentiation in this class, but we can already see some examples here in the dorsal LGE before these neurons get to the striatum. Next slide. In addition, this transcription profile looks a lot like a profile found in the amygdala, these intercalated cells of the amygdala. And Kenneth Campbell's lab had some elegant studies in mouse describing this TSHC 1 migratory stream to the amygdala forming these intercalated cells. And we can see this stream here as well in primate. And based on the continuity of migratory streams in that last slide and transcriptional profiles, let us to suggest that these are actually the same initial class migrating to multiple populations. But in our sampling, we found that class in the cortex. And so do we also see these neurons migrating to the cortex? And that was in macaque, you could see it. And also in human, you can see these cells. And it looks like they're migrating to superficial white matter, this eccentric spining or unlike signature in primate cortex. And we, it's very rare, but we have not yet seen that population in rodent. Next slide. In addition, this class also migrates to the olfactory bulb. And it looks to us that these dorsal LGE leg cells that express SP8 and Fox P2, but not the CGE marker NR2F2, merge with the rostromigratory stream here or merge at least with the stream to the olfactory bulb to go to the periomerular layer. And so I've gone into a lot of detail and jargon about these different cell types because this TSHC1 class is a great example of a single initial class that radiates broadly to many different regions of the telencephalon. And when we think about it from an adult or terminal class perspective, we're considered to be different cell types, but at least the same transcriptional program, it's hard to say without lineage tracing the exact same progenitor, but the same transcriptional program generates this diverse array of cells and is also generating a white matter population in primates. So then returning to this other class that looks transcriptionally quite similar to adult born granule cells of the olfactory bulb. And when we look in the primate brain, indeed we see the markers of this class in the rostromigratory stream forming parenchymal chains. But when we take a coronal section here, next slide, we also see the same parenchymal chains radiating out at the head of the caudate into the cortex. And so this set Matthew off on a sort of search, where are these going in the cortex? And this is about three or four weeks after cortical neurogenesis has stopped for excitatory neurons. But in the anatomy, it's a little challenging here, the dorsal LGE wraps around the head of the caudate. So you can see the rostromigratory stream here, but you can also see dorsal LGE cells here migrating medially towards the cingulate cortex. And next slide, we see these cells are actually bounded by th positive fibers, likely coming from the ventral midbrain, and we only see them interior to these fibers. We don't actually see th expression in these cells themselves. And I'm going to come back to the th. It's very sparse here the size of the circle corresponds to the fraction of cells and the color corresponds to the intensity. But the th was highly specific for this population. So we'll come back to that. Now, if we take an oblique horizontal section, we can see that these cells are also migrating caudally towards posterior cingulate cortex, and maybe even further caudal. And they're also forming these same parenchymalutane like structures that are used to reach the olfactory bulb and rodent. Next slide. And so what about in human? This one sagittal section summarizes, I think, quite well what we're seeing. There is this ancestral distinct migratory stream to the olfactory bulb. But we see the same class of neurons expressing mice to an sp8. Also forming this arc migratory stream recently described in primates by Mercedes Prettys and a Traver as Bulya, and Matthew identified this offshoot of the arc. We're calling the arc interior cingulate cortex that we saw in the last slide. So where do these cells end up? Cells expressing this combination of markers end up in the deep white matter of cingulate cortex. And do we see these in mouse? Next slide. In mouse, you can find this population. There's a beautiful study with an HDR3A reporter mouse line that shows you actually see this population in the deep white matter and deep in the white matter and deep cortical layers in mouse. But you don't see the same histologically distinct migratory stream. And next slide. We see in our sectioning that the vast majority of these cells are in the rostrum migratory stream going towards the olfactory bulb in mouse. And so that suggests a model to us where there's a conserved initial class of neurons that forms a primate specific and histologically distinct migratory stream to reach the deep white matter. And in rodents, it's only a few cell bodies from the rosa LGE to the white matter and deep cortical layers. But in primates, these cells have increased their abundance and forms these very distinct migratory streams. Next slide. So I wanted to come back or Matthew wanted to search further for these TH positive neurons that we saw a signature of early in development. And we looked now postnatally. Here's the remnant rostrum migratory stream. I think we skipped this slide, maybe. And you can see the same TH positive cells. You can see a fraction of these cells are expressing TH. And then in the next slide here in the olfactory peduncle, you can see many TH positive cells and cells with this molecular signature go on to form the TH periglomerular cells in the olfactory bulb and traverse the olfactory peduncle. But Matthew looked very closely at this section. And if you go to the next slide, we'll look at the clavistrum and found these examples in the clavistrum of these TH positive myc2 secretogogin PaxX cells very sparsely in the striatum and in the clavistrum. And then next slide, along the striatum, these almost formed a wreath or boundary around the striatum. And so Matthew has called these the striatum laryotom neurons for this wreath that they form. And so when do they get there? When do they mature? Next slide. They reach the edge of the striatum at day 120, which is already quite late, three or four weeks after cortical neurogenesis. But even at this stage, we don't yet see the processes. So they arrive and mature quite late in telencephalon development. Next slide. And so what about in humans and do they persist throughout life? Here's an 88-year-old, a section for an eight-year-old brain sample, and you can see these in the external capsule and along the striatum in humans as well. Next slide. And do we see these in rodents? We see the expression pattern for this or the immunoreactivity of this combination in the olfactory bulb and olfactory peduncle, but we never see it along the striatum or in the classroom. And that suggests to us it's human specific. And so to summarize this section, we suggest there's a kind of triple homology that these deep white matter neurons in humans come from a homologous initial class to the adult born granule cells that represent the vast majority of adult neurogenesis and rodents. Next slide. And that these striatum or autum neurons appear a molecular homologous to these PAC-6-TH positive periglomerular cells of the olfactory bulb that are actually dopaminergic. And then coming back to the 261 class, this class radiates to generate a diversity of cell types in striatum, amygdala, and even in primate white matter. And it looks to be homologous to the other class of periglomerular cells, these cubline in one box P2 cells. So to summarize that model, in terms of the migratory streams, there are these streams shared with rodents of these three populations to the olfactory bulb, as well as the eccentric spining neurons that are conserved between primates and rodents. But then there are also these potentially primate specific ESPN like cells in the superficial white matter. Next slide. And there are these deep white matter neurons where there's a homologous class in rodents, but not a homologous migratory stream, and we don't see them at the same abundance. And then finally, there are these primate specific striatum or autum neurons. And next slide. And so this would represent to us this kind of second model of it's a conserved initial class of neurons that is shared between primates and rodents, but it is being moved to primate specific locations where it may have adopted new gene expression signatures and functions. And one of the things that as Matthew finishes his PhD, we're really excited about is trying to explore when along these migratory streams, this nice two pack six initial class differentiates based on destination. Next slide. And so why might these olfactory bulb neurons be redistributed? And we're calling this kind of reduce and reuse hypothesis that the relative reduction of the olfactory bulb compared with the dramatic expansion of the cortex and underlying white matter as well as the dorsal algae progenitor zone that sits adjacent to the cortical excitatory neuron progenitors may allow for these neurons to be co this initial class to be co opted for new functions. And next slide. And, you know, we often think about cortical expansion in terms of the expansion of neuron number and gray matter, but the white matter has expanded super linearly compared with the gray matter and is the latest to mature region of our brains taking several decades to mature in the prefrontal cortex. And we find it very interesting that the same initial class of neurons that is involved in plasticity of the olfactory bulb and the major source of adult neurogenesis that that same initial class is redistributed to white matter regions of the enlarged primate brain. And so together so far, I've presented these two contrasting examples of of self evolution. In the first case, a unique transcription factor code specifies a qualitatively distinct initial class of neurons, these tack three neuropeptide expressing neurons. And in the second case, a conserved initial class of newborn neurons is redirected to a new location, particularly these stratum laureatum neurons. Next slide. Given how rare it is to see a qualitatively distinct initial class on and how common post mitotic fate refinement appears to be from initial classes to terminal classes, we would predict that post mitotic fate refinement due to extrinsic factors could be a potential source of species specific cell types as these as the cortex and other destination regions have elaborated in evolution. But we also think that new cell types are likely very rare in more recent human evolution since our divergence with a from a common ancestor with chimpanzee. And so in the remaining time that I have, I'll talk about some of the strategies we're using to study the evolution of conserved cell types. And this would be changes in gene networks and conserved cell types, the teaching old cell types, new tricks. And so in our lab, we're still interested in potential causes of cortical expansion. And you know, I know people in this audience have done some of the work in this area as well, but we're also interested in these trade offs and the cost of large brains. And in combining, you know, these comparative single cell genomics approaches with evolutionary genetics approaches, where we might decode not yet, where we might decode the adaptations of specific cell types to this large brain environment. And, okay, now, so one of the places that we think this trade off from unequal scaling may be most strongly manifest is the ventral midbrain. Next slide. These a few hundred thousands of ventral midbrains up from allergic neurons form these vast projections to stritem and as well as to cortex. And their targets have expanded disproportionately with their source number. If you compare a human brain to a recessed macaque brain, the volume is about 17 times greater, but there's only two or three fold increase in number of these neurons. And so we're exploring the hypothesis that these neurons could represent a vulnerable joint. And in addition to this unequal scaling, there's some studies suggesting that there's increased innervation to certain areas like the medial caudate in humans, even compared with chimpanzees and gorillas that these innervation could be related to increased pro-social behaviors or flexibility in humans. So the combination of unequal scaling and new functional requirements could potentially create these vulnerable joints. And these neurons themselves, next slide, have these incredible cellular specializations where their single axon can be over a meter long, these bushes in the stritem. And they have very high energetic demands. They have pacemaker activity and very high calcium flux. And we want to test the hypothesis, are there cell intrinsic changes that would increase connectivity in humans as compensation or for new functional requirements? And also, are there compensatory adaptations that allow these neurons to take on greater energetic burdens? And could these compensatory adaptations point to potential targets or therapeutic factors that we could further harness? And next slide. And we're interested in these neurons as well because they're involved in these disorders that are enriched in humans and rare or absent or even impossible to define in other species. So next slide. How do we, how can we study human specific changes in metro mid-range populations? And for this, this is the first time I'm discussing this project, we're really lucky to have Sara Nolbrant join the lab. She trains in the Parmar lab where she developed some excellent dopaminergic neuron protocols that are now entering clinical trials. And in our group, she's established this phylogeny in a dish where we can generate dopaminergic neurons in metro mid-brain neurons from human chimpanzee orangutan and rhesus macaque. Next slide. And with this, we're trying to test this hypothesis. Is this a case of teaching old cell types, new tricks? And based on, you know, these combination of trade-offs and next slide. So we want to ask, have cell intrinsic changes in connectivity, gene expression, and bioenergetics evolved in these dopaminergic neurons? And this is a project that is in progress very much. For the connectivity questions, next slide. Sara has generated these assembloid models where we can culture ventral mid-brain from one species with cortex or striatum from another species and compare the intrinsic and extrinsic influences on connectivity. We're also looking at this from the gene expression evolution perspective. Next slide. And for this, it's such a challenge to do large sample sizes with these comparative IPS projects, but we're also really interested in cell intrinsic changes. So we are culturing intraspecies and interspecies pools and organoids where we can really tease apart intrinsic and extrinsic factors as well as scale up to many individuals. So Sara has made these pools now of 17 individuals from four species. Next slide. And here's a look at her protocol. These are from interspecies examples. So this is the 17 individuals here. And we can get very specific ventral mid-brain patterning. Next slide. We can follow these organoids through development, and we have strong T8 immunoreactivity, particularly in fibers that bound the organoid. Next slide. And we can take these out 100. I think she's taking them out to 200, 250 days. Next slide. And we can start to look at the cell type diversity and start to compare gene expression divergence across species. And just really quickly, here's the example at day 40. There's about 20,000 cells from human and 28,000 from chimpanzee. This is from two independent replicates. And I should say in both our replicates, we only started with one macaque and one orangutan individual. And they seem to have differentiated their pattern correctly, but they differentiated earlier such that they're quite underrepresented at this stage. And on the other hand, we have relatively even representation. We managed to keep the seven or eight human individuals through this stage in at least six of the chimpanzee individuals. So the human and chimpanzee cells actually culture well together and maintain their representation. And this is a UMAP projection. We have not aligned the species just for the purpose of showing you human versus chimpanzee, but the cells do align quite easily with standard methods like batch bounds carrying nearest neighbors. And so these cells show markers of the neuronal lineage. There's the progenitors on this side and the differentiating neurons on this side. Next slide. And you can see here that we've captured a significant proportion represent this LMX1a lineage. And if we follow this through, we can see markers of maturing dopaminergic neurons in both human and chimpanzee for all these markers. Interestingly, we have a branch, this or a path here that is the ventral mid-range dopaminergic neurons. And then we also have a subthalamic nucleus neurons in both human and chimpanzee. So we're sampling more of the ventral mid-brain diversity. But that will let us ask whether specific cell types have been substrates for gene expression changes and to really isolate the specific changes in the dopaminergic neurons. Next slide. All right. Next slide. And so if we do the simplest analysis, just principal components across the time points along this LMX1a lineage, we can see that the major axes of variation relate to differentiation from the almost pure progenitors at day 16 to maturing neurons. Interestingly, there's still some progenitors at day 100 that may be stalled or may retain neurogenic potential at that stage. And then the second axis of variation relates to species. And you can see the few orangutan and macaques cells that are in these initial runs. So we are repeating this now. And we can see here, you can see these kind of conserved maturation trajectories, the sequence of expression you would expect as these neurons differentiate. But then next slide. We can also identify species-specific changes. Here's an interesting example of shared expression in the progenitors. But then specific, I think this one is actually a down regulation. Yeah, this is a human-specific down regulation in this case. And right now, we're focusing on genes influencing calcium buffering and physiology. And we're seeing some enrichments here that potentially could support the initial hypothesis. But it's still early in this analysis. Next slide. And so I wanted to talk about this as an example of how we think about studying the evolution of cell types along the human lineage after divergence with apes. And so we are taking these comparative approaches using IPS derived cell lines. And we have found that the gene expression divergence we see in the IPS lines corresponds and overlaps quite well with gene expression divergence we observe in primary tissue samples between human and macaque. And the primary tissue validation is important to us. We've also found that we can really find tissue-specific gene expression changes. About 85% of the changes we saw previously in cortex, we would not have found looking at just IPS or fibroblasts. We can use this data to identify intersects with candidate mutations influencing genes with specific expression profiles. Here's a splicing change that we previously found in a regular mitosis. And also to try to identify candidate pathways that differ between species. And in this latest project in progress, we're looking at the evolution of selective vulnerability through this lens. And we've mainly focused on gene expression next slide. But we'd really love to expand to other multimodal accounts of species differences and get to cell behavior and physiology. And the tavernal lab over here is kind of leading the way in looking at the comparative functional maturation of these in vitro-derived neurons using these types of methods. And then to try to couple that as best as we can with primary tissue validation. And that's why collaboration and using these clinical samples becomes so valuable as a ground truth or correspondence for the in vitro models. And then the other strategy we're taking that I didn't have time to talk about today, but next week potentially we'll talk about is using genome engineering strategies to study human-specific changes in the appropriate genetic and cellular context using these models. And next slide. And so with that, I want to acknowledge the people who did this work and, you know, Matthew, who's here in the audience, really shaped the evolution of inhibitory neuron projects. He had the idea for these patterns and really led all aspects of it. We're fortunate to collaborate with his co-mentor, Jimmy Yee, on some of the computational analysis, and Tom Nowakowski. And then we're so lucky to be next to Mercedes Peretti's, who identified the ARC migratory steam originally and with her student, Caitlin Sandoval. And then Sarah Norbrent is leading the dopaminergic neuron comparative project. And Brian in the lab, I think, has generated his inner large panels of IPS cells previously from his work in New York Glod's lab that are widely used. And in our own lab, he's also expanded these panels. And then Sophie Solemma at UC Santa Cruz, who generated the orangutan lens, and then the whole lab. So thank you. And apologies for some of the tech mishaps here, but I think we got through it. You hear me? Yes. Thank you very much. I think so these, wonderful seminar for presenting such exciting data, including a lot of unpublished work. So thanks a lot. And the floor is open for questions. Oliver. Wonderful presentation. Alex really enjoyed it. Particularly interested also in the latter part and potentially also my own background, as you may know. I was curious, based on the selective vulnerability hypotheses, the two questions essentially. So as you have kind of the ground truth, as you say, in the primary tissue, you can make comparative analyses in vitro compared to the primary tissue. Do you see the true diversity of dopaminergic neurons within the eventual midbrain? So A9 versus A10 specific dopamine neurons, because obviously there's a selective vulnerability between those subtypes. And then while the change in expansion is obviously an interesting question, I think evolutionary, but I was curious from a clinical perspective. As far as I understand, there's actually quite a lot of redundancy in the amount of cells within the eventual midbrain before Parkinson patients develop symptoms, right? So I think we know from transplantation studies that you can lose up to half of your dopaminergic neurons within the midbrain before you become symptomatic. So is there perhaps a selective vulnerability or a problem in scaling in specific subtypes? Or is it a connectivity? How do you see that kind of within the hypotheses like this, that you can actually lose so much of your midbrain dopaminergic neurons before becoming symptomatic? All right, thank you for the question. So the first question, how much of the diversity of dopaminergic neurons do we recapitulate in this culture system? And I think that question also gets into can we expect an in vitro system to recapitulate aspects of selective vulnerability that we might see, you know, in vivo or in tissue? And, you know, we have some markers of A9 versus A10. We don't have control over that patterning yet in our protocol. And different lines vary, you know, we're doing 17 lines through this protocol. And different lines vary in their wind activation, which influences, you know, how caudal they are along the ventral perpate. So we are actually doing, we're bracketing the wind exposure. So everything I showed you there was from three different three different shear concentrations for wind activation. And so, you know, I believe we're spanning both identities. We still have, despite many cell sequenced, and the majority of them being in the LMX1A will lineage, we still have limited sampling of maturing dopaminergic neurons. So it's harder to see the full subtype diversity. There's this beautiful study from the McCosco lab, even showing a primate specific population in substantial nigra that expresses Calvinin1. And it's hard for us to map to that diversity yet with our cell number in terms of whether we would expect the in vitro system to recapitulate differences in vulnerability. You know, it challenges how mature can the neurons get in vitro. There's some arguments that, yes, we can. So there's studies of explants of actually our friends, the Th periglomerular cells from the olfactory bulb versus substantial nigra explants. And in culture conditions where you dissociate and then culture those cells in vitro, you can indeed see differences in the vulnerability. So, and, you know, in our hands, we can do these, you know, wrote known treatments or other bio energetic stress treatments. And we can see selective vulnerability of the Th neurons compared with other cells in the organoid. So I think it is possible to model this in vitro. I do think there are some limitations to the maturation of these neurons. The nice thing about the ventromid brain compared with the organ cortex is these do mature more quickly than the cortical neurons. And then, you know, the other question is, you know, I've talked about this unequal scaling of the volume of targets with the number of neurons themselves, but, you know, the volume of targets is also going to influence the overall external arborization and axon length. And, you know, the question is there's a reserve. It's not like we're on a knife's edge. And, you know, I would argue that, you know, the most recently evolved traits are often vulnerable to dysfunction. But there already are compensatory adaptations such that it is not generally pathological in the population, especially pre-production. So if you know people with, you know, knee problems or back aches or worried about childbirth or had wisdom teeth out, you know, all these things relate to recently evolved traits like bipedalism, changes in public morphology or brain size. And, you know, the argument would be that to the reserve capacity is lower. And whether that is, you know, I think maybe simplistic to think of it in a neuron number and probably more related to the, you know, bioenergetic stress and arborization and the, you know, that I found at least one example in the literature of high penetrance mutation that could cause Parkinson's reported in a rhesus macaque colony. But the idiopathic cases and the common cases without great epidemiology, you know, in non-human primates notwithstanding, you know, are far more rare and unobserved. So I, you know, I do think that increasing strain has been put on this system even in the last six million years and that the IPS organoid models would give us a test kit to follow that up. But, you know, it's a hypothesis we're testing now. And I think, you know, we hope to functionally interrogate some of the genes that differ between species to see if they could answer something. I think there's two parts of it. There's cell intrinsic changes to accommodate more connectivity, essentially even more functional requirements for connectivity related to these. You know, Marianne Roganti has this kind of dopamine driven hypothesis about human brain evolution and social conformity. And then there's also just the unequal scaling weakness. And so that we, we are attempting to test both of those hypotheses with the system. But it would be fun to drill down further on that. At the price of one. Incredibly cost effective. Thanks. It was very exciting to listen to your talk. Thank you. I would have two unrelated questions. Let me first ask the first. So about the olfactory bulb and would you then try to correlate across all mammals, not just primates and rodents and not just carnivores, right? Like, I mean, really all of them to see the, if this, your hypothesis would hold true. Thank you. So the question is we've put one possible explanation, this reduce and reuse hypothesis. And so how do we test that further? And that's something Matthew is wrestling with now as he wraps up his PhD. And we're thinking about a few different ways of testing that, including some functional experiments in rodent to try to kind of force the scaling. And then also a kind of comparative association, which isn't going to get to causation, but could add some support for it. And we're exploring both. You know, I think it's hard to get that many brain samples, especially at the stages when these streams are present, but we're working on it. Yeah, because Farad seems to have a huge olfactory bulb. I remember just so, but yeah, it would be interesting to test that. Okay. So the about the mid brain and basically interspecies organized. I was wondering there if very kind of technically, when you have so few macarons and orangutans contribution, is it possible that the, you know, the conditions in which you grow organoids are very much human and chip favorable? And if so, could then, you know, what would interest me is, could these extrinsic factors that then influence on cell fate could be different and differentially affect the different species? Yeah. So the question, you know, has a few parts, but the, you know, have we optimized our culture methods, you know, for human and chimpanzee? And is that depleting the macaque and orangutan? And then another layer of that is, you know, are there some signals, you know, and I think, you know, there's studies of competition between species and in chimeric culture that are ongoing now from, you know, June Woo's lab, for example. And, you know, could it be that there are, you know, humans are human or human or hominid, maybe promoting factors that are not there for the resys macaque? I will say in the lab we've worked very hard to build the new stem cell medias and differentiation conditions from the ground up that are optimized to work across species and to reduce variants within species. And when I started this doing the reprogramming, I was using the human protocols and my human positive control always works great. And I really struggled. And, you know, so we've taken a lot of time to rethink that. And if you grow, we grow these in isolation and the macaque and orangutan organoids grow really well. But I don't think it's a fair comparison because in these first couple of runs, we only had one individual from each of those species. And so we only seeded them at maybe 10% of all the cells. So stochastic factors or other things could have influenced the loss. And so it's something we're repeating now in the additional replicates. And I'll get back to you. But I will say those, they, even in this case, they still patterned properly, but they grow very well, you know, when separated. But I wouldn't conclude yet that there's some sort of competition based on, you know, only performing this twice and they're being underrepresented in the initial start. We did a more even proportion by individual rather than by species. We were most focused on the human chip comparison to start before we go to isolate what's derived on the human lineage. And we wanted to make sure we had that representation. Okay. Thanks. So the first is related to the ventral midbrain and this idea of this higher iteration of the dopamine neurons being one of the underpinnings of pro-sociality. And I was wondering, first of all, what you think is the best evidence for that? And if you think about Parkinson, of course, depending on the progression of the disease, there are of course elements of social restriction, but which are usually thought to be secondary to the other symptoms of the condition rather than primary feature. So I was wondering what what your thoughts were thinking about this higher innovation of dopamine neurons as a correlate or pro-sociality. And the second question relates to the last part of the talk, which was also very interesting. And I was wondering whether, I mean, as you know from when we initially spoke in Cosplay Harbor, we've been doing a lot of multiplexing among different individuals in organized paradigm. And so I was wondering to which extent you always see a more or less even distribution among individuals of the same species. And if you have sort of benchmarked how often, especially at very advanced stages, you actually maintain the distribution of individuals that you had at the beginning of culture. Thanks. All right. So the first question is what is the there's this intriguing dopamine driven brain evolution hypothesis. And there are, if you look at the fossil record, it suggests that human sociality predated a lot of the brain expansion that has been observed in terms of group sizes or reduction in sexual dimorphism. And so that suggested that maybe there were some driver mutations in social conformity and a social affiliation that were kind of prerequisites for additional changes in brain expansion. And so what is then the evidence for the social conformity side? So there's neurochemical evidence in the ratio of dopamine and serotonin levels and innervation to norepinephrine compared across humans and great apes that suggest lineage specific changes. And then there's some limit evidence for increased dopaminergic neuron innervation in certain brain regions. But you know, I think there's these experiments are tremendously difficult to get matched samples between human and nonhuman primate and similar postmortem intervals and just to quantify these features that are really hard to quantify. And our hope is that an orthogonal method using stem cell deriving or modulatory neurons might give us insights from the gene expression and even causal genetic change level ultimately one day as a field, which could then be linked back to test this hypothesis through additional methods. On the technical question, we were very worried to invest this much money in single cell sequencing as a young lab that we would be sequencing just one individual. And actually our orangutan line grows really well and we were especially worried that one was going to take over. We pre-screened all the lines for p53 mutations. We cultured many but not all of the lines to establish that they had roughly similar growth rates and patterning for density. Sara did her a heroic amount of work to get to this point. It's really amazing and speaks to the training she already had before she joined the group from the Parmar lab. And the representation, what I was amazed about is the representation of the human and chimp cells was almost identical to the starting conditions, especially in our second replicate. You know, I think out of the eight humans, there's a distribution. They're not perfectly evenly represented, but they're all retained through day 40. The chimpanzees, I think we lost one individual and another one is much lower representation. But some of that speaks to intensive pre-screening that we did in terms of, you know, we didn't want p53 null lines or, you know, lines of strange growth rates, but also, you know, we had a larger panel of lines to choose from thanks to Brian and his heroic efforts reprogramming these and also optimizing media at the state of the cells. When you start, the differentiation is so important. And, you know, I think how we actually do the pooling. And yeah, it was certainly nerve racking when we were doing the de-multiplexing. If it just ended up being an experiment on human or something after all that work, we did some validation at that time. But, you know, I think it is a really exciting frontier for folks in this room where human genetics meets stem cell biology and a lot of the problems of scale that have challenged us, you know, in the last decade in this field are, you know, going to be met, including through the pooled culture approaches. So the population in addition by watching the native approaches are, you know, extremely exciting and really get at the kind of promise of the genetics, especially the genetics of cell intrinsic differences. And, you know, I tried growing organoids from 20 different lines as a postdoc and, you know, man, it was hard to do in a rate format from a technical perspective and just kind of whack a mole with different individuals keeping up. So then if not, we thank you again a lot, Alex, for this wonderful visit. Thank you.