Yale Genetics Seminar 11/1/22|Dr. Nicole King, PhD| A History of Hypotheses on the Origin of Animals

very exciting Nicole about your talk thank you it's very nice to see you it's everyone Jen is everyone in from the waiting room should I start yeah you're all set yeah okay so I'll go ahead and introduce Nicole and get started um so I'm very excited to have uh Nicole King here today um for our seminar which was postponed from September so I'm even more excited that we were able to reschedule it um so Nicole is an hhmi investigator and the professor of molecular and cell biology at UC Berkeley is um before I get in you know she'll tell you about some of the current things that she's working on um she did her PhD actually in gene regulation and drosophila I believe um and that was my undergrad your undergrad okay I understand um and then did a postdoc with Rich loser where you had at least one publication HD I'm on SO okay anyway he's one publication on um on bacillus subtlest that's right um then a postdoc with Sean Carroll okay there um got into sort of founding the field of of um kind of flagellate biology and using that to dissect the origins of multicellularity in animals um and so this has been just an amazing series of studies that you know something to kind of admire and live up to um that uh has been both technically and conceptually Innovative in setting up montesigo brevocolas as a model organism and and sort of establishing what this can tell us about how animals became multicellular originally and one of the sort of amazing things about you know Nicole's body of work is this like ability to balance um sort of small biology and big biology and think both about the molecular and genomic insights as well as the sort of evolutionary and organismal insights that you can get out of the system which I find really inspiring and has just been um an amazing set of Publications to read um and has given us a a glimpse into how animals became animals which is also just really cool she's of course accumulated many honors including few scholar she was a MacArthur fellow she's a member of the French Academy of Sciences as well as the US National Academy of Sciences and in addition to all of the the scientific innovation well this is scientific as well but I think has been an example of how to set up sort of a new field and make it ensure that it's open and and sort of supportive scientific culture which is also very inspiring um so um with that I'll I'll turn it over to her and looking forward to a really exciting lecture thank you so much it's uh it's a real pleasure to be talking to all of you I wish I was there in person um maybe I'll just riff off of a couple of things that Luma just said one is one is actually when I started uh studying choanoflagellates Peter Holland and I organized a small meeting I think there were only 11 of us and we we all agreed that we would be better you know the field would grow and Thrive if we all worked together and supported each other and collaborated and so right from the bat that was part of the culture and we've continued to meet every two years and the community has only grown and it's just a wonderful friendly supportive group um so that's really been great um the the second thing I would say is really for the trainees um and that is that this talk today is going to be about our single-minded pursuit of one question and what I'm going to show you is that that single mind we have continued to fail in our single-minded pursuit of that question and yet along the way I think I've made some really exciting discoveries so um and that's sort of something I learned from Rich losick uh and that is that if you keep your eyes open and your mind open and don't ignore the unexpected uh uh results that you can be taken in um really exciting New Directions and so please don't judge me at the end of this talk when you see that we actually have it uh accomplished our original goal but um instead hopefully Glory with us in the um the other findings that we've made along the way so um so today actually I've changed the title and I'm going to be focusing on um or uh the phenomenon of mating in choanoflagellates and this is interesting because one of the first uh incidents of cell differentiation that's thought to have evolved in the animal cell lineage or animal lineage was the differentiation of somatic cells from gametes and so by studying the phenomenon of mating and unicellular organisms we hope to gain some insight into how that process originally evolved on the animal stem lineage okay so let me just back out and give you some big picture um I think many of us are fascinated by the incredible diversity of animals and animal forms um and animals really are you know I think of them as variations on this theme of multicellularity and it's it's incredible you know we have animals swimming in the oceans and flying and uh you know crawling across the Earth and they make a living in very different ways you know um some of them are filter feeders some of them are predators um it the diversity is remarkable and it really has been a major focus of the field of evolution and development how do we get so much diversity but there's something interesting about animals and that is that they all share a single common ancestor and in a way we see that reflected in the way that animals develop they all Start Life as a single cell the zygote and through cereal rounds and cell division with the cells remaining clonal largely having the same genomes um they arrive at these very different forms and so really animal life every generation is a transition to multicellularity now that's at the the level of the individual but in my lab we're interested in the the big picture how did animals evolve multicellularity in the first place can we reconstruct the biology of the last common ancestor of animals and then back up in time and gain some insight into how multicellularity itself first of all a Millennials leading to animals and how mechanisms for regulating cell differentiation and the coordination of function within the multicellular animal how did that evolve and I won't go into details but let me just say that this transition to multicellularity has happened over and over again within eukaryotes so for the purposes of this talk we're focusing specifically on the transition to multicellularity that led to animals now um had it been possible I would have loved to have done this work by referring to the fossil record because there you have an absolute you're actually looking back at time but unfortunately the fossil record cannot or does not preserve the small soft-bodied organisms that were the progenitors of animals and even if it did we wouldn't necessarily know it and even if we knew it it wouldn't give us insight into cellular and molecular mechanism so instead what we've done is turn to living organisms as a window into the past and in particular we're focusing on a group of organisms called the quantiflagellates and the reason is that these are the closest living relatives of animals and recently by sequencing the genomes and transcriptomes of many different choanoflagellates we've learned that they encompassed a remarkably diverse group whose genetic diversity actually Rivals that of animals and so I've you know intentionally drawn these boxes to be approximately the same size and so just as we can compare the biology of animals um and gain insight into their last comedy ancestor we can compare the biology of quantiflagellates to each other and learn something about their last common ancestor and then pulling these comparisons back in time we can learn something about the organism from which they evolved and think about what might have changed along the stem lineage that separates animals from all other life pardon me so if you haven't looked at a clinoflagellate before let me just give you a quick introduction um the cell body of a corner flagellate is about five microns so it's about the size of the yeast cell and uh every corner flagellate has a actin filled collar of microvilli that extends from the apical pole and a long flagellum and you can better see this in a live image this is a high speed camera or a high speed Imaging that we've slowed down and um what happens is that flagellum beats back and forth inside of the collar and that generates currents either propelling the choanoflagellate through the water column to swim or to pull material out of the water column up against the collar which allows it to capture it and so they actually are um uh really voracious bacterial Wars they're an important part of the um the food web in the oceans for essentially capturing bacterial prey and making that carbon available to larger sized organisms so I mentioned that Quantum flagellates are incredibly diverse um uh but there were very few people studying them so we've really focused on developing a small number of model choanor flagellates that are experimentally tractable and the star of this talk is going to be the the Quantified which we named for the fact that it makes these really beautiful rosette-shaped um uh uh colonies and these colonies are to me reminiscent of early stages of animal embryogenesis so we were very interested in understanding the how these form um at the same level of detail as we see when we study animal embryogenesis okay and so over um uh the decades starting you know first with the description of choanoflagellates but then really taking off with the sequence thing of the first point of flagellate genome that was published in 2008 um we've been working to develop tools to make uh estrusetta and other Corner flagellates experimentally tractable and so for instance with astrozetta we can control cell State transitions in the laboratory we've established forward genetics so we can isolate mutants um in 2018 David Booth developed a technique for transfecting choanoflagellates was which was remarkably difficult to get off the ground um and then in 2019 Pablo burkhart's group um published a 3D ultrastructure of estrusetta in including colonies and so this has helped to reveal in particular the secretory pathway and how that works polarized secretion and choanoflagellates and then very excitingly in 2020 so very recently David booths while still in my lab established crispr genome editing and that has been foundational and it's remarkable you know again so hard we were working on it since I started my lab getting some sort of um genome editing going and now with its establishment it's just incredible all the things that are being done not just in my lab but in other Joanna flagellate Labs as well so I think it's really in a um uh accelerate research on these organisms in the in the years to come so why you know I told you quantiflagellates are closely related to animals um but uh why should we be particularly interested in them and one of the reasons is that they help us gain insight into the ancestral functions of genes that are critical for animal biology now when I started the lab I was particularly interested in identifying the animal Innovations what is it that distinguishes animals from everything else and these are the types of genes you see these are many you know famous developmental Regulators for instance um but what was surprising is that many of the genes that were in textbooks as being animal uh animal genes are actually conserved in quantiflagellates and so that means that they were also present in the last common ancestor with Quantum flagellates and these include things like c-type lectins you know adhesion proteins like adherins and immigrants there are collagen repeats um and I'm particularly later in the talk going to focus on g-protein-coupled receptors but this is just to make the point that some of your favorite genes may actually be in choanoflagellates and that raises interesting questions about what they might be doing at a unicellular level before they became involved in obligate multicellularity in animals okay so I'm what I'm going to do is first give you a very brief introduction to the life history of s Rosetta to give you some context for um uh the biology that I'll be discussing I'm then going to describe um two unexpected discoveries that we made in the past years and these are that bacteria regulate both mating and multicellularity in cholana flatulence and I'm I'm essentially even though that work has been published I'm essentially introducing it again here because it motivates um our study of quantiflagellate G perching coupled receptors and this is very exciting new work really uh more data are coming in every day um and so you know this morning I was adding in new slides so I'm excited to tell you about that okay so as I mentioned um s Rosetta can form colonies uh but it also um makes a very happy living as a single-celled organism and so one of the interesting things about it is how does it transition from being single celled to Colonial and it does this through a process of Serial cell division just like a zygote dividing over and over again to become an early stage embryo but these are highly polarized cells and so what happens is they divide along the apical basal the not talking well apical basal axis and uh and then the two cells will remain together and this will continue with the cells reorienting to establish the um a two-dimensional essentially plate and then they will pop out into the third dimension um uh with further rounds of cell division and you can see that here this is a founding cell um and then through multiple rounds of division it produces this three-dimensional Rosette so we've been very interested in understanding this and you know one thing you might be wondering is how do these cells stay connected and one way they stay connected is that through incomplete cytokinesis they leave behind an intercellular bridge and another thing that happens is they oh here's a tem of one of these intercellular Bridges showing these mysterious plates that we've yet to um identify but one of the other things they do is they secrete extracellular Matrix into the middle and I'll show you in a moment that genetic screens have shown that key members of The extracellular Matrix are essential for rosette development so we think it's these two features these Bridges and the extracellular Matrix that are essential for building the Rosette but um for now I want to really focus on this question of Regulation so what is it that controls whether these cells will continue to divide as single cells or whether they'll switch into um a pathway that leads to multicellular development and of course we're interested in both extrinsic cues and also the genetics that contributes to this transition but when I first started studying uh as Rosetta I was really frustrated in this effort and the reason is that um although it was isolated from nature as a rosette when I brought it back into the lab it was primarily in the single celled form so hopefully you can see these single cells and you can probably also see the bacterial prey that we have to grow them with they have they there has to be live bacteria there for them to grow and so this is um exceedingly frustrating actually my uh my postdoctoral fellowship um was The Proposal was that I was in a separate colonies from single cells and somehow you know uh for the for the older members of the audience I was going to use subtractive hybridization to identify uh genes that were specifically upregulated in one cell type and not the other younger ones don't even know what that is anymore um and uh anyway I could never get enough Colonial material to do anything um and so uh really kind of set it on the back burner until I came to Berkeley and then to Berkeley um we started working on trying to get control over the bacterial diversity um for genome sequencing and interestingly there one cocktail of antibiotics we used actually preserved enough bacteria for them to eat but blocked all rosette development so in this condition we never saw rosettes and that raised the interesting possibility that cues from the environmental bacteria may help to induce rosette development and and that actually turned out to be the case so that was you know we had we had been changing all these other uh features of the culturing conditions pH you know various nutrients that we were adding in temperature you know you name it we were trying it but um despite having trained in a bacterial genetics lab it didn't occur to me that bacteria might be the the key feature and so this result got us thinking about the bacteria and so we um isolated different species from the environmental condition and added them one at a time here and it turned out that one of the cultures was sufficient one of the bacteria was sufficient to rescue Colony development and this changed everything first of all it emphasized the importance of uh host microbe interactions in the life history of US Rosetta and secondly it gave us really straightforward uh control over this process in the laboratory so the the responsible party here is this bacterium alborophagus which makes these really beautiful um orange colonies on plates and this was exciting because it was co-isolated with the choanoflagellate so um so it seemed to be you know ecologically relevant partner um we could grow cultures just with astrozetta and algoraphagus as prey and it was sufficient food and it's related to bacteria in the vertebrate gut and so what we learned about the interaction here might actually have some relevance for um interactions between vertebrates and their their gut microbiota so we definitely were very excited about this um and we collaborated with a chemical ecology lab John Clardy at Harvard Medical School to identify the compound that regulates Colony development and this turned out to be an extremely difficult process um largely because well for two reasons one is that the inducing compound is a lipid um and and it was really tricky to separate it and the second is that it's present at exceedingly small levels in the bacteria and so we had to grow up we meaning the clarity lab had to grow up vast quantities of Al Gore phagus to get enough material but it was worth it because this is a fascinating molecule this is what we've named riff one for rosette inducing Factor one and it's a member of a class of molecules called sulfonolipids named for the sulfonic acid head group um but these are interestingly related to sphingolipids which are a critical signaling compound in animals and again um uh somewhat poorly understood in terms of how this signaling works so we definitely wanted to pursue this further foreign the question that has really been driving us and the one that I mentioned that we have still not succeeded in answering is what is the receptor for the rosette inducing Factor how do choanoflagellates sense these reset inducing factors and how does that signal get transduced to change to switch from being unicellular to Colonial and so what I'm going to tell you is our persistent effort to identify the receptors for the rosette inducing factors and then how that's led us in a number of really exciting uh uh directions none of which have actually answered this question so there's the setup um okay so the first approach um was spearheaded by a Really Brave graduate student tare 11 who came to the lab and said she wanted to set up forward genetics and choanoflagellates and I thought that was a spectacular idea you know I I think of for genetic screens as the gold standard for linking genes to phenotypes but there were some really critical barriers that I thought were going to prevent her from success and I told her I said you know as your Mentor I'm not sure that I can in good conscience encourage you to do this because I don't think it's going to work and she broke it down she's like these are the barriers and this is what I'm going to do to overcome them and sure enough she succeeded on every one of those so it was um a real tour de force so this is the way the screen worked she would grow up you know one key challenge is that the queen of flagellates don't grow on solid plates so they have to be grown in liquid media and what that means is that it's not straightforward to isolate clones um but what she would do is mutagenize in liquid media then quickly isolate clones by limiting dilution into 96 well plates um and these 96 volt plates contained the rosette-inducing factor and so then she could visually Screen through each of the plates find the ones the wells that had cornoflagellates in them and then ask uh were they capable of forming rosettes or were they defective in some way and just to get a sense of you know how laborious this was you know she screamed over uh um 29 well let's see I'll do the quick math 36 37 000 uh clones whoops and uh only got 18 Newtons but these mutants actually fell into some interesting classes so here on the left um for comparison is the wild type so when it's not induced it can be unicellular or it can form these linear chains of cells and when it's induced then it forms these beautiful rosettes and what you can see is that there are a range of different phenotypes under the induced and the uninduced forms this one was particularly interesting because it was perfectly normal in the uninduced state but then entirely defective in Rosette development um many of the others were defective under both conditions including this one these clumpy cells in class D they would Clump up in the un-induced state and in the induced State and when we uh you know one of the neat things about rosettes is if you introduce Shear uh the rosettes are entirely robust to that you can even Vortex them and they stay together whereas these clumps if you Vortex them or or even look at them the wrong way they fall apart so it's a much gentler interaction here than here and so chair 11 uh um sought to characterize the the genetics of this phenotype and then later uh another graduate student Laura Wetzel really focused on this class okay the problem was that um although we had mutants and we had interesting phenotypes we didn't yet have a straightforward way to connect genotype to phenotype and some of the key reasons were that we didn't know if quantiflagellates made it and in fact despite having been first identified in the mid-1800s no one had seen queen of flagellate mating um and even if they had they might not have known what they were looking at so we didn't know if coinoflagellates could mate we didn't know if that mating could be induced in the lab and if they did mate we weren't sure we could isolate the meiotic products and without that we didn't know if we could map the genetic lesions we really needed the mating to essentially separate out the causative mutations from uh uh from the background mutations and keep in mind um you know I think that that that's the best way to do it anyway but we didn't have crispr or any genome editing so any other approach you might think about we didn't have at our disposal so really mating was a key barrier um so Tara decided to take that on also and um and what's clear is that in many microbial eukaryotes stressful conditions can induce mating and so she started um applying different stressful conditions to corner flagellates and sure enough um starvation it turned out was sufficient to induce mating um but even when the cells were starved meaning was rare and this helped to explain why uh it hadn't been observed before so in this whole field of cells you can see that they're starved they look you know they're pointy and they look unhappy relative to the other cells you've seen so far but of these only two are about to mate these two right here um and generally we found that the cells differentiate in terms of size with a small cell and a large cell that we've provisionally named you know the male and the female gamete and so um if we zoom in on those two cells we can see how mating occurs um so the cells do this little dance the male cell binds toward the apical pole of the zone and then the cells fuse and so this process um has led us to you know identify what we're calling the Hallmarks of mating and quantiflagellates um and in this uh what happens is the cells find each other the membranes then fuse after that you get nuclear fusion recombination and then to return to the haploid State there has to be a round of meiosis and Tara identified all of those steps in chlanoflagellates including evidence for recombination so that um one of the key uh things to keep in mind at this point now is that these quantiflagellates have Dynamic life histories so I've told you that a single cell can become multicellular in terms of the rosette and when the rosette inducing factors are not present it can also form these chains but um this is all asexual and these kind of flagellates can also land to uh produce these fecaled cells ordering starvation they form these small what we call fast swimmers but Tara uncovered another part of the life history which is sexual reproduction in which the haploid uh cells confuse and then uh produce diploids that are CK and then they can return either into the asexual part of the life history or continue through a sexual reproduction pathway okay so the discovery of mating then made it possible to map these rosetteless mutants and so the first thing that happened is Tara was able to map one of these uh Class A mutants to a particular protein called the c-type lectin and you can see it in teal Here It produced this is the extracellular Matrix that I mentioned before and if uh it's this c-type lectin is not present the quantiflagellate will not become multicellular or it won't form these rosettes so that really pointed to um an important role for the extracellular Matrix and interestingly enough when Laura focused on this class she uncovered two different genes two different glycosceal transferases that were also critical for um the wild type rosette development as a quantiflagellate and these also are genes that are involved in sculpting The extracellular Matrix so these two findings were very exciting they first of all were a proof of principle that we could do forward genetics but also pointed to an important role for the extracellular Matrix that we really hadn't been focusing on before however neither of these two genes was the receptor for the rosette inducing factor and so I want to return to that hunt okay and here's where um now Ariel wozneka a graduate student at the time stepped in so we couldn't we couldn't use chemical approaches to tag the reset inducing Factor there were a number of reasons why that wasn't working former genetics was not rapidly leading us to the receptor although it's certainly an approach that's worth continuing to take um so you know in a Act of desperation in a way um we saw what if we can use um a comparative transcriptomic approach to identify genes that are specifically upregulated in uh Rosette uh rosette development cultures and not upregulated when they're not forming rosettes and because um the sorry because the inducing bacterium algorithagus is delivering lots of different cues to the quantiflagellates not just the rosette inducing factors we thought we could sort of eliminate those background ones by also comparing um s Rosetta cultures to cultures treated with other bacteria that induce rosettes so here what you're seeing is a phylogeny of different bacteria and then those that we know induce rosette development are shown with an X so the idea was Are there specific genes that are up regulated in all of these cultures in the quantum flagellate and not upregulated when they're exposed to bacteria that don't induce reset development so I hope that's clear um so Ariel was um or it was and is an extremely thorough and and thoughtful uh biologist and so um when she did these experiments every time she treated with a different bacterium she went back she didn't accept you know that what she'd been told that they don't induce for that development she went back and looked at them herself carefully and when she did that um something she saw something really weird in cultures treated with vibrio Fisher eye so sure enough you know we looked at vibrio fisherie a lot it's a a well-known commensal uh and symbiont with with different animals um we thought it would be super cool if it industries that development but it didn't um but when she looked right away so she she would add the bacteria and then rather than waiting 24 hours for is that development to occur she looked right away and this is what she saw um so on the left you see a culture that's been treated with um control bacterium that doesn't induce development or anything but Fisher eye when added caused the corner flagellates to aggregate into these clumps and these are these clumps are very different from rosettes because they're forming through different cells coming together so those of you who know digestelium you know it's not as beautiful as the tasty Lane but it's conceptually similar with these cells coming together and aggregating and so I still remember sitting in my office scenario coming to the door and she's like I there's something weird going on in these cultures I don't know what it is you know can you come look and I looked and and we spent a bit of time trying to figure out what might be going on because this wasn't um a behavior that we'd seen in the lab before again it had not been reported for Quantified bullets and we didn't really have a strong um notion of whether whether this was even physiologically relevant uh um you know that was like Ariel was like I don't know whether to keep studying it because I don't know if this is just some weird lab thing or this is something that is actually indicative of esperzetta's biology in nature um but then I remembered that uh many ciliates actually swarmed before they made and in addition um swarming is actually a prominent behavior of uh mating and animals and so you can see you know fish swarm these are these African lake flies that for these huge swarms bats swarm crab swarm that you know this is a common Behavior where uh members of a species uh increase their local density prior to mating and so we wondered whether that might be what's going on with Esther Zeta also and in fact that was the the answer and so it turns out that vibrio fisherai is not only inducing swarming and clumping but it's also that that is the lead-in to mating um and so here you can see two cells with their nuclei labels um their membranes fuse their nuclei migrate together and fuse and then uh remember our key question is do they uh undergo recombination and so she tested that as well and so here you see two strains with different uh um genetic variants and if she if she compared the outcome um or the genotypes of bacteria that came from this mixing these two together in the flask whether they were exposed to control bacteria or vibrio fisheri and sure enough with control bacteria she only recovered the parental genotypes but when she added bibrio fischeri now she saw abundant evidence for recombination and so that told us that vibrio fisher eye is producing a molecule that uh induces mating in the koana flatulate and so just to summarize that here this is a nice figure that um uh accompanied a News and Views piece and so the idea then is that when haploid s Rosetta is exposed to vibrio fischeri bibrio fisherie is releasing a molecule which we've named arrows and this is actually a chondroitinase and that chondroitinase interacts with s Rosetta triggering swarming and cells actually meeting up together the cells then fuse they become diploid and then through meiosis and recombination return to a haploid state so Ariel did not answer the question of what is the receptor for the resin inducing Factor but she found this really remarkable uh life history um activity and and this mating was actually in many ways different from The Mating induced by uh starvation because it happened almost right away within minutes instead of within weeks uh and the orientation of the cells was different with the cells contact each other by the cell bodies as opposed to um fusing at the the collar of the larger cell um and so this is an interesting phenomenon where we had essentially have these two different meeting pathways okay so um that used to be what I uh primarily talked about but I want to return to this hunt for the receptor because this is going to bring us to the new um data that I'm very excited about so this is the work of uh a postdoc in the lab Alan Garcia De Los Banos and here he is at um as we're trying to brainstorm and understand some new data he has that some of which uh Bears on what I've just been telling you so we when you look at the Genome of the clanoflagellate um if you had to guess what the receptor for the rosette inducing Factor might be the best possible candidate um are these g-protein-coupled receptors so why why is that our best candidate well in animals g-proaching coupled receptors first of all they're the largest family of cell receptors but importantly although they can respond to many different kinds of inputs they are the best known and best characterized receptors for lipids in general but in particular a special class of lipids called the sphingolipids and if you remember the rosette inducing Factor shares many chemical features in common with single lipids and so we thought the G protein coupled receptors would be a good candidate now before um uh coanoflagellate genomes and transcriptomes were sequenced it was thought that gpcrs were restricted to animals but in fact we now know that gpcrs have a much richer phylogenetic history so this is just a schematic of one particular class of g-protein-coupled receptors this is the adhesion g-protein-coupled receptors which have adhesion motifs in this extracellular domain a cleavage domain called a GPS and then the transmembrane domain and intracellular motifs and it turns out that gpcrs in general and adhesion gpcrs in particular are widespread across across choanoflagellate diversity and so here's our friends tell pingalico Rosetta right here and you can see that it has 19 total gpcrs and 12 of these adhesion gpcrs and so this was very interesting in addition if we look across eukaryotic diversity we see that animals have lots of adhesion gpcrs as do choanoflagellates and other unicellular close relatives of animals um but the the prevalence of gpcrs drops off dramatically as you get further away on the phylogenetic tree so all of this let us be very interested in not only in gpcrs in general but adhesion gpcrs in particular and what I'm going to do is focus on one particular class of gpcrs that we find is conserved across at least nine different choanoflagellate species so this uh gpcr has EGF domains um uh near the interminus it has a lamb G domain and importantly it has this GPS domain that's conserved and for reasons that should become clear um we've named this ppcr in US Rosetta clumpy so here again is the full length gpcr and Elan was able to generate edits at two different places um one right here so this one will be called one through five fifty three and one right here which is 1 through 95. and the key thing I want to tell you is that um these two edits have the same phenotype in every case and so we think these are knockouts okay so what you're going to see is the cellular behavior of wild type cells here and then two different uh mutants and what I hope you'll be able to see is that these cells actually aggregate together over time so here are single cells and now you can see these big clumps starting to form okay perhaps reminiscent of something that you've seen recently um but we were surprised because plumpy is an adhesion gpcr so we expected um lots of adhesion not gain of adhesion um this is just to say that uh Elon Quantified this and again as you um move through time with the wild type on the top and then the two mutants you see that you accumulate these large clumps and then you can quantify them and you can see the wild type in Black does not Clump up but these two mutants do and essentially at equivalent rates okay so that that was unexpected um is is clumpy important for growth and we can find no difference in the growth curve of the control versus these two clumpy mutants is clumpy important for rosette development um it turns out it's not so um here's wild type on the top no induction or with rosette induction and you can see Wild type forms beautiful rosettes as do these clumpy Muse okay and it's Quantified over here showing that they are essentially equivalent omvs are outer membrane vesicles and this is how we deliver the bacterial cues so no difference so so far the only difference that we can see is this aggregation behavior um but it doesn't seem to affect the cell adhesion that's required for rosette development so how are these clumps forming they're actually forming when cells are adhering through their collars so these are not adhering to their cell bodies the way that vibrio induced mating cells were adhering these are adhering through their collars and Elon actually went through and measured the number of contacts of cells to collars cells to cells or callers to callers and what he found is that the vast majority of the interactions were collar collar or collar cell and in work I'm not showing you when he removed the collars he found that that completely blocked cell aggregation now it turns out that this color collar interaction actually is similar to the type of interaction that we see in cells that are mating in response to starvation not ones that may in response to the Rio treatment and so here you can see pictures of the mutants or the controls control cells that have been starved and when Elon did the same type of analysis of the number of contacts um when he looked at Starved cells this is a control edit here he saw that they interacted in the same way so primarily through color collar and collar cell body so this certainly was very interesting and unexpected um and so he began to wonder whether in fact he had uncovered a negative regulator of mating could it be that by knocking out this gpcr in fact he was unleashing mating under conditions where mating didn't normally occur and so to test that he um he developed an assay for Cell fusion um that allowed him to detect rare Fusion events through using facts and so the idea was um to use cells that were either labeled in the cytoplasm with mtfp or in the nucleus a histone tag uh um uh with M cherry and then after 20 hours measure the number of cells that had both fluorescent signals and so here's one example here where you can see um the the cytoplasmic fluorescence the nuclear fluorescence and then the overlay so this is in Wild type cells and the question was if you did the same with these mutant cells what would happen and pardon me in fact there was a dramatic increase in the number of cells that were fusing here we're seeing the percentage of few cells of the control versus compait and you can see that there's a dramatic and significant difference in the number of cells that are fusing in response to this uh clumpy mutation and so that suggested in fact that clumpy might be related to The Mating pathway and so the next thing that uh um Elan did was to look at which genes were upregulated and down regulated in response to deletion of clumpy and so here what you're seeing um uh are the uh logful changes of genes in control versus clumpy and what I hope you can see is that there are 29 or sorry 28 genes over here that are significantly down regulated and 59 that are significantly up regulated and when he looked at what they were interestingly the vast majority of those that were down regulated were retrotransposons um which whose regulation is thought to be involved in gametogenesis and many animals um now in terms of up regulation uh there was an important number of them involved in cyclic GMP signaling and Iran has a whole other Story related to this um to this deep dive on these genes um but what I want to talk about right now is the fact that there were also genes that are known to be involved in cell adhesion and Cell fusion and gametes and one of those was is a homologue of hap2 which is an ancient gamete fusogen and eukaryotes and so this is just to show all of the eukaryotes that uh that Express have to and use it uh dirt for gamete fusion and so the way hap2 works is it sits between the membranes of gametes and it it helps to set up the Cell fusion that allows these cells to fuse and you can see here chlamydomonas has have two that's expressed near the apical pull of the cell which is where Cell fusion occurs during mating and uh Clearwater bonus and also in tetrajaimana we see enrichment of hap2 um uh in the area in which these two cells have fused and so um what Elon has shown is that this clumpy mutant um well let me back up and just say in Wild type when wild type cells are grown in high nutrient conditions they don't mate um they have very little collar adhesion very little Cell fusion and very low expression of hap2 and other Regulators of gametogenesis but when he uh deletes most of the clumpy uh protein or Gene what he finds is now he gets rampant clumping of cells which is a phenotype that we associate with mating he gets increased collar adhesion increased Color Fusion or Cell fusion and increased gamete gene expression and these phenotypes match with the mating phenotypes that occur when we starve as Rosetta and what we don't know at this point is what happens to the clumpy Gene during starvation but this is certainly something that he's investigating now and so with that um uh I'm going to close and just say uh thank you to you know incredible lab who kind of held it together during the pandemic and were joyfully emerging from the other side um and I'd be very happy to take questions that uh you know bear on the many different threads that I uh threw out there to you and uh and I think that all bear on trying to understand how Environmental um cues coupled with intrinsic genetic circuits help us to understand how cell differentiation evolves so thank you all thank you oh that was great thank you and uh maybe let's see if uh trainees have questions first great raise your hands um virtually raise your hand or put a question in the chat um so uh looks like we all right thank you so much for a wonderful talk I really enjoyed it you just light up my whole day I have two questions the first question is does the arrow change the expression of have to I was just wondering the connection between the arrow that you found in your bacterial screening and which the capture that you found in the cellular mechanism of matching and then my second question is in one of the videos you show I think is I-95 the clump so like basically the question I'm trying to get to is I'm interested in the swamping behavior that you mentioned it looks like some of the colonies has more uh movement than the others the sound of the colonies stay still but some of the colonies seems to move around in the aggregate more I'm just curious about whether that means there's a need for the mental differences between the colony or do you think that was just so the Brendan and then it's more population uh differences thank you so much thank you um this is actually a great opportunity for me to introduce Alan who I've invited to come along so maybe he can wave his hands I I just had him turn on his uh camera um so in terms of your first question is Eros turned up regulated in response or sorry is clumpy upregulated following treatment with Eros I actually don't think we know um but that's why I asked Alan to come along if uh uh he has do you know if we've have you looked no we don't know and I think yeah you were asking for app2 as well and we we same we have no idea uh but that's definitely something that will be interesting to try to know for sure yeah um great question and then the other question I think you were referring to Wild type um cultures that have rosettes that behave in different ways and we actually see a lot of difference in Behavior depending on how many cells are in the rosette so the large uh rosettes tin if you think about it there are all these competing forces with these flagella but the rosettes are not perfectly spherical or perfectly symmetrical and so what you can see is that depending on how uh you know how far they are off of symmetry you'll see different behaviors in response to and another thing that happens is sometimes the colonies actually collect lots of bacteria on the collar they're really good at prey capture and that will make the ways in which they swirl and move through the water even more chaotic um so I don't know that we know like is that is that an Adaptive thing has there been selection on it or is it just a secondary um response to biophysical uh features of the rosette we don't know um let's see Dominica is that are you next perfect hi that was phenomenal talk we were talking about it while you were talking about okay thank you um so it's really interesting to see that multicellularity is not an obligate part of development that you can be induced in cultures and I'm wondering it's my understanding that the bacteria are things that kind of quantify to its prey on um and I've heard that being used as an argument for metabolism as an inductor of morphogenesis right so unicellular to multicellularity right I just wonder if they're preying on bacteria and they're in like in taking them in do they need receptors or is it possible to do like condition media with what things bacteria produce I don't know how that would work in college yeah so we saw it we thought it was going to be a metabolic story um but it's not actually because we can't we can do conditioned media but we can also just take that lipid at very very low concentrations and that alone is sufficient to induce rosette development it doesn't seem to change the metabolism because they're eating a different bacterium um so they have plenty of nutrients uh and um all they need is this one little cue and we when we look at the concentration that is required it seems to be far more consistent with a model of having of there being a receptor um than with a metabolic model um in addition there's another uh small molecule that we've isolated uh which we've named uh inhibitor of rosettes and like what was it ior and it essentially is a hydrolyzed version of Rift one and it looks like it's acting as a competitive inhibitor so that also I think is consistent with there being some sort of receptor um but maybe there's no receptor and we're just hopefully going to keep learning cool things along the way I don't know just thank you thank you Bloom I'll let you go ahead and tell me who to I think we have young men had his hand up uh and then all right okay it's very inspiring to talk he's gonna make me you know the light of the whole days about the memania development so I was um uh so curious about just why it's uh the specific and uh lipid makes this kind of a multicellular development you know it's it seemed like it seemed like this lipid is a great I mean uh uh how to say in light uh uh how to amplify the still going to make them together right so I mean what's the receptor that in gpcr receptor or some other receptor of this specifically to make this kind of uh signaling make this sale aggregate oh we we can say the the developments yeah uh I'm not sure I entirely understood the question but I I think I have a few things that might contribute to understanding the one is that it turns out that riff one that particular molecule I mean it's a highly specific interaction if we change the stereochemistry of that molecule at all it doesn't work so so there's some sort of very specific interaction but it's actually a very poor inducer of rosette development and uh so it does induce but at very low levels and so Ariel wozneka the grad student I mentioned actually went back and discovered that there are there are additional lipids um and she identified two others one was similar to riff one and we called it rif two very creatively um and the second is a lysophosphatidyl ethanolamine that has no inducing activity on its own but when mixed with the riffs great they synergize and and it essentially recapitulates full induction those three lipids together um so uh and we don't understand that at all um so that's that's one source of specificity that occurs uh and and boosting of signal the other one that's very interesting is work by um a former graduate student um uh Ella Ireland in which she found that if you actually mix the mating-inducing signal Eros with the rosette inducing ones that greatly amplifies rosette induction also um and that we still don't understand um and I I'd be loved to hear people's ideas of why that might be the case um but it seems that essentially she adds she adds them both together they mate really fast and then they form these really big beautiful rosettes even at very low concentrations of the rosette-inducing factor but for example water you can't Inhibitors use the drugs like to inhibit a specific signaling to see whether it's blocked this kind of uh even you add the lipids there you block it right we've done a lot of inhibitor studies um and so far have not found an inhibitor that is specific to the process of rosette induction um but it's a good idea you know what I take that back uh nothing thinking about it um a former postdoc in the lab um uh Flora Rio de janeira did a really beautiful chemical chemical genetics screen um and the first phase of that is now on bioarchive in which she was looking for Inhibitors of cell proliferation but she has a second wave of screen in which she has identified specific Inhibitors of rosette development and she hasn't yet fully worked that up so I can't I don't actually know which inhibitors uh they were but she's in the process of working she has her own lab now at Stanford and she's working up those results now so that's actually that's a very good point okay thanks and another question I'm sorry about two questions and I thought this is sexual um I'm a sexual reproduction a sexual reproduction in the in the real estate formation the similar amount and uh also also the uh for the sex show they're making together and the weather there from the synaptomama complex for that recombination okay um we do not have enough resolution these are very small cells and we can't visualize even individual uh chromosomes at this point um we think they have over 50 chromosomes um and we just have not looked at that level of uh resolution so we don't know um and I I the first question was about whether aggregation and Colony development are the same thing or yeah I mean as sexual and sexual the real estate development I mean the same is similars or not no they're completely different so rosette development is clonal and it's through cell division and aggregation is cells coming together that are not related to each other necessarily oh what my question is the sexual the minister because if you stop aging them sexual they make of each other and then they will move forward to this guy roles in the structure but if you inducing the real estate as sexual reproduction they were also from the real estate structure I mean both subjects kind of roadside structure is a stimula or not out there let's share the stimulant feature yeah well rosette development is independent from mating oh so they are not yeah um okay got it yeah okay thank you so much I think our art was next yeah so uh biochemists often uh go to The Brood force uh sort of uh Avenue and it seems to me that you could make riffs synthetically and and make it radiochemically hot and it'll it'll be still functional I'm sure of that yeah and and just use that to just Chase it down through chromatography um I mean I love that idea 30 experiment that would be time consuming but I you'll definitely get I would assume you can get conditions where uh hopefully riff isn't itself metabolized but is is stably bound to its receptor yeah I love it I um unfortunately I can't get any of our collaborators to uh to isolate that for us um uh yeah so it's it's actually it's an extremely inefficient purification [Music] um and uh and the synthesis I'm trying to remember the the synthesis is extremely inefficient also so unfortunately that that has not been an Avenue that we've been able to go um we've also our collaborators have been trying to tag the um tag the compound through something oh yeah no I'm sure I'm sure yeah that's not active um no but the inhibitor is so a tagged inhibitor is active and so that is one possible route that we'll take in the future but yeah it's a new it's a new generation I I've spent my time in the hot room how many steps of synthesis is it I I'm afraid I don't remember okay sorry since I didn't actually do it so okay yeah thank you though all right Lynn hi Nicole thanks for a great talk um excellent I have a question about you said you didn't have resolution to see chromosomes and I'm wondering about cell divisions because I'm curious about the difference between linear chains and rosettes and is there for example are the linear chains also connected with Bridges can you Vortex them and they'll stick together and then does something happen about the cleavage planes when the rosette is induced so that they form the more three-dimensional thing rather than the chain right so can you watch that happening I guess this question so the linear chains are connected by Bridges um but it is not a robust connection so they will fall apart in four text um and we don't really I think there's more work to be done but this is my view of what's happening during reset development I think that when they divide they remain you know it's it's symmetrical and you get two but what they're doing is they've secreted this shared extracellular Matrix and I think that extracellular Matrix tightens up um and that pulls the basal poles together and tilts the apical poles apart um and that that view comes from a lot of very indirect research and I think you know what we what we haven't done is a really careful examination of um what happens when we treat with uh enzymes that might cleave The extracellular Matrix will that you know prevent that um one of the challenges we have is that it's actually really difficult to keep the colonies in place under the microscope to watch the full process of rosette development but um yeah these are open questions that I think we really need to get back and and delve into more deeply great thanks thanks I have one more question um sure yeah but I was wondering what happens if you um if you mix clumpy knockout with wild type do they just fuse with each other or will they fuse with a yeah I took that slide out a lot did do that experiment and they actually will fuse or I don't think he's tested yet if they fuse but they will adhere to the wild type cells um so it seems like it it doesn't have to be bi-directional it's enough to have a sticky mutant and and it will stick to the wild type cells um okay any other questions if not um let's thank Nicole again um and thank you all for the great questions that was fun um and well I guess at this point log out of here and there's a separate you have a break a little break um okay I'll put link for the meetings later outstanding okay I look forward to talking to all of you take care if you want um I know you were scheduled to have a 15-minute break and it's now a five minute break so and I'm the next meeting so if you want a little more time we can oh no it's fine I'm just gonna go and make a little more coffee and then I'll meet you sounds good okay take care bye

Channel: Yale Genetics

Views: 2,407

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Id: QPGReWULBls

Length: 68min 45sec (4125 seconds)

Published: Mon Dec 12 2022