 today to introduce to you Kim, Kimberly Anne Haupt, better known as Kim. And some of you at least know that Kim grew up in Michigan, a smallish town I think, in the outskirts of Detroit, or at least on the outskirts of Detroit, maybe, I don't know, probably doesn't matter. And she went from there to Ann Arbor to get her Bachelor of Science at the University of Michigan, and she got there a major in biochemistry and a minor in German. And it was during her undergraduate years that she started doing research. In fact, she started as a freshman in a lab doing research, and I think it was rather prescient that she started working in a lab that was working on RNA. And she worked in other labs but I think RNA got into her blood. And when she joins the IPIV program soon after finishing her bachelor's degree, and we were very lucky in my lab that she chose to come and do her thesis research with us. So her project, as you'll hear, has continued the theme of the RNA world, but been very different in that when she was, as an undergraduate, she was doing protein purification and in vitro assays, and now she's moved to the, trying to understand how molecules work inside an animal. So in an in vivo context. And this is really, I think, at the frontier of biochemistry today to go instead of being in a test tube to actually assay the activity of molecules inside animals. And so that's what she's been doing. In my humble opinion, I think her thesis is really magnificent. She has done three very meaty projects over the course of her thesis research, and that is really a lot. So in chemistry, three projects have, that's no big deal, you can do that in the weekend. But in biology, it takes a lot longer trying to figure out how molecules work inside the animal. And she really has done an amazing job of coming, doing three projects that have led her to go from a point where we really knew virtually nothing about the molecular basis of how things are working to having really good ideas of how things are working. And I'm not going to tell you anything what she does. That's her job. But I just want to tell you, pay attention because it's really cool. So she managed to do so much by, you know, all of the great things that are Kim. She's really smart. She really is incredibly motivated. And she also has a work ethic that is a killer work ethic. And those all have been incredibly important in managing to do these three meaty projects. In addition to that, she has been working as a member of a team and more recently as the leader of a team. And these projects have been, would not have been possible without that team effort, but they would also not have been possible without her leadership skills, which are remarkable. So in parallel to this research, she's been a major contributor to the IPib community. She won the Sigrid Liermo award, which goes to the graduate student who has done the most that year, is recognized by her fellow students and the faculty for being a major contributor to the IPib community. So she's really done that as well. So Kim has been a model student. I'm going to start crying. And a model member of the IPib community as well. She's been a leader at many levels throughout her graduate career. And I think she's been growing in that role as well. She's a good, become a good friend. And it's really bittersweet to introduce her for this final chapter in her thesis research. So with that, Kim, help. All right. Can everybody hear me okay? Okay. Whoops. So thank you all so much for coming today. I am so blessed to see so many faces in the audience. So much support for me in this room. And I really, really appreciate all of you that are here. So I'm here today to tell you about my thesis research. I've studied stem cells in the small nematodes, the elegans, which you saw crawling around during my introduction. And I'm going to begin with a short primer on stem cells, what they are, and why they're important. So most cell types that we think of in the body are specialized for a specific role. So for example, red blood cells serve to deliver oxygen to the tissues in the body. The gametes role is to take our genetic information into the next generation. The role of stem cells, on the other hand, is to remain unspecialized. Stem cells are flexible and plastic, and they're responsible for generating those more specialized cell types that we think of. And they do this through a process called differentiation. Stem cells are important in a number of biological processes, including growth and development of organisms, the maintenance of tissue over time, and repair and regeneration of tissue upon injury. So in addition to differentiation, stem cells can also undergo a process called self-renewal. And self-renewal is the mechanism by which stem cells make more of themselves, more stem cells. And it's this self-renewal process that has been the focus of my own work. So key to understanding my research is the idea that stem cells must and stem cells and self-renewal must be regulated. In a wild type animal or in a wild type tissue, the balance between self-renewal and differentiation is even such that neither population of cells can overtake the tissue. However, if there's too little self-renewal or too much self-renewal, that balance is upset. And as an illustration of this, I'd like to show you some images of a mouse small intestine. So in the first image I popped up there, these are the villi in the intestine, the little fingers that grow into the lumen and absorb nutrients. You can see that they're uniform in size and have really regular spacing. When there's not enough self-renewal or if there's too much, the tissue morphology changes a lot and it has implications for the function of of the tissue as well. So it's clear that understanding the controls of this process of self-renewal has implications for how tissues develop and grow. And thinking about that in the context of regenerative medicine is certainly a frontier in terms of clinical research today. And then the question that I've sought to answer is how is the process of self-renewal controlled in a poster child of stem cell research, this small nematode worm, C. elegans. It's about one millimeter long, so this is pretty magnified for you guys to see. A couple of aspects about C. elegans that have been really useful for me in my work are the fact that it's transparent. So it's great for microscopy and cell biology experiments. It has a simple body plan and the it's very amenable to genetic manipulation. So we have been able to do, we've been able to manipulate the molecular regulators of stem cells in vivo on a nucleotide by nucleotide basis, which has been extremely powerful. And finally it gives us an example an opportunity to study stem cells in vivo, so in the context of an organism. So the stem cells are found in the germline, which I've outlined here in white. There are actually two arms and you're just looking at one outlined here. They're symmetrical to each other. They're U-shaped tubes with one end closed. And at that end is where the stem cells are located. The germline stem cells are GSCs, as I'll refer to them throughout this talk. Move away from this closed end of the tube towards the opposite end. And it's that movement that launches them into differentiation and eventually they become gametes because they're germline stem cells. We can get a closer look at the germline tissue by extruding it from the gonad, I'm sorry, from the animal. We can cut off the head or the tail of the animal and pop the germline out. And what it looks like when we do that is this image. So the polarity of the gonad here is from the distal end to the proximal end. At the distal end is where the germline stem cells are located. And then as we move towards the proximal end, the cells enter into differentiation and eventually differentiate into gametes. So something that's critical to know here is that the germline stem cells and the cells close by to them are mitotic. So they're in the mitotic cell cycle and it's the entry into meiosis that is the start for differentiation in this tissue. So with that I'd like to give you some context for what we knew about how self-renewal was controlled in this animal, in this tissue, when I came to the project. So the first mechanism of germline stem cell control is signaling from the germline stem cell niche. So in C. elegans, I'm going to turn the lights down really quickly, sorry. So the stem cell niche is a microenvironment. It's a concept in stem cell biology. It's a microenvironment that houses the stem cells and tells them to stay stem cells and not to differentiate yet. In C. elegans, this is a single cell and I have blown up, it's located at the very tip of the germline where the asterisk is on the picture. And I'm showing below a scanning electron microscopy image that shows the niche cell. It's like a little octopus that sits on the end of the germline signaling to the stem cells that they should stay stem cells and not differentiate yet. And we know that in this tissue it's notch signaling from the niche that's essential for the process of stem cell maintenance and promotion of self-renewal. And downstream of notch are two genes activated transcriptionally by notch signaling called LST1 and SIGL1. And when I joined the Kimball Lab we knew that LST1 and SIGL1 were really important and that they played a big role genetically but we didn't have any idea what these genes and their protein products were doing in the germ cells. The second mechanism of germline stem cell control is a robust RNA regulatory network comprised of two genes called FBF1 and FBF2. They're very similar and collectively we call them FBF. FBF promotes self-renewal and inhibit differentiation and we know some stuff about how they work. They are RNA binding proteins from the PUF family. They are crescent shaped and they bind to the 3-prime UTR of transcripts in the cytoplasm. They are post-transcriptional regulators of RNA stability. It's known that PUF proteins and FBF work with partners to mediate RNA regulation. However, we didn't know what those partners were in the context of self-renewal when I came to the lab. And in fact this drawing that I'm showing you of FBF bound to a single RNA is actually a really narrow view. What's actually going on in the tissue is that FBF is a network hub. It binds more than a thousand RNAs and regulates them in germ cells. May regulate them in germ cells. So I've told you now what the regulators are that we knew about in stem cells. Next I'll describe the genetic evidence for the importance of these regulators. All of which disrupt the self-renewal to differentiation balance. So C. elegans when they're born as larvae have two germ cells which proliferate to about 2,000 germ cells by the time the animals reach adulthood. So I've already introduced you to what a wild-type germline looks like. I'm showing you again here. The germline stem cells are located at the distal end next to the niche and there's an even balance between self-renewal and differentiation in this tissue. When we disrupt notch signaling as one of the regulators of self-renewal we get a really drastic phenotype. So you can see below the germline is really small and contains only a few cells that have differentiated into sperm. So what happens in these animals is the two germ cells that the animal is born with divide only once or twice self-renewal fails and the animal the germ cells in the animal differentiate to sperm very early in larval development. LST1 and SIGA1 similarly. These two regulators are redundant so a mutant of either one alone has almost no germline phenotype. However, depletion of both of them simultaneously we get the same phenotype as a notch mutant with that four to eight germ cells precociously differentiated into sperm and no germline stem cells present in adults. And finally FBF1 and FBF2 these regulators are also redundant so a single mutant of FBF1 or FBF2 gives essentially a wild-type germline with germline stem cells but depletion of both simultaneously in that double mutant we get almost no germline stem cells. However you'll notice that this germline is quite a bit bigger than the other two above it and it contains about a hundred germ cells in adults. This is because FBF mutants proliferate through larval development as normal and then when they reach the larvae to adult molt then the germ cells differentiate so self-renewal can occur in larval development but fails in adulthood. So if we look at this data another way and we plot on the y-axis the total number of germ cells produced in these mutants versus wild type and then on the x-axis developmental time you can see that self-renewal is not occurring in LST1, SIGA1 or notch mutants but does occur essentially indistinguishably from wild type until later development in FBF mutants. And so this phenotype suggests the difference the discrepancy between these phenotypes suggests that there's another regulator present in the germline that we've not yet discovered that functions during the larval development in FBF mutants and I sought to figure out what it is. So in my talk today I'm going to tell you three vignettes about these self-renewal regulators that culminate in a molecular model for their relationship and the mechanism that drives self-renewal in these stem cells and it suggests a complete mechanism by which notch can promote the stem cell fate. So my focus through this talk and in my thesis research has been LST1, the gene, another graduate student Heiji Shin did parallel experiments to everything I'm going to tell you today with SIGA1 and the results with respect to my talk today are essentially identical so that'll play into the model that I present at the end but is important for me to tell you up front. I'm just going to show you data from LST1 alone. So I begin with my characterization of LST1 as a pivotal self-renewal regulator. LST1 encodes a protein. It encodes actually two isoforms of a protein, a long form and a short form. We did some experiments that showed that the long form is the one that's important for self-renewal and so that is my focus through this talk. The long form LST1 protein encodes predicted nanos like zinc finger at its c-terminus but otherwise does not have any predicted domains aside from some intrinsically disordered regions of hydrophilic and charged residues. One of the first things that we wondered upon discovering that LST1 was important for stem cells was where is the protein expressed? So to interrogate that question we used CRISPR Cas9 genome editing in order to epitope tag the LST1 locus. This entails a gene specific guide RNA forming complex with the Cas9 endonuclease to cause a double stranded break in the LST1 locus and then we introduce a repair template that encodes homologous sequence and an additional 3xv5 epitope tag. The result is a edited locus an edited LST1 gene that contains a 3xv5 at its 3 prime end and the result of that is that in the animals with the edit we get LST1 protein with an epitope tag on its c-terminus and we're able then with this reagent to visualize the LST1 protein in the tissue when we do that we find that LST1 is strikingly restricted to the germ line stem cell region of the gonad. You can see that it's enriched at the tip of the germline and if we look more closely I hope you can appreciate that this protein is cytoplasmically expressed in granules with these beautiful perinuclear punkta decorating the nucleus and that it's restricted to about 35 to 50 cells in the very tip of the germ line. So we wondered whether or not this restriction was important for germline stem cells and LST1's function there so we generated a single copy transgene to test this question that would express LST1 ubiquitously throughout the germline. You can see the results of that expression in the images on the screen and the phenotype when we assayed the LST1 ubiquitously expressing transgene worms was quite striking so here you're looking at the the DNA in the nuclei of the germ cells and in the wild type control there's productive differentiation into gametes and in the LST1 ubiquitous strain we get production of no gametes and in fact the germline appears to be full of mitotically dividing cells suggesting that LST1 can drive a tumor full of mitotic cells. So we assayed this animal more further to characterize the identity of the cells in the tumor and what we found is that indeed the cells throughout the LST1 tumor are mitotically dividing so if you look the white cells are expressing a mitotic marker and in wild type animals that is restricted to the distal end of the germline where the stem cells are found but in the LST1 ubiquitous line there's white cells throughout the germline tissue. Furthermore we looked at a differentiation marker in green. The differentiation markers normally very low at the distal end of the germline and increases as we move approximately and the cells begin to enter into the differentiation program and start their miotic entry and in the tumor in contrast we see low differentiation marker throughout the tissue suggesting that these cells are not differentiating and they are indeed continuing to mitotically cycle. So what this tells us is that LST1 is a really potent self-renewal regulator and that expression of this protein alone is sufficient to drive the mitotic cell cycle in germ cells and this was really exciting. This isn't a phenotype that you get on accident so we knew that this is a really special regulator and the next question that we had was how is it working? How does it do this? How does it make this tumor? So I'll tell you now that LST1 works as a partner with FBF but next in this next section of my talk I'll share the data that led us to that conclusion so interrogating the relationship between LST1 and the downstream RNA regulatory network. So one of the first experiments that we did to try and ask about the function of LST1 was to ask whether or not the tumor requires FBF for its proliferation. So we performed genetic experiments taking our tumor and asking whether it was working through FBF or through some other mechanism and we tested this question by genetically eliminating FBF1 and FBF2 from the background of the tumor and the phenotype that we saw was that no tumor forms and therefore LST1 must require FBF for its self-renewal function so even if FBF is ubiquitously expressed in the cells or even if LST1 is ubiquitously expressed in the cells it requires FBF1 and 2 to drive the mitotic cell fate. So with that we knew that LST1 was working through FBF so I've added an arrow to the pathway and there's two obvious ways we think that this relationship could exist first LST1 could be regulating the expression of FBF but we tested that hypothesis and it wasn't the case so we turned to the function of FBF and asked whether or not LST1 might have a role in modulating the function of these RNA binding proteins so recall FBF binds to RNAs a canonical FBF target in the germline is the gene gold 1 it is repressed in the distal end of the germline in wild type animals and my pink bracket indicates this repression this is the signature of FBF regulation when we look in an FBF mutant the repression is relieved and gold 1 is expressed so we asked whether or not LST1 might also be regulating gold 1 or might play a role in the regulation of an FBF target and we did this by looking at gold 1 RNA and gold 1 protein the wild type control on the top you can see is not really different from an LST1 mutant disappointingly however LST1 and SIGL1 function redundantly and when we looked in an LST1 SIGL1 double mutant in the bottom row here you can see that gold 1 RNA and gold 1 protein are both significantly upregulated compared to the control so this suggests that yes LST1 and SIGL1 do regulate gold 1 and this can occur even when or this so there's a failure to repress gold 1 even when FBF are present in that tissue if LST1 and SIGL1 are gone in that last row there so we thought one way that LST1 might be having this effect is by partnering with FBF to modulate its activity so the next question that we asked was whether or not LST1 could interact with FBF so some elegant in vitro studies of two other FBF partners showed that the binding interface for partner FBF interaction was comprised of the residues the amino acid residues KT X which stands for anything L and so that inspired us to look for interaction sites that might contain this motif in the LST1 sequence and indeed we found two KXXL motifs that were quite similar to that known motif and we decided to test whether or not these sites might facilitate an interaction with FBF we used an assay called yeast to hybrid and without going into too much detail I'll just briefly describe how it works we express in yeast fusion proteins of FBF and LST1 with activators of a reporter gene when FBF and LST1 can interact the reporter is turned on and when they fail to interact the reporter does not turn on giving us a negative result so we did this assay with a wild type LST1 construct and we found that FBF2 specifically in this assay and LST1 could interact when we mutated either the A site we called the first site or the B site to alanine's the interaction with FBF was not disrupted however when we mutated both sites there was a failure for LST1 and LST1 and FBF2 interact by this assay so this was exciting and seeded LST1 as a potential partner for FBF in self-renewal and GSCs and so the next thing we did was tested whether or not this interaction was relevant in vivo so we had done our previous experiments in yeast and now we wanted to move the idea into the worm so i'll remind you i will not remind you of anything i didn't tell you this yet we built the mutant the AB mutant and it didn't have a phenotype on its own but when we tested it in a single one mutant background we found that the animal made only four to eight germ cells per per animal which is indistinguishable from an LST1 total deletion mutant so these couple of residues are really important for LST1 function in the animal so from this data we conclude that LST1 is a partner of FBF and its interaction with FBF is required for self-renewal so finally having established a relationship between LST1 and FBF i'll share how we identified gene X to complete the molecular or the self-renewal hub so we knew that some gene X might exist i want to remind you of the data for that so the FBF12 phenotype is quite different from the notch or LST1 single one phenotypes we normally grow the worms at 20 degrees and this phenotype typical difference is obvious at that temperature it's even more obvious at 25 degrees which is this is Celsius 25 degrees where actually some germ cells in the tip of the FBF12 mutant gonad fail to begin differentiation and continue to mitotically divide as the animal is an adult so there must be some other molecule in these animals in FBF12 mutants that's able to promote self-renewal in the tissue so we had a hypothesis that it might be another puff protein so C elegans has 10 puff proteins in its genome and two that are closely related to FBF1 and FBF2 are puff 3 and puff 11 so some really these two genes are 90 identical and really beautiful work from Tom evans group showed that puff 3 puff 11 mutants are have oocyte defects but don't have anything wrong with their germline stem cells there's no phenotype there and so we decided to test whether puff 311 might have a role in germline stem cells in the absence of FBF1 FBF2 and to do this we performed RNAi against puff 3 puff 11 in an FBF12 mutant background we did the experiment at 25 degrees for the most striking result and what you can see is that very few germ cells are produced and we get only 408 in an adult animal upon knockdown of all four of these puffs we overlay that onto the graph that i showed you earlier FBF12 puff 311 phenotype is enhanced to nearly the same defect as notch and LST1 single one so we can conclude that puff 311 have a role in promoting self-renewal during larval development and we've also done some experiments to test whether or not they're important in adults and indeed they also are responsible for the proliferation seen in adults at 25 degrees in FBF mutants so in addition to their redundant genetic role for self-renewal FBFs and puffs have molecular similarities as well so both of them are able to interact by LST1 with LST1 by yeast 2 hybrid and they also by some beautiful work from marv wickens lab have identical RNA binding elements suggesting that they might bind similar targets in the animal so these molecular similarities along with the similarities between LST1 and SIGL1 led us to the following model of an RNA regulatory hub for self-renewal consisting of four puff proteins that directly bind RNA and also interact with puff partners LST1 SIGL1 and its formation of this complex and its respective regulation of RNAs that promotes the stem cell fate in GSCs so this model defines RNA regulation as the central mechanism for germline stem cell self-renewal in this system and together these six factors completely account for all of the germline stem cell self-renewal that we see so you might be wondering to yourself why do you need so many factors why is there so much redundancy and we think what's going on is that redundancy in this hub lends robustness so first of all it's important to make gametes for producing the next generation of worms and so it makes sense that evolution would want to safeguard this process another idea that we have is that during times when much proliferation is needed such as during larval development or during under stressful conditions such as high temperature the self-renewal process needs more puff power in order to effectively promote self-renewal and more factors doing the job guarantee species survival under those more maybe more stressful conditions so our description of this hub as central to GSC self-renewal leaves a lot of questions to be answered about how it is actually working so it's a start but a few things that we're really curious about are which RNAs are regulated by the hub what are the functional effectors that are working alongside puff and its partners so in vitro work and work in yeast has showed some putative effectors but we don't know yet which ones are actually important in germline stem cells and finally are all of these puffs and partners created equal or do they have specialized roles there's evidence to suggest that fbf1 and fbf2 actually have quite a bit of specialization and so we're curious whether or not the same will hold true for puff 3 and puff 11 as well as ls2 and single one this is just the tip of the puff iceberg understanding puffs their partners and the RNA regulatory networks they control has implications across biology puffs are really broadly important so first off puffs are really well conserved they're found from yeast all the way to humans and broadly they have roles in stem cells including germline stem cells neural stem cells hematopoietic stem cells which produce blood cells and immune cells and in also in planaria neoblast so planaria are a model of regenerative biology and the neoblasts are the cells that are responsible for that regeneration so the role of puffs there is really interesting and they also have a lot of functions beyond stem cells as well puffs are known to be important for neuron function across across organisms they've been implicated in some cancers the immune system response they're also important in development and for gametogenesis in the best model organism the worm so we're really just learning we're just starting to learn what puffs are doing in these systems and C. elegans germline stem cells can serve as a model for the study of these proteins potentially the hub that we've discovered might be a paradigm for understanding puff function in biology more broadly so what i've told you today is that lst1 and sigoan are pivotal regulators of stem cell cell renewal in the C. elegans germline they work with puff proteins to comprise a hub of RNA regulators that completely accounts for cell renewal in the C. elegans germline and if you walk away today remembering only one thing i hope it's this the regulation of cell renewal is really important and if it's misregulated the tissue and functional consequences are drastic so with that i have a lot of people to thank but i first would like to start with the people that contributed to the work i've presented today so first i have to say thank you to judith my advisor for her support compassion advice and contagious passion for science thank you for your guidance but also thank you for giving me the space to find my own way thank you to my thesis committee for their role in my growth as a scientist and to laura robin and kate for everything they do for ipib and the biochemistry department with regards to the work that i presented here i'd also like to thank my colleagues in the kimball lab first and foremost heiji shen who's pictured on the slide we collaborated for years on the lst1 sigoan project and her hard work and thoughtful ideas were instrumental in in the success that we saw together the others that i've listed here amy alan and kim law all contributed data that i presented today and many other people contributed reagents and time and energy to these projects it has been an honor and a privilege to work alongside such brilliant and generous people in the kimball and wickens labs these past years thank you all so much for the mentorship the critique the advice and the passion that you've shared with me i'm deeply grateful to have been part of this group and lucky to count many of us friends i've also had the privilege to mentor smart and curious undergrads post-bacs and grad students and to each of you specifically thank you you taught me as much about people as i taught you about pipetting also to the current kimball lab members i'm sorry about the monopolization of the break room computer for the last six months thank you for your understanding i also need to say thank you to my family many of whom traveled to be here today i'm so grateful for your support in all its forms phone calls texts visits to madison vacations meals help with my plants and seemingly bottomless advice and patience finally thank you to my friends roommates classmates hiking buddies and teammates for your friendship truly my community in madison has brought me so much love and joy thank you to everyone who reached out in the last days weeks and months to cheer me on i really appreciate the support thank you for your attention i'm happy to take any questions you might have hi yes so we know that it's dispensable for self-renewal but it does affect actually the abundance of lst1 mrna so and the protein as well so we think it has some regulatory role perhaps an auto regulatory role but it doesn't seem to be important for binding fbf whether it enhances the binding that i can't say we haven't done those kind of in vitro experiments but it's not import it's not essential in the worm certainly good question hi yeah yeah great question yeah so um they are they live i okay so to answer your first question do we see differentiation in other cell types so we use specifically a promoter that would only give expression in the germline mostly we think and we didn't see any changes when we used another promoter where we tried to use a more ubiquitous promoter so we think that perhaps lst1 expression alone in those tissues is not enough but perhaps its partner puff is not there in sufficient quantity to give us that phenotype we haven't tested those hypotheses yet do the animals live they do they live until adulthood sometimes the tumors proliferate so much that they explode but they're otherwise they're okay yeah they don't produce they don't produce any progeny as you might expect because there's no gametes but yeah yes so the question is do the is a pairing between a partner and a puff required or is there possible um multimerization we don't know the answer to that question um we definitely think that the fact that there are two binding elements in lst1 is an interesting potentially uh seeds an idea that potentially it could maybe bind two puffs at once but we we don't know the answers to those questions yet we're really curious to learn definitely and the intrinsic disorder domain as well might um whether or not dosing amounts of intrinsic disorder into the complex could change its dynamics yeah yeah perfect so the question is lst1 exist in humans the answer is not that we know of it's not conserved outside not obviously conserved outside of nematode worms however it's very possible that there are partners of puffs that don't look similar to lst1 but serve the same function in other organisms any any burning last questions so you'll have to wait a couple hours yeah they're doing okay yes thank you for asking