University Place

cc >> Welcome everyone to Wednesday Nite at the Lab. I'm Tom Zinnen. I work here at the UW-Madison Biotechnology Center. I also work for UW-Extension, Cooperative Extension. On behalf of those folks and our other co-organizers, Wisconsin Public Television, Wisconsin Alumni Association and the UW-Madison Science Alliance, thanks again for coming to Wednesday Nite at the Lab. We do this every Wednesday night, 50 times a year. Tonight I'm delighted that you get to hear a talk by Lindsey Traeger. This is a talk I've been waiting for three or four years ever since I heard that she was starting to work on the genome of the electric eel. We're going to hear more than just about the genome, but also some of the mechanisms at the cellular level. Keep in mind that voltage is pretty important to cells of all sorts. But when you get to crank out volts by the 600's, that's pretty cool. It's also deadly. I think we're in for a treat tonight. It's very cold out. I think we're going to get warmed up by hearing about some of the stuff that the electric eel can do. Lindsey grew up in Cold Spring, Minnesota. She went to the University of Minnesota at Duluth. She got a BS there in 2007. I must say, Duluth is probably one of the few places that's colder than Madison tonight. Then she worked for three years for a food science company by the name of Danisco. Than last four years she's been here in the genetics department at UW-Madison studying the electric eel. Please join me in welcoming Lindsey Traeger to Wednesday Nite at the Lab.

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>> Thank you, Tom, for that introduction. And thank you all for coming here. I know it's frigid out there. I would like to be on my couch wrapped up in a blanket right now. But here I am. I'm really excited to be here talking to you all today. I am lucky enough to have a project that's actually really exciting to talk about and actually really fun for me to talk about. The science I'm going to be showing you all today is really the culmination of the efforts of about 15 scientists that are spread all cross the United States. This is in preparation too for a paper that we hopefully will be putting out towards the beginning of next year. This is all kind of bright and new. What I will be explaining is how this project started off as a wide attempt at molecularly characterizing certain tissues in the electric eel and how it really became a lot more than that as we progressed in this project. Before I actually begin I'm going to show you an outline of where I'm going to be taking you in this talk. First I'm going to go over some background and history about the electric eel and also some other electric fishes. I'm going to talk to you about how this works physiologically in the electric eel. And I will hopefully convince you that this process is fundamentally interesting to study. Then I'm going to get into some background on my research, some interesting results that I have to share with you, and then briefly touch on future direction. This is a photograph of Dear Eelinore which is an eel that we first housed in the lab when I first joined about four years ago. Eelinore is an electric eel. The scientific name for an electric eel is electrophorus electricus. If you've heard about electric fish before it's likely this is the type of fish you've heard about. You've probably heard about the electric eel. For those of you who've never heard of electric fishes before, what do I mean when I'm saying electric fish? I mean literally a fish that is capable of generating electric discharges around it's body. Dear Eelinore here, being an electric eel, she's about two feet long in length. She's not full-grown. She can generate about 200 volts of electricity. Electric eels are not actually eels. They are part of a larger family of electric fishes that are native to South America that belong to the order of Gymnotiformes. There's about 200 different species of these fish. Like I said, all of them are native to South America. All of them are capable of generation weak electric fields using electric organs. So the electric eel is actually unique among Gymnotiformes because it can produce these high voltages. All of these other fish-- Let's see if I can find my curser. The electric eel here is pictured on the top. All of these other fishes are Gymnotiformes. They all have this long, undulating tail in common. They are actually quite a beautiful fish to see swim around. All these poor fish on the bottom here are all weakly electric only. It's measure in millivolts. You can't detect it with your hand. The reason they've evolved these electric organs is to allow them to navigate and communicate in murky waters. Electric organs are made up of a specialized cell type called electrocytes. They are developmentally derived from skeletal muscle. This is an important point that is going to keep occurring throughout my talk is that electrocytes are differentially derived from skeletal muscle. This is important to remember. Like I mentioned, the vast majority of electric fish are only weakly electric and measured in millivolts. They generate these discharges from a single weakly electric organ. They just have one organ and they're only weakly electric. So the electric eel is unique because it can generate these high voltages and it actually has three electric organs. They are the main electric organ which is used for creation and defense. It is strong voltage only. It has a Sachs' electric organ which is weak voltage only. Again, this is used in navigation and communication. Then it has a third organ which is called the Hunter's electric organ. Depending upon where you are in the fish it is either weak or strong voltage. These fish can generate about 100 volts per foot of tail length. They can get to be upward of five or six feet in length, so they can generate, you know, about 600 volts maximally. This is the body plan of an electric eel. What you'll see here is that the first 1/5 of the body is dedicated to head and internal organs, while the remaining 4/5 of the body is really just a cattle prod. It's really just devoted entirely to electric organs. Like I mentioned previously, electric organs are made up of electrocytes and they are developmentally derived from skeletal muscle. They share some characteristics with skeletal muscle even though they are a completely separate cell type. Unlike skeletal muscle they are non-contractile, but like skeletal muscle they are activated by a neurotransmitter called acetylcholine which is released from a neuron that comes and attaches to the cell. So how does this work? This is a cartoon showing kind of how an electrocyte in an electric eel looks. The first thing you'll see is that there's a huge amount of asymmetry with these cells. It has one membrane face that has a nervous connection coming to it. We say that this side of the membrane is innervated. The opposite membrane face has a huge amount of surface area to it. It has a lot of invaginations in the surface. The innervated membrane face, like I mentioned, these cells are activated by a acetylcholine. In this innervated membrane face it is chock full of two different kinds of sodium channels. The first is an acetylcholine-gated sodium channel. The second is a voltage-gated sodium channel. The channels in this membrane work, when they open they allow sodium into the cell. Sodium flows across the membrane and into the cell. In the opposite membrane there's actually sodium pumps which work to remove sodium from the inside of the cell. The way that this process works is that when acetylcholine is released from the neurons, acetylcholine goes and binds to the acetylcholine-gated sodium channel which allows some sodium to flow into the cell. You get a slight change in potential, or a slight voltage change within the cell. This causes a second type of sodium channel to open which is called the voltage-gated sodium channel. When this opens a massive flow of ions flows across the membranes. You get a massive flow of charge in one direction. Because, and you'll see this on a different slide that I have set up later on, these cells are arranged massively in series and in parallel, and because all of them are activated simultaneously by acetylcholine you get sum of charge in the form of these sodium ions flowing in a single direction across the cell. Because so much of the body plan of the electric eel is dedicated just to the electric organs, they're dedicated only to creating these voltages. That's how the electric eel is capable of generating such high voltages. But what's truly amazing about this process is that the ability to produce electric discharges isn't unique to only Gymnotiformes. It's actually evolved independently six times in fishes. This is an abbreviated tree of different orders of vertebrates. Each red line here contains a lineage that gave rise to a fish that ended up being electric somewhere on there. It's not really important that you know how this tree works, but what I would like to point of on this tree is the six branches and what they are. There are electric torpedo rays. There are electric skates. There is a large group of weakly electric fish that are native to Africa. I will be talking about these later in my talk. I will refer to them as momyrids. There are electric catfishes. Here are the Gymnotiformes on the tree. Then there are stargazers, which are actually both venomous and electric. They're pretty terrifying. Electric fish aren't only interesting to just scientists or biochemists. They've actually been a source of inspiration throughout human history. This is the Narmer Palette. It represents some of the earliest hieroglyphic inscriptions ever found. It's about 5,000 years old. What I want to show you this is up on the top. This is the two sides of the Narmer Palette. Up on the top here is a box. When you zoom into that box what this is is a phonetic representation of King Narmer, his name. Here is a catfish, it actually is an electric catfish, and a chisel. When you combine, phonetically, the sounds for these word, 'nar' and 'mer,' you end up with the king's name, King Narmer. 5,000 years ago in ancient Egypt they were already interested in electric fish. This is an electric catfish. In addition there are stories from ancient Egyptian times where they referred to the electric catfish as "He who has save many on the sea." This is because when a fisherman would cast out his net occasionally he would pull in an electric catfish with him. Electric catfish have very strong voltage similarly, on the same level of the eel really. You can imagine if you're trying to pull in a net using a wet pole or the net itself is wet, you might get a pretty severe shock and accidentally release all of the fish that you have in your net. And so it's an appropriate name, "He who has saved many in the sea." In addition to that, actually the electric organs have been used in the first battery design. This is a voltaic pile as drawn by Alessandro Volta. It's from the 1800's. What this is is a stack of alternating copper and zinc disks that are separated by brine, or saltwater. And actually the design of this voltaic pile was modeled off of the electric organs of the electric eel and also the torpedo ray. You can see some similarities here when you look at the zoom-in cross section. This is an electric eel, a cross section of an electric eel's body. You can see how massively in series these electrocytes are stacked. It was this type of model that was used in the initial design of a battery, interestingly enough. In addition, electric fish had their debut in On the Origin of Species by Darwin. Electric fish actually represented a special case for Darwin. He could recognize the utility of the strong voltage organs that he could see and detect with his hand, but at the time there was no way to detect weak voltages at all. He recognized these fish had very similar organs to these fish that he could detect. Of course they were weakly electric and he wasn't able to physically detect it. He wasn't able to make sense of this. Why would a fish have an organ that looks very similar but no matter what we do we can't perturb this animal enough to give us a shock. So he said, in On the Origin of Species, "The electric organs of fishes offer another case of special difficulty, for it is impossible to conceive by, what steps these wondrous organs have been produced. But this is not surprising, for we do not even know of what use they are." So the electric fish have a pretty interesting history. Why are we even interested in studying the electric eel's electric organs? From my perspective there are a couple of reasons. The first is that this will provide a tremendous amount of insight into the evolution of complex traits. This is a theme that has been thrown around a lot, in particular by one of our main collaborators, James Albert from the University of Louisiana-Lafayette, who works a lot in especiation and biogenetics of electric fish, and also other fishes. In addition to that, studying electric organs may offer society new ways of generating and storing electrical energy. This has actually been described in the literature in a number of places. One of my favorite places is a computational approach at designing artificial cells or biobatteries. This was done by -- in 2008. Why would you want to do this? I mean, you can think of many reasons, but the first one that comes to my mind is that pacemakers are powered with batteries. If you could somehow create a biobattery to replace pacemaker batteries that could be a huge human health benefit. The first step in this is to really characterize molecularly electric organs. This has never been done before. Like I mentioned, this is a large collaborative effort. It really started off as a study of the electric eel. From the electric eel end of things we worked to sequence, assemble and annotate the genome of the electric eel, electrophorus electricus. In addition to that we studied gene expression on various eel tissues. The tissues, we interrogate eight of them total. They were the tree electric organs, skeletal muscle, heart or cardiac muscle, brain, spinal cord, and kidney. There were eight total. Before I get into the nitty gritty of those experiments I just want to go through some technical background. The approach that we used to study gene usage in our different tissue types is a method called RNA sequencing. In particular we were sequencing messenger RNA. If you look at the central dogma of molecular biology, you start with your genome, which is comprised of DNA. In your genome you have genes that ultimately are going to lead to the production of a protein. You have your genes, and to be able to use you genes your cells requires certain machinery to get in there and make a copy of this gene. To do that it's a process called transcription. It takes a gene and makes a copy made out of RNA. This is messenger RNA. When I say gene expression I'm meaning measuring levels of mRNAs for particular genes. I guess this is an important point. When I talk about gene expression I mean the level of mRNAs in the different tissues. So from the mRNA level things are then translated into proteins, and proteins are actually the functional unit in a cell. It is common practice to measure the differing levels of RNAs in tissues to try to say something about the physiology of that cell. For instance, if a cell needs a lot of a particular protein to function-- I already mentioned the voltage-gated sodium channel when I was describing how the electrocytes function. You can imagine that the electrocytes need a lot of that protein. You would expect a lot of that mRNA for that protein to be present. Oppositely, if the cell does not need a protein, you might expect little or no mRNA for that protein to be present. For the electric eel, like I said, we did the genome assembling annotation. We also did RNA sequencing of eight tissues. After we had an idea about how each of the approximately 25,000 genes in our genome were expressed in our eight tissues, we wanted to be able to say something about each tissue type specifically and generally. One question that is important that we wanted to answer was whether there's a unique set of genes utilized in electric organs compared to skeletal muscle. The reason we want to do this is because we know that electric organs are developmentally derived from skeletal muscle. So we want to know what's different between the two cell types. The approach that we used was a computational one. We clustered genes together by similarity of expression. In other words, we used an approach of grouping things that were similarly expressed in electric organ tissue and not really expressed anywhere else, or expressed in heart tissue and not really expressed anywhere else. This is an example of some of the graphs that were output from this analysis. It's not important that you understand what these graphs are showing you, but just to walk you through one of them, this is cluster nine on the bottom here. What this graph is showing you is, in the Y axis is expression value. In the X axis we have our eight different tissues. It starts spinal cord, brain, kidney, heart, skeletal muscle and then Hunter's electric organ, Sachs' electric organ and main electric organ. The electric organs are in yellow in every graph. What you can see here, this red line is median expression value. What you can see is that there's a really low expression value for all tissues except for the three electric organs. What cluster nine represents is 211 genes that are highly expressed in the electric organs and not really expressed in any of the other tissues that we studied. Likewise then, cluster one, you can see, the red line is low for everything except for skeletal muscle and also heart. Cluster six is a cluster that represents genes that are highly expressed in skeletal muscle and the three electric organs. Cluster seven is a cluster of genes highly expressed in skeletal muscle, heart and the three electric organs. So to answer the question that I posed in the previous slide which was whether there was a unique set of genes utilized in the electric organs compared to skeletal muscles. The answer is yes. We have cluster nine. These are genes uniquely expressed in the electric organs. And we also have three other clusters, the first that is unique to muscle only, and cluster six and seven which is a combination of electric organs and muscle. The next question that we wanted to ask is whether there was gene functions that were enriched within these clusters. I'm going to explain that on the next slide. One way of approaching the question as to whether or not there's functional enrichment in genes in these clusters is to use an approach called gene ontology, or GO, for short. What gene ontology analysis is it's a series of controlled vocabularies that you can apply to your genes of interest that helps simplify things greatly. Instead of dealing with-- I told you there was 211 individual genes in cluster nine. By using the GO terms and assigning GO terms to these genes I can reduce that vocabulary significantly. Instead of having to deal with trying to understand 200 things, I'm going to be understanding maybe 50 things. And I can look for enrichment with in those 50 things, because the vocabulary is very controlled. So we assigned GO terms, or these general terms, to each of our genes. These, in turn, described a general function, and we can look for enrichment of these general functions within our clusters, or within our co-expressed genes. Why would I want to do this? I want to know something generally about what is unique about each of these clusters. I want to know generally what is unique about the genes that are only expressed in the electric organ. This is a graph of the output. Again, the graph itself isn't necessarily important for you to understand. But I'm going to walk you through our findings for our understanding what was functionally enriched in our different categories. On the top here, you recall, there was a cluster that was specific to muscle only. When we look at our enriched terms from this category we see things involved with muscle contraction and also cytoskeletal proteins. These are things that we expect to be there, like actin-binding is present, or, let's see what else. There's also calcium transporting. These GO terms are important for the known function of muscle, so it's important that we're seeing what we expect to see from our genes expressed in muscle. Next we had our cluster that was skeletal muscle and electric organs specifically. This represents genes that are still shared between the electric organs and skeletal muscles from which they were developmentally derived. We're seeing GO terms involved with sequences specific DNA bindings. These are things like transcription factors. Things that are regulating the production of mRNAs. We also see our friend the acetylcholine receptor. I mentioned the acetylcholine-gated sodium channel. This is coming up in our functional enrichment analysis. Next we had our cluster that was skeletal muscle, heart and electric organ. We're seeing GO terms involved with metabolism coming up. This wasn't something that we'd really thought about before doing this analysis. We hadn't really thought about the metabolism at all, and that electrocytes and muscle might be sharing a fair bit of their metabolism with each other. So this was a nice finding. In addition to that we're still seeing GO terms indicating, still, shared functions between electric organs and skeletal muscle in the form here of calcium binding. Finally, for our electric organ specific category, it's really dominated by GO terms involved with transmembrane transport, which, like when I talked about how electrocytes function, all of those ion channels and ion pumps, those are all transmembrane transporters. So it makes sense that we're seeing this enriched in our electric organ group only. To recap what I just told you, our electric organ group is enriched for genes involved with genes of transmembrane and ion transport. This is consistent with what we know about the importance of this function in the over all physiology of electrocytes. In addition, muscle and electric organ are enriched for genes of all this cell metabolism and this was something that we hadn't thought about before doing this analysis. It suggests that electrocytes and muscles still share a lot of there metabolic characteristics. Next, muscles and electric organ are also enriched for several transcriptional regulators. It suggests that a lot of the underlying, perhaps, structure of the cell or some sort of homeostatic mRNAs are regulated the same way in electrocytes and muscle. This isn't necessarily surprising given that electric organs are developmentally derived from skeletal muscle. Most importantly, this gives us a general sense about what makes electric organs different than muscle and also the same as muscle, what is still conserved between these two independent cell types. Moving on to our next big analysis that we did. We wanted to know whether there were electric eel genes that were showing an increased rate of evolution in the eel compared to other species. In other words, we wanted to know whether there were eel genes that were evolving at a rate that was significantly different than you would expect when you looked at the rate in other fish. The reason we want to know this is, you can imagine that there might have been genes that evolved specifically for form and function of electric organs in the eel. We wanted to kind of interrogate this a bit. To go back to our central dogma, like I said, it moves from genes in DNA space. These genes are transcribed into mRNAs. Then these mRNAs are translated into proteins. Proteins are actually comprised of amino acids so they're amino acid sequences. There is actually redundancy built into the DNA code. What this means is if there's a mutation within your DNA you can imagine what kind of a disaster that would be if that would effect the underlying protein structure. But the cell has a way to deal with this, and that is just redundancy within the DNA. Sometimes you can make a mutation in the DNA and it won't have any impact on the underlying protein sequence. You can leverage mutation rates both in the form of rates of change that do not effect the underlying protein structure, one. So rates of change that don't effect the protein structure. And two, rates of change that do effect the protein structure. By comparing these rates in the electric eel compared to the rates seen in other fish, you can start teasing apart which genes maybe undergoing accelerated evolution in the eel. Then once we-- I forget how many we pulled out. I think it was something like 500 genes that seemed to be undergoing accelerated evolution in the eel. We look for functional enrichment, again, in the form of gene ontology like I just demonstrated on the previous slides to look for if there's specific functions that might be changing in the cell. Again, it is not important to understand what this graph is showing you, but it's another way to demonstrate GO enrichment or gene ontology enrichment. The graph is structured-- Where's my curser? There it is. The graph is structured such that you go from very non-specific GO terms to more specific GO terms as you travel down this graph. You can see certain nodes here are rectangles in different colors. The ones that are in rectangles are statistically significant GO terms that are enriched in our data. Really, the take home message on this graph is that the graph is dominated by this large group here which is ion channels and ion transporters. What this is saying is that in the eel genome there is a lot of accelerated evolution happening in the functional category of ion channels and transporters. This is a pretty interesting finding for us. In addition, there's some cell signaling, there's also some transcription factors that are coming up. Overall, this analysis made us pretty excited. The next question to ask is whether genes are evolving in our tissue-specific clusters. Recall back from a few slides we had one group that I called cluster nine which was just genes that were expressed in the electric organ, and they were expressing in the electric organ at a high level. The question was whether we had genes in that cluster that were coming up as experiencing accelerated evolution. The answer is, yes, we do. What are they? Well, we have our voltage-gated sodium channel coming out at the top. This is vital to the physiology of electrocytes. It's previously been demonstrated to be undergoing positive selection in other electric fishes, but it's great that it came up in our analysis. In addition, we have a number of transcription factors including transcription factors that are important in muscle development. It might mean that because the transcription factor here is important for muscle development, it might mean that there's mutations here that give it new function in the electric organ or different functions compared to something that might be expressed normally in a muscle cell. But overall this just suggests that there is evidence that there is evolutionary selection in genes that are important for the form and function of electric organs. Because these genes are the ones that are highly expressed in the electric organ. Presumably if something is highly expressed you'd anticipate that it might be important. Now I'm going to switch gears entirely away from mRNAs and start talking about something entirely different, micro RNAs. Just to get back to our central dogma here, micro RNAs are short RNAs, so micro RNAs. They are transcribed but they are never translated. They're purpose is to regulate at this step here. In a very sequence specific manner they bind to mRNAs and cause them either to be degraded or to not be able to be translated. They essentially turn off the affect of a gene at this step. We did micro RNA analysis in the eight tissues of our eel as well. Micro RNAs in the electric eel, or in any electric organ really, have never been interrogated before. So this represents something that's pretty novel in the field. There's a couple questions that we wanted to answer. First is whether there were novel micro RNAs expressed in the electric organ of the electric eel. And the second is whether these novel micro RNAs are playing a role in the form or function of an electric organ. This is one of our candidates that has come up in our analysis. This graph on the left here is showing you, on the Y axis, expression, and then on the X axis is our eight different tissues. This micro RNA is virtually turned off in brain, spine, kidney and heart. It has a really low expression value in muscle, and a really big expression value in the three electric organs. We're hoping that it's important for electric organ form or function. Interestingly, I didn't talk about this potassium channel gene, but there's a potassium gene that's vital to the function of electric organs as well. This micro RNA is actually transcribed in the reverse orientation in the middle of this potassium channel gene. It's kind of interesting. We then set out to computationally look at whether it seemed as though our micro RNA could be targeting some of our mRNAs for degradation in the eel. Remember I said these micro RNAs go and they bind to mRNAs and cause them to be degraded or cause them to not be translated. We employed a computational method to look at whether our micro RNA could be binding to any of our mRNAs in the eel. Preliminary evidence suggests that, indeed yes, our micro RNA may be targeting mRNAs. In particular, mRNAs with roles in muscle development. This is pretty exciting for us. Because electric organs are developmentally derived from skeletal muscle the fact that we have a micro RNA that seems to be targeting muscle specific function would give us a lot of hope, really. I mean, you can imagine this micro RNA is turning off muscle specific things. So perhaps it's playing a vital role in preventing the electric organ from becoming muscle and actually staying electric organ in the pathway. I've just shared a lot of information with you. Just to recap, I have more for you, but just to recap where we've been, I showed you that there's a functional enrichment of genes highly expressed in the electric organ that are specific for transmembrane transport for instance. I've showed you that there are genes that are showing evidence of accelerated evolution in the electric eel, and that they are functionally enriched. As well for these ion transporters and transmembrane transport. I've showed you that we have a novel electrocyte specific micro RNA that may be targeting genes that are known to function in muscle physiology and development. As I've really tried to rein in here, electric organs are developmentally derived from skeletal muscles, so one huge question that we have to ask is, how do the electric organs compare to skeletal muscle with respect to their expression of transcription factors involved with muscle development? That seems like a natural question to ask. So back to our central dogma. What are transcription factors? Transcription factors act at the level of transcription. They bind to specific regions of the DNA, and they either enhance the production of mRNA or they turn off the production of mRNA. Transcription factors regulate the production of mRNAs and they're very specific about which mRNAs they're working for or against. What I'm showing you here is really the start of the bread and butter of our paper. This is a heat map. This is comparing the expression of several muscle-specific transcription factors in electric organ compared to muscle. I'm sorry our paper isn't published yet so I haven't put the gene names up here. But they're not important for this discussion right now. What you can see is in this heat map there's a cluster here that is really red. These are transcription factors that are really high in the electric organ compared to skeletal muscle. It turns out that these transcription factors function very, very early on in muscle development. In fact, if you have a system that you can perturb the genetics, if you experimentally up-regulate some of these transcription factors you prevent muscle development from happening. Now you can imagine maybe the presence of these transcription factors prevents the muscle pathway from taking place and keeps these cells on an electrocyte-specific pathway, perhaps. Conversely, these transcription factors in this cluster here are involved with late muscle development. They are off in electric organs compared to skeletal muscle. This is what we would expect, right, because we have-- our electric organs are not skeletal muscle, so we would expect that the transcription factor is important for late muscle development might not be on in our electrocytes compared to skeletal muscle. That is mostly true. Interestingly, there are a couple here in the electric eel that are actually highly expressed in an electric organ. The one on the bottom here is the one that I showed you, it is experiencing accelerated evolution in the eel from our analysis. That's pretty exciting. Similarly, we have a cluster on the bottom here of transcription factors that are involved with body patterning. We're not sure what this means yet. We're not sure if this means that there's a new function here for these genes in, maybe the development, or the function, of the electric organs. Or we're not sure if it's just important for some reason that their expression is retained in adult differentiated tissue. But regardless, this is an exciting finding for us. Okay. I've just showed you that the electric organs in the eel highly express transcription factors that are involved with early muscle development. They lowly express transcription factors involved with late muscle development. Really thus far we've only looked at the eel. This might be an electric eel-specific phenomenon and it might not have anything to do with any of the other Gymnotiformes electric fishes. Really, that's the next logical progression in the study, to ask whether these transcription factors are expressed similarly in other Gymnotiformes. That's what we did. We did RNA sequencing on two other Gymnotiformes. This is sternopygus macrurus and eigenmannia virescens. We did RNA sequencing of the skeletal muscle and electric organ in these fishes. Remember that these two fishes only have one electric organ. Where'd I go? These two fishes only have one electric organ. We wanted to know whether these two fishes express these transcription factors in a similar way. We found that, indeed, they actually do express these transcription factors similarly. This result suggests that the expression of these transcription factors is important for Gymnotiformes as a whole and not just the electric eel. These transcription factors are important in the Gymnotiformes that we've tested here in their development of electric organ. Great. So they are evolved in Gymnotiformes and it's not just as electric eel phenomenon. But I've told you already that electric organs have independently evolved multiple times. Could it be that these transcription factors are important in different lineages even if they have independently evolved electric organs? Let me remind you of the tree. We have, with these red stars next to them, we have our Gymnotiformes down here. We also have our African weakly electric species up here that I'm referring to as momyrids. This is the lineage that we interrogated. We looked at one fish in this lineage here, --. Again, we did RNA sequencing of skeletal muscle and electric organ, and asked whether they expressed transcription factors similarly. The answer is, yeah, they do. These results suggest that the expression of these transcription factors is important for electric organs in independent lineages. Then we have two independent lineages that are expressing things very similarly in their electric organs compared to skeletal muscle. It suggests that possibly there's a common evolutionary tool kit in use here in the independent evolution of these electric organs. This is really exciting for us, obviously. You can imagine how excited we are. Just to summarize what I've talked about today. The electrocytes that we've studied here express a unique repertoire of genes and it is molecularly distinct from muscle. Expressing a unique group of transmembrane transporters and genes involved in receptor signaling. This is a result of our gene ontology analysis. That's where I'm coming up with this point here. Our data suggests in addition that transmembrane transporters, as well as transcription factors may be undergoing accelerated evolution. In addition, electric organs are expressing novel micro RNAs. I showed you one of them which seems that it could possibly be targeting genes that are important for muscle development, which is exciting. And finally, I've demonstrated that we have two independent lineages of electric fish that are sharing expression patterns of key transcription factors in their electric organs. This suggests that evolution utilized a common genetic tool kit when developing electric organs from muscle. So my future direction, because I'm a fourth-year graduate student right now. I have a couple of years left, and in that amount of time, right now, we're actually actively looking in other independent lineages. Right now in the pipeline we have more RNA sequencing data coming back from a third lineage, an electric catfish is coming. We're really excited to see that. In addition to considering other independent lineages, I'm also interested in trying to investigate differences in the three electric organs in the eel themselves. You noticed, I really didn't talk at length about what's different about the three electric organs and that's because we don't really have any biological replicates for this data. And we have no plans to get additional biological replicates to do RNA sequencing on. Instead I'm going to be looking for protein expression and modifications of those proteins as a means to try to explain some of the physiological differences that we seen in the three electric organs themselves. In addition to that, I'm going to leverage what I know about the genome of the electric eel to try to target and study specific genomic regions in other Gymnotiformes, and try to understand speciation and things like that. As far as long term directions go, this is really just the tip of the iceberg. Possibilities are endless as far as where this can go. Can we create an electrocyte in an organism that doesn't have one? That's kind of-- Perhaps we can if we know what transcription factors we need or what micro RNAs we need to express in muscle. Perhaps biobatteries are in our future. I mean, it's going to be several years off, but you know, but perhaps. In addition to these more out-there ideas, this research is really going to help scientists understand the evolution of complex traits. Because we have multiple independent events giving rise to something seemingly very complicated, the independent evolution of electric organs from skeletal muscle. In studying this we're going to be able to glean some insight about the evolution of complex traits and also vertebrate evolution in general. With that, this is our entire Eelista group, as we call ourselves. There's about 15 individuals here. You can see that they're spread all over the US. These are all the people I've been working with on this project for four years. In addition, this is the Sussman Lab. In particular, I work with a guy named Jeremy -- who is a brilliant -- and kind of taught me the ropes on how to do a lot of these types of analyses. And finally I'd like to thank my funding. And thank you all for coming today and letting me talk about my exciting work. Thanks.

applause