Regulation of Endothelial Cell Specialization | NAVBO Webinar

[00:00:05]good afternoon everyone and welcome to today's webinar sponsored by the navos education committee I'm Anar quvo from the University of Illinois College of Medicine and I will be moderating today's session it's my pleasure to welcome our speaker Dr Karen Hershey from the University of Virginia she will present her research entitled regulation of endothelial cell specialization we will also welcome Dr Nichol um Chavin chapkin a postto also at the University of Virginia he will monitor today's question we have something new to announce and I'm sure you've received something on your email our webinars and journal clubs as well as other online activities are now listed in the navo Events app available exclusively to Nao members be sure to download the app and keep track of nao's online events throughout the presentation please type your questions on the question box in the control panel these these questions will be answered at the end of the presentation after those questions are answered provided that there's time attendees will be able to ask any additional questions in real time if you have a question after the questions have been typed and read please raise your hand by clicking on the hand icon on the left side of your control panel you will be recognized and mute your mic and ask your question this webinar is being recorded and archived at the nabo website for future use so we have today Dr Karen Hershey she's an ajun professor and a member of the cardiovascular research center at Yale and also the alumni professor of Cell Biology at the University of Virginia school of medicine and a member of the cardiovascular research center at ubaa the hersel lab is primary interested in vascular and hematopoetic development they are focused on elucidating Regulators of endothelial and blood cell commitment differentiation and specialization as well as modulators of endothelial cell cycle state during brand development without further Ado welcome Dr herie all right thank you so much Anar for that uh introduction and I'd like to thank a minute i' take a minute to thank uh bernardet and um and sharen and all of the education committee at navbo for organizing these webinars and also a special thank you to bernardette for organizing so many activities that are now available to us as members of navbo helping to keep us connect Ed during these very challenging times um and also I'd like to thank ahead of time Nick for monitoring all the questions at the end of the talk and uh finally thank all of you for tuning in for this webinar I really really really wish I could see all of you um and I hope that we can get together in person soon hopefully for the annual Napo meeting in the fall in October um so until then I hope you all stay safe and well and I hope to really see you soon um so when I get into my talk I'm going to turn my camera off because the connection is more stable that way um so I don't think you can see me anymore but I am here okay oops all right to me all right so as an R said um today I'm going to tell you about some of the work that we've done trying to understand the regulation of endothelial cell specialization um and just as an overview of what my lab does U we've been very interested in understanding various aspects of blood vessel formation and we've been studying this predominantly in the mouse embryo model system and we try to understand Regulators of different steps in this process and determine whether or not um similar mechanistic steps um occur in human development in development of human um endothelial cells and homat Aquatic cells as well and so we um have human PL poent stem cell models in the lab including human es and IPS cells and we can generate human endothelial and hematopoetic cells that we can also then use in tissue engineering and regenerative medicine uh projects that we are involved in such as this H very interesting organs on chip project that's headed up by godana at Colombia and so this is just an overview of the various steps that we think are necessary for the formation of blood vessel structures and we have been very interested in several of these aspects including the differentiation of endothelial cells from mesodermal precursors um their specialization into subtypes that enable them to have distinct functions and then also the acquisition of the vessel wall which is mediated by endothelial cells that recruit Mangal progenitors and when these two cell types interact it transforms the Mangal progenitor into parasites or smooth muscle cells that will make up the surrounding vessel wall and to today we're going to focus just on endothelial Cell specification and I'm specifically going to tell you about studies we've done trying to understand hemogenic endothelial cell development um and so we know in the developing Mouse embryo that one of the first places where blood vessels form is in the extraembryonic yolc which is shown here and this tissue acts as a connection between the placenta and the developing embryo inside the Yol saac and when vessels first form there in this capillary uh plexus that then has to be remodeled into a circulatory Network and this is not a weird thing that happens in the yac um vascular Plexi form in other um as other tissues are developing in the embryo proper as well and during this remodeling into a circulatory network is when there is specification of arterial and Venus endothelial cells and these cell types end up with different structures that enables them to have different functions and in this yok saac tissue at the time when arterial and Venus endothelial cell specification is happening there's also specification of hemogenic or blood flow blood forming endothelial cells and so this happens first in the yac and then slightly later in development in the placenta and then within the embryo itself in a region referred to as the aorta gonad mineis region or the AGM and we're going to talk more about that um and so this generation of blood from the endo oium we think requires two different steps the first is the hemogenic specification of endothelial cells that didn't previously have blood forming activity and then the formation of multi-lineage hemat aquatic stem and progenitor cells from the hemogenic endothelial cells and my lab has been most interested in this initial um process and we're trying to understand what are the unique cellular and molecular characteristics of hemogenic endothelial cells how do they differ from endothelial cells that don't have blood forming activity what are the mechanisms that enable them to acquire this special U characteristic and phenotype and are the mechanisms then applicable to the specification of other endothelial cell types um and so we I'll talk about some of the work that we've done but I first want to briefly review um uh hematopoetic development in the mouse model and show you the timeline relative to vascular development and so in all mammals including humans there are two waves of blood production and the first is referred to as primitive hematopoesis and this is uh predominantly the uh generation of immature arthro blasts and it occurs only within the Yol Sac tissue and then a day later in development definitive hematopoesis begins and this is the production not just of red blood cells but also myoid and lymphoid cells as well and this occurs initially within the yolac tissue also but then in the placenta and then in this AGM region of the embryo and so hemogenic endothelial cells are specified in all of these tissues and they generate hematopoetic cells but it's the ones that are generated in the AGM that are thought to be true hematopoetic stem cells as opposed to multi-lineage progenitors that are produced in the Yol saac and the placenta uh and this was uh discovered by taking these cells and transplanting them into lethally irradiated recipients and the cells that form in the AGM are the only ones that are able to rescue the formation of all blood lineages and those that form in the yoak and the placenta don't really form lymphoid cells very well and so all of the hematopoetic stem and progenitor cells that form in these tissues then migrate into the fetal liver as it forms later in development and then um at last into the fetal bone marrow slightly before birth which is about 20 days for most Mouse strains and so um the formation of hemogenic endothelial cells in all of these tissues and their generation of hematopoetic stem and progenitor cells is extremely important for postnatal hematopoetic um postnatal hematopoetic development and the process of formation of all blood cell types that we need postnatally um and so I'm going to tell you about some of the mechanisms that drive hemogenic endothelial cell development but I first just want to show you um this happening uh in the tissues themselves and so this is transgenic line that we made in collaboration with Mary Dickinson at Baylor College of Medicine and I'm going to show you two movies uh this one is hemogenic endothelial cells giving rise to blood cells in the Yol saac and on the next slide it's um the cells in the AGM region and in both cases these um in these mice endothal cells are labeled with M Cherry which is driven by an endothelial endothelial cell specific region of the flick one or V receptor 2 promoter and in this case case the blood cells are labeled with the promoter from lmo2 driving gfp and lm2 is a transcription Factor that's necessary for definitive hematopoesis and so when I play this movie hopefully you will see the red endothelial cells giving rise to Green blood cells as shown here and they pop off into circulation of this remodeling yolac plexus we'll see another one giving rise here and so the blood cells are formed from the endothelium and they immediately enter circulation in this remodeling yolc tissue and there's a slightly different process that happens within the AGM so in this case again the endothelial cells are labeled with M cherry and the blood cells with gfp and this time driven with the Epsilon globin promoter and you'll be see as I play the movie that the blood cells are being generated from the red endothelial cells and in this case in the AGM the cells the blood cells that form do not immediately pop off in into circulation they are generated and they divide and they form what's referred to as an intra aortic cluster um and this Niche this cluster or this Niche for these cells May in fact be one of the reasons why the progeny um have different potential so when the Yol sack of blood cells pop right into circulation whereas here they develop a niche that's thought to be important for their for their maturation and so we and others are trying to understand how the micro environment Within These different tissues dictates the hematopoetic progyny it may also be that the hemogenic endophilic cells themselves are molecularly different and so we're very interested in addressing that as our other labs um and so we spent a lot of time trying to understand the phenotype of the hemogenic endothelial cells in both the yolac and the AGM and by doing a lot of Flo cytometry and Colony forming assays um we found that the um endothelial cells that have these characteristics represent hem enic endial cells in both of these tissues and so these cells Express FF receptor 2 or flick one ckit which is the receptor for a stem cell factor and they do not express um proteins typical of hoptic cells like cd45 um they also um have an SP or side population phenotype and this is uh similar to what occurs in hematopoetic stem cells in adult bone marrow um so these cells overexpress multidir resistant pumps that en Ables them to extrude small molecules including DNA binding dyes that can be used then for their identification and isolation um in the Thea flow cytometry and so if we isolate endothelial cells that have these Collective characteristics um from either the Yol Sac or the AGM and we plate them onto fibercon we could see that they'll form a small endothal cell colony and these cells have endal cell function and that they could take up dii a culated LDL um they also Express Enos which is not shown here and then if you expose the cells to cyto kindes and growth factors that promote uh blood formation you can see this explosion of blood Colony forming activity so this is a colony of ocytes this is a colony of granulocytes and macrophases myoid cells and this is a multi-lineage colon that contains granulocytes aryes macrophases and monocytes and so very importantly a single hemogenic endothelial cell can give rise to a multi-lineage colony suggesting that these cells then give rise to multilineage hematopoetic progenitors um and so we spent a lot of time trying to understand now that we had sort of a working definition of the cells and these tissues we could then try and understand um what are the processes that uh regulate their development and we found that retinoic acid is a really important initiat factor in this process of hemogenic endothelial cell specification and I'll tell you um more about these uh studies um and we we were very interested in retinoic acid initially because a long time ago uh we were doing studies of the effects of retinoic acid on arterial venus uh formation and we found that in um embryos that lack active retinoic acid and so these are mutants that um have deleted retin aldhy dehydrogen a 2 which is an enzyme that converts vitamin A or retinol from maternal circulation into active retinoic acid in the embryos so embryos that lack this enzyme are essentially retinoic acid deficient and in these embryos we find that vascular development is disrupted and this is a lethal defect um and one of the problems is that there is lack of vascular Remodeling and so El plexus will form but is never remodeled into a circulatory Network and we found that the underlying problem in these uh mutants was lack of endothelial cell cycle control and so in the absence of retinoic acid there was reduced expression of cell cycle Inhibitors including p21 and p27 so in the absence of these Regulators the endothelial cells spent more time actively cycling and less time arrested in G1 State and so I'm going to come back to this issue in a minute but this was the underlying problem in these mutants and so we wanted to know if arterial and Venus specification is not happening happening properly what about hemogenic specification particularly in this Yol sack tissue and so we looked at this using the definition of the cells that I previously um explained and we found that in these embryos that are retinoic acid deficient there is a significant impairment in the development of hemogenic endothelial cells and then also their generation of these multilinear AG um hematopoetic progenitors and interestingly in these mutants they could be rescued by providing um exogenous retinoic acid active retinoic acid so they have receptors they just cannot um generate active retinoic acid themselves and so when we do this um and we could do it in two ways taking the embryos out and putting them in embryo culture and then giving them exogenous retinoic acid or we could feed the pregnant mom retinoic acid in the her diet which is then transmitted to the embryos and so we can do this either way and we can see a significant Rescue of hemogenic endothelial cell development and then also their um development of multi-lineage hematopoetic progenitors um and so we went on to do a number of other studies to try and understand what is retinoic acid doing and how is it promoting the formation of these hemogenic endothelial cells and so this is a model of what is happening particularly in the Yol saac um but the same processes happen in the AGM and so we know in this tissue which is fairly simple um there's a visceral endoderm layer on the outside and below is the mesoderm where endothelial cells and blood cells will be forming and we know uh that retin aldhy dehydrogenase 2 is expressed specifically in the endodermal layer and it generates aoic acid which then signals to a subset of endothelial cells in the mesodermal layer below that um highly Express retinoic acid receptors and downstream of retinoic acid signaling is the upregulation of ckit the receptor for stem cell factor and downstream of CIT is the activation of Notch and this leads to the upregulation and function of p27 which is a cell cycle inhibitor and these steps are necessary then for the upregulation of other Regulators including Bruns brunks one which is a transcription uh factor that was found by n spec and other groups to be essential for the generation of hematic stem and progenitor cells from hemogenic endothelial cells and this process of hoptic stem and progenitor cell production is very complicated and involves a number of other Pathways that different investigators have identified and we're still trying to understand how all of these Pathways um are coordinately regulating this generation of hematic stem and progenitor cells from the hemogenic endothelium and my lab has been particularly interested in this initial step of uh hemogenic specification and trying to understand the role of p27 and uh cell cycle control in this process and so I'm going to come back to this in a minute but I also just want to say quickly that um even though we have been very interested in understanding what promotes hemogenic specification um there also important signaling Pathways that limit how many hemogenic endothelial cells form and this is really really important because when there are too many hemogenic endothelial cells and too many hematopoetic stem and progenitor cells they actually become uh lineage skewed and they produce more myoid cells than other lineages and this causes um hematic defects postnatally and so this is work that was done uh we we did in collaboration with um diona Casper in Stefania nicholes Lab at Yale um and so what diona found is that microrna miror 223 is very important for putting the breaks on this process and it does this through the regulation of En glycosilation of proteins that endothelial cells Express that are involved in this EHT process and so I'm not going to say anything more about this um diona actually did a journal club for navbo in January to um talk about this elegant work and it was it's very complicated study and diona did a great job explaining all of the int intricacies of this regulation and so just a shout out to um who's going to be starting her own Lab at Dartmouth soon and to Stefania our awesome collaborator at Yale and so I highly recommend that you check out Dion's Journal Club to discuss this work okay so getting back to this issue of p27 and its regulation of hemogenic endothelium I just want to show you um a little bit of data from those studies um first um this is so these data come from endothelial cells that were isolated from yog sacks of either wild type or uh retin aldhy dehydrogenase knockout mice or littermates and we were looking at the cell cycle distribution of these yac endothelial cells at e8.5 when definitive hematopoesis is happening and so this is the distribution of endothelial cells in different cell cycle States in while type situation these are the ones that are um arrested and these are the ones actively cycling and when we look at the endothelial cells cell cycle distribution in the mutants you could see that there are less that are arrested and more actively cycling which was uh similar to what we found in the phosphohistone 3 standing that I showed you earlier um and so this impairment of cell cycle control was also associated with this significant suppression of the development of hemogenic endothelial cells and when we could express back p27 in the yolac endothelial cells and we could do this using um embryo culture and a lentiviral vector that expressed p27 back to Wild type levels um we were able to um restore cell cycle control in the endothelial cells which wasn't that um surprising but then we also were able to rescue hemogenic endothelial cell development just by controlling cell cycle with the expression of p27 and so this was extremely interesting to us and I I just want to show you this very simple diagram of cell cycle regulators um just to remind you what these proteins do um and so p21 and p27 are members of this Kip siip family along with P57 and they function to inhibit cdk2 or four and six in this G1 phase of cell cycle and um their activity prevents the cycl the cdks from um interacting with cycl proteins and other proteins not shown here and the collective function is to arrest cells in this G1 State and not allow them to um transition through this g1s restriction point so when p21 and p27 are active the cells remain in G1 they don't enter S phase where they would undergo DNA synthesis and eventually get into um M phase where they would undergo mitosis so they stay in this G1 State um and so we wanted to know well if p27 in cell cycle control is so important for hemogenic specification is the cell cycle different for these cells compared to endothelial cells that don't have any blood forming activity and so in order to get at this question uh we adopted a different Mouse model and these are the fuchi mice and they um Express two different uh fluorescent Fusion proteins uh um uh human part of the ginine protein which is fused to mvenus and the CDT protein which is fused to M cherry and so you can see in this time lapse image taking from the uh previous paper that when cells first divide neither of the reporters is expressed but then as cells remain in G1 phase there's an accumulation of the CDT protein um and the cell nuclei are red um and then if the cell's transition into s-phase the um CDT protein is quickly degraded although they are transiently yellow expressing both reporters and then once the cells are in s phas and all the way until they divide they they are then expressing um uh ginine driving M Venus and then the cells divide and then neither reporter is expressed again and so we uh isolated the AGM from these fuchi mice and we isolated the endothelial cells in this tissue and we used flowcytometry to figure out what is the distribution of these different cell cycle States in endothelial cells that are hemogenic versus those that are not and so this is uh uh fetometry based on the red fluorescence versus green fluoresence and when we do these studies we found indeed that the hemogenic endothelial cells there was a significantly higher proportion of these cells in G1 compared to non-blood forming hemogenic endothelial cells and this was really interesting to us because stem cell biologists have found that this phase of cell cycle uh G1 is really important for the commitment of embryonic stem cells into different lineages um so during this phase there are a lot of differences in the expression of transcription factors their accumulation changes in chromatin structure and other changes um that are not seen in the other cell cycle States and they also found that it's really important where in G1 phase the the cells are if they're early in G1 they're more responsive to signals that would induce mesoderm or endoderm differentiation and if they're later in this phase they're more responsive to ectodermal um deter perence and so this G1 phase is thought to be a commitment window enabling phenotypic changes and so what we found in our studies is G1 arrest is necessary for hemogenic specification but it's not sufficient so there are other factors extrinsic factors that are regulating um the endothelial cell phenotype just um specifically within this commitment window and so we're trying to figure out what are the factors that promote hemogenic specification when the endothelial cells are are arrested in this G1 State um so we're also trying to understand the role of G1 arrest in the specialization of other endothelial cell types and I could tell you that there is a significant role in arterial venus specification and um this work was presented by Nick our question moderator and he did a short talk for navbo uh just last week about this topic and so I'm not going to present his data again but um I'm sure he'd be happy to take questions about his work um and so for the rest of the talk I'm going to address some other questions that we had so we had learned a lot about the regulation of hemogenic specification in the mouse model system so we wanted to know if we could apply these insights to the generation of human hemogenic endothelial cells from either human es or IPS cells which would then could potentially serve as a source of human hematic stem and progenitor cells uh for clinical therapies if this process could be properly optimized um and so we were able to generate a while ago endothelial cells from both human es and IPS cells but the system that we had um put together was not very efficient and it required a murine feeder layer and the culture conditions were fairly undefined and uh that wasn't really a good way to look at molecular regulation and so we wanted to know if we can optimize a system now where we use human stem cells in a very defined system without a fe feeder layer and without any serum products so that we could understand um the regulation of hemogenic endothelial cell development and human endothelial cell development in these stem cell models so this work was taken on by um Jing my former postdoc who actually managed to graduate in May during lockdown um but she finished these studies before leaving which was great um and in her work she ended up adapting uh a stem cell culture system that was previously published from other labs and in this system human es cells or IPS cells are plated um in a monol layer on major gel and so with very defined factors and no murine feeder layer and there's no uh requirement for the formation of embryoid bodies as well and so in this culture system the es CS are first treated with gsk3 inhibitor which uh promotes the formation of primitive streak cells which you can see um in this culture system is a significant decline in plur potency markers um like socks 2 very early in development and then the Primitive streak development is um indicated by the expression of brachi or T and then um to the culture system is added basic fgf or fgf2 which promotes the formation of mesodermal progenitors and you can see that um this is reflected by the expression of hand which significantly increases during this time and then to promote the formation and the propagation of endothelial cells from the mesodermal progenitors um bmp4 and vfa are included and you could see that there is a significant increase in the expression of endothelial genes like cdh5 which is V cerin and there are also expression of other genes that are enriched in endothelial cells such as eb2 and fb4 but um we collectively when we look at the profile of these cells we don't have there's no clear mature signature of endothelial cells and they're really what we consider to be primordial or primitive endothelial cells and we can do this very efficiently in this system and generate about 85% of endothelial cells in this protocol and then we wanted to know if we provide retinoic acid to these primordial endothelial cells at this stage would we promote the formation of hemogenic endothelial cells and hematopoetic cells and so um interestingly and luckily we did see that there was this increase in the expression of rung SW which I mentioned earlier is the critical regulator of hematopoetic stem and progenitor cell development um and Jing was also noticing that there are blood cells that are being generated and um and and she could notice these in the supernate of the cultures and so she isolated the cells that were forming in this culture system and she did cytospins and gim of staining to show uh by morphology the different types of hematic cells that are formed and then she could also gather back the adherent cells and the cells in the superan and stain them with antibodies that are specific for different hematopoetic lineages and then we can quantify the proportion of the different type of hematopoetic cells that are forming in this system um and so I just want to say up front that we did not make a huge effort to try and characterize all of the hematopoetic cells that formed in this system what we are really focused on was defining the phenotype of the cells that were generating the hematopoetic progenitors and so in these studies what we did was we then isolated all of the cells that formed that were cd45 negative and so we wanted to get rid of all of the cells expressing cd45 that were already committed to a hematopoetic lineage and when we did this and we looked at the um hematopoetic potential of cells that were cd45 negative in these methyl cellulose based assays that I mentioned earlier we could see the generation of different types of hematopoetic colonies rytes myoid um myoid cells um and also these uh multi-lineage hematic colonies including Gran sles orther sites monite monocytes and macres um and so we wanted to Ser to further fractionate the cd45 negative cells and see if we could further Define and enrich for blood forming activity and so uh from these cd45 negative cells we then further fractionated them into um cells expressing a variety of cell surface proteins that are seemed to be enriched in either endothelial cells or hematop putic cells and what we found is that those that are um coexpressing cd31 and are that are negative for cd45 that's where there was um a high enrichment in this Blood forming activity which is very encouraging to us because cd31 positive c45 negative is a flow cytometry definition of endothelial cells and so we wanted to further fractionate this population and see if we can then further enrich for blood forming activity and so ultimately I'm not going to go through all the data but ultimately we found that we could um achieve an 82 fold enrichment by adding additional markers so uh VF receptor 2 or flick 1 kdr and the human system ckit cd34 and the absence of V catarin and cd45 and so um this is our working definition of human hemogenic endothelial cells u in this system and we can isolate these cells and plate them in um on a single cell level into these colon Colony forming assays and show that they can give rise to all of these different kinds of hematopoetic progenitors and so with this definition in hand then we wanted to know if retinoic acid is really promoting their development in our inv vitro system and so we did a similar uh culture as say as I showed you earlier where the cells at day five were either kept in control conditions or were treated with retinoic acid and you can see this uh significant enrichment in the formation of these hemogenic endothelial cells and then this is suppressed when we add an inhibitor of retinoic acid signaling um and so retinoic acid is promoting hemogenic endothal cell development but is it doing it in a manner that is um dependent on cell cycle control like we found was necessary in the mouse and so to get at this question we had to um adopt a new um embryonic stem cell line and this is the H9 line expressing this Fuji reporter so it's the same reporter that I showed you that is expressed in the mice um where we can determine by flow cytometry the cells in these distinct cell cycle States and so Jing used these es cells and subjected them to the same culture conditions that you previously defined and then we could identify which cells are in what cell cycle State and when we compare the hemogenic endothelial cells to endothelial cells that are forming but do not have blood forming potential we could see that there's a significant enrichment of hemogenic endothelial cells that are in early and late G1 compared to non blood forming endothelial cells and then a significant decrease in those that are transitioning into sphase and under undergoing active cycling um and so the next question was well if these cells really are in a different cell cycle state if they're enriched in G1 like they are in Vivo in the mouse is retinoic acid functioning to promote G1 arrest of these es derived human es derived endothelial cells and this and is this necessary for their chemogenic specification um and so we did these studies where we treated again the retinoic the cells either in control conditions with retinoic acid or with a retinoic acid inhibitor and we find that indeed retinoic acid treatment is um in is causing an arrest in early G1 State um of the endothelial cells and interestingly this is not the case for this late G1 State um and there's a significant decrease in these cells that are transitioning um from G1 into S phase and then we saw this interesting increase in cells in sg2 and we're still trying to figure it out but we were very interested in this role of retinoic acid in promoting early G1 state in the endothelial cells so we wanted to know if this is necessary and or sufficient for this retinoic acid induction of hematopoetic specification um and so we did these studies where we again cultured the cells and the same culture system but this time at day five they were treated not just with retinoic acid but also with a cdk inhibitor so this is a cdk46 inhibitor that causes cells to accumulate in late G1 State and so in this case we are sort of forcing the cells to not be in early G1 but to accumulate in late G1 State and when we do this and then we treat these cells that are now in late G1 one more so than early G1 we see we lose this effect of retinoic acid and no longer is retinoic acid allowed to U or or enable to uh promote this hemogenic endothelial cell development so we concluded from these studies that this early G1 state is necessary for this hemogenic endothelial cell formation so then the next question is is G1 State actually sufficient for this retinoic acid induced hemogenic specification so in these studies we again use the CD K 46 inhibitor to cause cells to accumulate in this late G1 State and then we also used a cdk2 inhibitor which cause the cells to accumulate in early G1 relative to late G1 although not to the same extent as retinoic acid treatment and so when we do this and we look at hematopoetic um hemogenic endothel cell development in the absence of retinoic acid we see that neither um early G1 or late G1 state is sufficient for the per otion of hemogenic endothelial cell formation so we know from these studies that this early G1 state is necessary but it's not sufficient for hemogenic specification which is similar to what we were finding in our Mouse studies and so we're still trying to understand um what are the factors Downstream of retinoic acid that are um causing transcriptional activity um to change the endothelial Sate toward a Mantic transition and so this is our working model where we think right now acid is doing two things first it's controlling cell cycle and it's promoting an early G1 U state which we think is a permissive signal setting up a unique window of opportunity for self- signaling but we also think that retinoic acid is um changing the transcriptional activity um of genes that are necessary for this transition from endothelial to hematopoetic cells and so this is where we are now we're still trying to pick apart the molecular mechanism Ms um and we've done a bunch of omic analysis where uh we have some nice leads so uh that is it for my seminar I just want to take a moment to thank everybody in my lab um and particular Nick who is helping out today um and my lab at Yale this is Jing who managed to graduate during a pandemic and yinu who was helping out with the studies that were done in collaboration with diona in stefania's lab um and then uh of course thank you for funding to be able to do this and hopefully we'll be able to get again get together again soon postco okay thank you so much Karen um at this point I'll ask a question while we gather more questions so I was wondering um how much evidence do you have of the re retinoic acid effect being endothelial cell autonomous is it possible that is also acting on other cell types that are contributing to the endothelial suell Fate specific um oh let me put my camera back on okay good to see you I'm not very technically U proficient but um so yeah that's a that's a good question um and we showed in the mouse that it really is a cell autonomous effect and we could do that using retinoic acid uh signaling um Mouse lines so that we could see exactly where the signaling is happening so we know in the murine system it is cell autonomous and we um trying to figure that out in the stem cell model uh we think it is because if we isolate endothelial cells and treat them with retinoic acid we see similar effects but it doesn't rule out the possibility that retinoic acid might be uh changing the micro environment to some extent in the culture system thank you so much so at this point I'm gonna let Nick handle the questions thank you yeah great so we have um a few questions already so the first question comes from Kiri um he has it kind of early so I think it might be uh based on kind of the earlier uh slides but he asked um the reduced expression of cdk Inhibitors is interesting and makes sense why endothelial cells are hyper proliferative um is this phenomena independent of VF and vegfr2 signaling um yeah that's a a good question um so we didn't look for interactions with the ve signaling pathway specifically um in the mouse studies uh I think with so I can't speak to that I don't know if it changes the regulatory Network in the VF signaling pathway um and so I guess I shouldn't really um speculate about that but thank you for the question great okay so then the next question uh comes from Jesus Gomez um and question is a great talk impressive results have you tried to transplant the cd45 population so we done any transplant popul uh experiments I assume he means the ones that are generated from the ESL system think so yeah so we didn't we purposely didn't go down that road um I think in order to um generate hematopoetic stem cells that were transplantable our system certainly would need to be further optimized and other labs that have tried to do that specifically have shown that you know there are additional steps that are necessary once you have hemogenic endothelium and and they have blood forming potential in order to truly optimize the formation of transplantable cells requires additional steps and so we didn't really go down that road uh because we were specifically looking at the early specification events and it's and their and its regulation great um so another another question um from and I apologize if I mispronounced this uh bronos patski um the question is so great talk um which Notch genes and Notch ligans are first expressed in hemogenic endothelium so we know that um at least in the specification events that it it's Notch one is the receptor that's important Notch four is also present but it seems not to be playing a role from what we could tell and then um the lians uh we are not positively sure but I would assume it's dll4 which has been shown by other groups more specifically so in our studies we we didn't dissect the specific receptor liand um interactions we know for sure it's Notch one and we think it's dll4 um but I can't Ru out a rooll for Jag one as well great okay next question um from Leah Yin uh the question is great talk Dr Hershey um how about the effect of retinoic acid on Cell metabolism um is that related to the cell cycle thank you yes okay that's a great question so we don't know the answer but we're extremely interested in that and so the whole idea um that the connection between metabolic State and cell cycle state is really interesting and we're very interested in understanding how there's Cooperative uh regulation there we we don't know the answer but we um hopefully will soon yes hopefully um so the next question is uh from Vicky vouch um as uh Karen great talk um putting together this data with Nicks thank you um putting together this with my data uh did the heem have arterial venous characteristics in culture um and does early G1 allow for Venus uh oh sorry allow for Venus fate mesh somehow with C capacity yeah that's a great question so in Nick's studies well Nick could probably talk more about this himself but so we found that um Venus endothelial cell specification seems to be happening in early G1 also um and this is different from arterial specification which seems to be happening in late G1 and so in the hemogenic endothelial cells that we isolate in culture we did not see um arterial venus gene expression enriched we um we didn't really see those phenotypes at at that time I think it's a good question we haven't really addressed it specifically I mean now that we know early G1 is important for this process for hemogenic specification and Venus specification we have to figure it out better um and so hopefully we'll be able to do that we just don't have all the answers yet but thank youy hey all right so next question um from uh uh Patricia deori yes what is the source of the retinoic acid um the source of the retinoic acid in the embryo uh yeah I think so okay so we know that the enzyme that makes retinoic acid retin alide dehydrogenase too is expressed in the endoderm layer of the yolac also the endoderm in the embryo proper and so that is um where retinoic acid is formed the during embryonic development the vitamin A or the retinol the precursor to retinoic acid is um provided through maternal circulation so I hope that that's my postto advisor so I hope I answer that right probably um so then uh uh oh actually a quick follow-up question um because it that's just came in uh um oh sorry she says thanks oh is that retinoic acid acting on other cell types simultaneously um I think they mean during development yes this is a just a followup question um okay so we know in development we have retinoic acid signaling mice so the retinoic acid response element that is linked to Lac Z and so we could use these mice to determine exactly where retinoic acid signaling is happening in the mouse during this process and we see that the signaling is in the endothelium and it's not in other surrounding cell types so in our invitro culture system um we think also that it's the endothelial cells that it's affecting endothelial cells in a Cell autonomous way because we can isolate endothelial cells and show similar induction of the hemogenic phenotype in the absence of other cell types but we can't in that system we can't really rule out um that retinoic acid has somehow influenced the other fraction of cells which is very small but still there that are surrounding the endothelium so we have to do further studies to show cell um autonomous effects in vitro but we know for sure that it's a cell autonomous effect in Vivo great okay so moving on to the next question this is from um again I'm sorry Harish uh petti jnan and the question is what is the time scale in which the end of IAL cell transitions from early G1 to late G1 yeah that's a good question and so um again we don't know you guys are having great questions and we don't know all these answers but um we we'll try to get at that using um in time-lapse Imaging um in Vivo and we hope to do that um soon and be able to address that question um and so the it's interesting that the G1 state is the is the uh phase of cell cycle that is the most different in different cell types so um S phase and M phase tend to be typically the same duration but G1 and sometimes G2 are the uh states that are most variable so I think uh what we find will be endothal specific if we're looking specifically at endial cells and it could be different for other cell types yeah okay so next question is from Sha uh Leu um who asks uh how end uh how do endothelial cells to hemogenic cell TR uh sorry how EC to he hemogenic stem cell transition specification events um get shut down during later embryonic stages that's a great question too so um again we don't really know but it is very interesting that hemogenic specification occurs in different tissues during distinct Windows of development so I had mentioned that Dion's work in Stefan's lab has revealed you know some factors at least one factor that limits hemogenic specification uh to control the number of hemogenic endothelial cells that form so there there have to be other Regulators that completely shut the program down so that this processes doesn't go out of control and we end up with too many hoptic stem and progenitor cells which we know if that happens during development it causes postnatal hoptic defects so we're I'm not sure what what shuts it down but um I know that that's a really important process okay so the next question another question from Leah Yin um who asks how about the effect of cell cycle difference on the maturation of endothelial cells yeah okay so that's a good question too and so um you know initially we wanted to know is cell cycle playing a role in the initial specification of uh event or the maintenance of the phenotype and so Nick could probably talk to this more um we've looked at this more in the arterial venus model that Nick has been working on and found that even in adult tissues the same distribution occurs where Venus are and early G1 and arterial Endo cells are in late G1 and that's maintained so um we that that might be part of maintaining the phenotype of the endothelium in these distinct vessel types um but again we're not entirely sure What mechanisms are are at play great oh and I thought we had one more but I think it was H shal Le clarifying that um asking about yok sack and amm getting shut down um but I think that's I think you answered that good okay good then I think um that's all the questions we have from the panelists or sorry from from the attendees all right thank you so much thank you Nick for moderating the questions thank you Karen for your wonderful talk if anyone still has a question that they didn't get to ask you can send it to info@ na.org and we'll make sure that um Dr hery gets it um I think I see we have a raced hand um one second Ma pulet has a raised hand if you want to unmute yourself and ask the question live Mathilda would you like to ask your question live I saw that she unmuted maybe she would maybe she clicked it by mistake anyone thank you everyone um for being here I hope this was beneficial uh these webinars are brought to you by the naval education committee our next webinar as you can see in um um Karen's screen uh will be on Thursday May May 6th um entitled regulation of endothelial cell mural cell interaction during development by Amber stratman and um look for more information at the nabo news be um as usual please feel the valuation form at the end of the webinar when you receive it in a subsequent email and let us know any topics or speakers that you may want to see in a future um navo webinar thank you so much for being with us today thank you Nick uh for that awesome handling of the questions thank you to the audience for the great questions and thank you Karen for all the great science um everyone have a wonderful day thank you very much hope to see you all soon