 First of all I have to tell you that I'm not a neuro-informatician, so the talk I'm going to give you is probably a little bit different from other talks that we've been hearing from yesterday morning, but I'd like to tell you something about what we how we have found and what we have found recently using some other people's data set. And also today I'm going to focus more on cortex or it's not going to be thalamus, but which is more about the thalamus and cortex on circuit development. So I've been interested in looking for and then finding what is the molecular mechanism which is controlling the cortical area development. And as you can see here in a really cartoon that which is really summarized in the easy way that different species that basically they look quite different from size wise. And also if you look at the functional area, they look kind of very different, but at the same time you can also find a common area such as primary sensory cortex or visual or auditory cortex. And on top of that, during the evolution, they basically make the brain the feel bigger and also they make it more complicated such as adding new areas like for us, for those area which deal with the language processing or higher cognitive function. So basically I was interested for many years like what is controlling, what is the molecular mechanism which is controlling this kind of diversity of the functional cortical area. And for the early developmental time point, we actually now know that a lot of the gradient of the morphogen and transcription factors are actually mainly controlling like an anterior posterior and meteorolateral patterning. But moreover, what the question remaining is what is more and what is different among different species. And before we go into that, we actually noticed that even in the same conserved functional area such as somatosensory area, if you look those detailed structure and then circuit in different animal, you can actually find they are quite differently organized within the conserved somatosensory area. So for instance, some of you who are coming from Australia probably are familiar with this animal like platypus. If you look at their somatosensory cortex, you actually can find this striped pattern, which is a topographic representation for their mechano and electric receptors, which is having the striped pattern on their beak. And also if you look at the star nose mole, this is the animal which is mostly living in the North America. You can find they have very interesting structure on their nose, which is these appendix have like 11 and also other 11 appendix on both side of their nose, which they use like their hands basically, rather than their nose. And while they're hunting, they use this structure quite a bit. And also if you look, there's somatosensory cortex, you can also find pretty much some similar pattern of these appendix, which is like 11, you can find 11 stripes in one side. And most well known on structure you can find in somatosensory cortex is rodent spring. Here as you can find in the somatosensory cortex, you can find so called barrel cortex and somatosensory cortex, which is representation of these whisker, facial whisker representation that you can also find in thalamus while they're making connections to thalamus to cortex. So as you can see here, they're all these structures are all in somatosensory cortex, but depends on what kind of peripheral structure they have, they also have different kind of organization in somatosensory cortex. So we decided to focus on this question, okay, then how they can make this kind of different connectivity. The first thing we did is we thought that, well, once since they have this structural organization, we thought that probably and they which is happening in early, which is controlled in the early developmental time point, we thought that they should have some molecular mechanism for that. So we use Alan's brains atlas and gene paint and others on people's website and keep looking at whether there are any specific molecule expressing the barrel structure, especially in the barrel hollow, which you can find in here or in the barrel septa, which is in between those two barrels. And these are only for some example that we have found, but we actually found good numbers of candidate we can start to work on. Then we decided for today, we decided to focus on this molecule called BTBD3. So at this time point, when we've have found we decided to work on this molecule, we actually didn't know anything about this molecule. But the reason why we got interested in this molecule is, I'm sorry, it's really wiped off, but it's really clean in situ. I hope you can believe me. This BTBD3 expression pattern looks really interesting to us because they have very specific expression in a barrel cortex, as I explained, and the rest of the cortex pretty clean beside a singulate and also preformed cortex, but there aren't any in the visual cortex. And then why we got interested on this is when we decided to look at BTBD3 expression pattern and different species, because our question is how these circuits get developed in different pattern and different species, we decided to look BTBD3 expression in common marmoset. And when we look BTBD3 expression in neonate common marmoset, which was really striking. So beside some of the sensory cortex and auditory cortex, they had a really strong expression, very specific to the primary visual cortex, especially in layer 4 neurons, which is a layer they receive input from the thalamus. So if you, in the previous figure, again, that you can see that there's no expression of BTBD3 in mouse visual cortex, but they have strong expression in the common marmoset on primary visual cortex. So what it means, this is a question we decided to ask. But before we go into that, we had to find out what's the function of BTBD3 in the mouse on barrel cortex, because this is the animal which is actually easy for us to manipulate with. So once again, just to remind you like the circuit for the barrel cortex, basically these input from the whisker reach the brainstem, brainstem to ventral basal thalamus, and they reach a layer 4 cortical area in the somatosensory area. If you cross section here, please imagine this is layer 4 cortical neurons, and here is the somatosensory barrel cortex, sorry, bariloid. And from the bariloid, from each patch, they basically project thalamocortical axons reaching into the layer 4 cortex, and they make this kind of clusters that we can identify in later states as a barrel cortex. And for the recipients, for those cortical neurons, which is sitting inside of each barrels, like here, they basically are receiving input from everywhere, so basically they can keep their dendrite level rating everywhere. However, these neurons which are sitting on the edge of the barrel, like here, basically they have to change their morphology looking toward to the barrel hollow, because this is the side where they're receiving more input. However, there's no thalamocortical axon from the ventral basal thalamus reaching in between the barrel's septa. Instead, there's other axons which is coming running through from the posterior medial thalamic nuclei here, they run through and then reach to the superficial layer. Moreover, layer 2-3 neurons which sense from projection to the contralateral side of the cortex actually needs to run through in between this barrel hollow. So basically, for these guys, they don't want to make connection with these two different kind of projections, so they basically keep to stay away from this structure. So this is what's happening in the barrel cortex. And once again, I told you we got interested in this molecule, BTBD3, BTB domain containing molecule. I told you that at that time when we started working on it, we didn't know anything about its function. However, the protein family, which called BTB domain containing family, had like 200 family members and some of them are known to function as transcription factors and so on and so on. Basically, they had a lot of variety of the functions. However, we couldn't find any domain in BTBD3, which we can predict its function. However, when we looked at these two papers which was published a little bit before, they have found this other BTB POC domain containing family member called ABRA, has ability to control the dendritic morphology. So this actually made us to think like, well, since this BTBD3 is expressing specifically in the barrel on cortex, why we don't test, why we don't manipulate BTBD3 and test the dendritic morphology in the barrel cortex. So the first thing we did is generated BTBD3 SH construct. We did a lacquer operation into the developing mouse cortex. We did a lacquer operation at E13.5. This is the time when layer 4 cortex is generated. As you can see, quite specific expression is happening in the layer 4 cortex and was using this SH construct compared to the control side. You can see quite huge reduction of BTBD3 mRNAs happening here after SHRDA lacquer operation. So in the control brain now you can see like this is the one barrel and as I told you, these neurons which is sitting on the edge of the barrel needs to stay away from the barrel septa and keep their dendrite looking toward the barrel hollow. However, when they lose or knock down BTBD3 expression, then this is what's going to happen. Basically the dendrites start to ignore the barrel septa and then they elaborate their dendrite everywhere. Okay. So it looks like BTBD3 has something important to control the dendrite morphology and the barrel cortex. Okay. Then the next question, what about in the visual cortex, which we have seen strong expression in the common marmoset but not in the mouse? To test the idea, we decided to do the ectopic expression of BTBD3 in the mouse visual cortex and see whether they can change the dendrite morphology or not. So here's the experimental procedure we take. So again, we use a lecopration technique to put BTBD3, this time it's an overexpression, so it's functional BTBD3 construct, a lecoprated in the visual cortex again at E13.5 and then we raise them till P14. And then we did a monocular deprivation for these animals and then look at the morphology in between this binocular region and the monocular region. So the basic idea is if you do the monocular deprivation from the contralateral side, these monocular regions here are receiving from this side basically get more silence. However, this eye, which is in the contralateral side, open eye, I'm sorry, the ipsilateral side which still have their input coming in, the binocular regions are still active. So we can make the contrast of the neuronal activity here. This side is higher and this side is lower. So we looked at the neuron which is sitting on the border of these two regions. And in the control animal, basically, whatever you do, even if you do the monocular deprivation, these dendrites do not respond to neuronal activity and then change the dendritic morphology. So whatever you do, they have the symmetric pattern of the dendrite in the visual cortex. But when we put BTBD3 in the visual cortex, so basically we force them to express BTBD3 and what happens is they start to respond to neuronal activity and change the dendritic morphology. So with the Weacher-Magelin tracer, we know that the right side of the screen is receiving input from the open eye. So basically these neurons are responding to the open eye column and try to send the dendrite to the higher neuronal activity side, which probably they think they can receive more input from this side, but not from this side. So for the efficient circuit formation, probably it is worse for them to change your dendritic morphology in an early postnatal stage. So now in mouse system, we know that BTBD3 is functioning to control the dendritic morphology. It depends on how they are receiving neuronal input and make efficient circuit. But what about these animal which have strong expression in BTBD3 in the visual cortex? Is it also the same in these animal which make their visual cortex working more efficiently or not? That's the question we wanted to ask. So just to remind you, in the visual cortex for these animal like a marmoset or ferret or cat, they have beautiful ocular dominance column which you can't see in the mouse on visual cortex. So basically the ocular dominance column is organized by right side and left side input which make this striped pattern. And a critical period if you do the molecular deprivation like here, basically those open eye column gets bigger and the column which is receiving from close eye which gets smaller like here. So what about if there are any function for BTBD3, forming the ocular dominance column and also changing the dendritic morphology within the ocular dominance column. So of course the straightforward way to test this idea is if we can manipulate the common marmoset visual cortex, probably we can answer it right away. But at this moment it's not that easy technique. So we decided to use different species which do have ocular dominance column and we decided to use ferret. So this is actually animal which is very well known and used for studies for many years, having a beautiful ocular dominance column. So there are developmental time points a little bit different from mouse but we align it and to find a corresponding time developmental time point compared with mouse and decided to do electroporation on these animals. But before starting doing electroporation we had a check on BTBD3 expression on these animals as well and like common marmoset we found they have strong strong expression of BTBD3 in the somatosensory cortex and also strong expression in the primary visual cortex as like marmoset. So this part is actually put in one slide but this is our three months work to try to establish ferret in uteroelocropation but actually end up we end up using exactly same protocol with mouse so basically it was quite easy I have to say. So here you can find on elocropation technique which is exactly the same as mouse use a injected plasmid DNA whichever you want to use into the ventricle of the developing ferret embryo and then place electros in this way or whatever wherever you want to elocoperate and then zap and basically then put them back and then raise them and collect them till the age you know with them till the age you want to look at. So here's the example how the elocopation is working it's a little bit hard to see in here but this is the right hemisphere we try to elocoperate in wide area here's like a visual cortex somatosensory you can see some of them are already start making the wrinkle unlike the mouse brain and if you come using the different combination of plasmid you can also make it to label sparsely like here then you can get the single cell resolution to see the dendritic morphology in a better way. Okay so here's our real experiment what we had done again just reminds you this is the BTBD3 expression at p12 postnatal day 12 that there are expression in somatosensory and also strong expression in the visual cortex we generated ferret version of shRNA construct for BTBD3 to knock down these BTBD3 in the visual cortex so we elocoperate it e34 this is the time point when the layer 4 get generated in ferret and then we elocoperate it sh construct here and then you can find they greatly reduce BTBD3 expression specific in the visual cortex so you can do the target knock down and ferret. Okay so in control case here it's not the molecular deprived animal this is control animal we did a sparse labeling of visual cortical neurons was using elocopration you can see these are symmetric pattern of the dendrite in control animal but if you do the monocular nucleation to make the ocular dominance shift in one side or the other and what happened to those neurons which is sitting in between those on column open eye column and close eye column what happened is basically they are receiving input from one side but less from the other side so they change your dendritic morphology towards the open eye column so here the right side again right side of the screen we know that was Weecher-McLennan injection to the retina it's an open eye column so they shift their dendritic morphology okay so finally on BTBD3 knockdown case what's happening here is basically they stop responding to the neural activity and they cannot change the dendritic morphology anymore so I think this is a really convincing data that BTBD3 is basically BTBD3 is important to change the dendritic morphology toward the active the site which is receiving more active accents or input and also the function of BTBD3 is conserved among different animals so depends on where you have expression of BTBD3 you can change the circuit formation in the different area part of the brain for instance some of the sensory cortex in mouth visual and ferret on also in the marmoset and also if you think how these animal live basically these guys for those people who's working on the visual system in mouse I'm sorry to say but their visual system is not that great I have to say I'm not saying they're blind but compared to the sensory cortex and their tactile they are actually I have to say like tactile is sort of more sensitive to than that and especially when they're it's this early postnatal stage when they're born they basically need their whiskers to find things and the directory and everything like that and also their olfactory system as well so I actually have a belief that they that's the reason why they have strong expression in the preform cortex as well and and these animal actually they as you know these animals have a ocular dominance column they actually rely on their visual input quite a bit from the very early postnatal stage especially common marmoset if you look at them their eyes are already open from their day one and they respond to the visual input and then also they're highly socialized animal but they vocalize pretty well so they need to well that we say they chat a lot because they really communicate each other not for only with their family but with other family which is separated in cases they really chat each other and they hear pretty well very sensitive to the sound so probably that's the reason why they have this expression in the auditory cortex as well so it kind of makes sense that they have bdbd3 expression to the area where they really do heavily rely on their very early postnatal stage and probably that's a kind of key for them to to get better viability okay so now i would just want to using like so one two minutes i'd like to show you what we're doing right now is so basically to answer our question is like a how the different cortical areas are developed or how they organize that in even in the same year how they get organized in different way if we can understand how bdbd3 gets their expression in different area like this probably we can answer some of those question and that is one of a project we are working on on top of more detail mechanism how bdbd3 is functioning the other thing we are working on right now is this is just one of the example that how we are processing our research and we actually noticed that was looking on other some people's data and also for the marmoset we are doing all of the institute by ourselves and then tried to build up our own data set and what is important and what also we noticed that is actually the expression pattern i mean gene expression pattern is very different quite different in marmoset and rodent was a very simple comparison so here's just the collections for the autism risk gene just probably i thought that many people get interested in this kind of thing but if you just simply compare those expression pattern you may notice and so this is the visual cortex is conserved area again but you can find they have very different expression pattern not only because their layers are organized in different differently but different subsets of neurons are having expression or not having expression of the same gene so basically we say we say like you know the genes are very conserved and it's different species but till you look at the detail expression pattern probably we don't know what exactly or how exactly their function different way in different species and thinking probably that's the reason why it is causing such a different organization and a circuit organization in the different even in the same area in a very different way so this is kind of project we're processing father again i'm not the neuroinformatician but we are definitely appreciating all of these data set and using in these kind of way that glad i had a chance to talk to you about this okay so here's my last slide here's all of my lab um the past members and um collaborators here for the mouse strains and here's um the funding thank you very much thank you very much for that i was looking at your barrel cortex data it struck me that what you've described is beautiful for the lamniscoll pathway but of course they are the parolamniscoll and possibly extra lamniscoll inputs as well do you believe the same rules apply in those cases as you've shown for the lamniscoll inputs so um i think you're talking about this pathway from the ventrobasal and posterior medial pathway right well um i didn't know at this moment that from what we have right now but recently Dennis Jabuddin from um Geneva um university published about that how the bpm neurons and pmm neurons are controlling all those lamholo neurons and septin neurons in the molecular basis so it could be so what we're thinking right now is this molecule bdbd3 which is expressing the barrel hole is controlled by the vpm neurons so if what he is saying is pom neuron also have something to control the turn on barrel septin neurons probably they do have similar mechanism like who gets there and who turns on the gene in the post um synaptic neurons i don't know i'm probably not answering your question but we don't have those data and this is what we are thinking right now yeah thank you wonderful talk and especially the last part because i do think that there is i'll show something an evidence for species specific species differences in gene expression and so i would like if you could elaborate a little bit more on marmoset why is it hard to do a neutral electroporation and i think it's an important question because there is a discussion at least united states whether marmoset should be used or macaque monkey and there are marmoset has many advantages right so and you are probably world expert on this so what are the problems with using a neutral cooperation in i mean because it's small brain i mean for primate it is very small it's like a rat size even in adult right so to be honest we tried few times but it was not straightforward somehow they i mean we think you know it's small and it should be easy to handle but we did a few procedures and then you know we fail basically okay so that's the one thing we we didn't succeed on doing a neutral like operation right and why we're using marmoset in our research thought that's your question or yeah yeah so yeah so especially for the people who's working on developmental biology they're you know the first generation turning the generation is one of the reasons that why you know we want to work on it because each time they have three or four offsprings you know and they once they are they make couple they're pretty monogamous so they stay like that for like 10, 20 years like that so they're very productive so that's one reason once you get colony you know they always generate offerings and the third thing which I don't know it's like you know to be a you're an atomist you know it's not a good thing to say but it's a brain's very simple so because probably you notice that they don't have many gyrus so which is really easy for us to find specific areas and also specific layers and those kind of things so for people who worked and mouse for many many years actually it's very complicated for us to look at um like a macaque brain and human brain for instance so even finding which location was really difficult but I found doing by myself marmoset it's very easy doing that so those kind of re-multiple reason you know is the reason why I'm interested in thank you hello on one of your gene expression maps I noted that this gene B3 seems to be expressed also in the mouse hippocampus and certain areas can you say anything about its potential function role and whether it also appears in other rodents at the same location that becomes good to be but um one thing is you know it's also no one that in hippocampus they change the dendritic morphology depends on the input so one one thing easily if people can think is probably they are doing a similar you know thing but also if you look at different species they have slightly different expression pan so on top of that they might have different functions but honestly I didn't know about that I'm waiting for our knockout mouse to come to see you know when probably you can get better idea or a little bit faster way so you're waiting for that and I think you're asking for those questions it's not only in hippocampus they also have strong expression in celibala just for your information yeah but yes no do you know what triggers what is the activity dependent trigger of the expression of this so the expression expression during development so the expression of btbd3 is not controlled by the neuronal activity once accents come in then they get turned on so we're working on that mechanism so what what is activity is doing they change their translocation so go they go into some we first we thought that they were going into nucleus but looks like they're going into some subcellular domain which is a unsolvable fraction so like let's say centrosome or something like that so they basically change the you know the the trans the translocate this protein probably we're thinking about probably to the centrosome which is actually a stem of the gender right father control ebics genase or some ROG DPA activity control the dendritic morphology that is our guess thanks for great talk thank you very much