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Meet the Scientist: My Path to a Career Studying U ...
Meet the Scientist: My Path to a Career Studying U ...
Meet the Scientist: My Path to a Career Studying Uterine Development and Function
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And so it's my honor to introduce our Meet the Scientist speaker today, Dr. Francesco DeMeo. He'll be introducing himself via his slides because he's gonna be telling us about his career path, but I can just briefly introduce him. So he's currently a senior principal investigator and he's the chief of the Reproductive and Developmental Biology Lab at the National Institute of Health Sciences in Research Triangle Park in North Carolina. A couple noteworthy items about Dr. DeMeo. In 2019 he was elected to the American Association for the Advancements of Sciences. He's a fellow in biological sciences and he'll be talking to us today about his path to a career studying uterine development and function. This is designed to be an interactive session so Dr. DeMeo will present for about half of the time and the rest of it is wide open for you to ask anything and everything of him and I hope you'll learn during that Q&A session that he is very candid and straightforward and he'll tell you exactly as he sees it. We also have online audience and I'll be reading those questions as they as they come in. So Dr. DeMeo, thanks for agreeing to do this and we look forward to learning about your career. So I'd like to thank the organizers of the committee to of the meeting to invite me to talk about my career. I'm not dead yet and I guess it's the Monty Python goes. So this is for the group of people here. We put the us in uterus, right guys? So I'll tell you how I got into a career in uterine biology. I work for the government so I definitely have nothing to disclose. Do that every year. So I've got my so like Lee before me we both graduated Cornell University as undergraduates although a little bit different times. I then went to Michigan State and I at Cornell I really got enjoyed the reproductive biologists in Cornell in the late 70s. It was Bob Foote, Bill Hansel, Harry Van Tienhoven, Bill Wimsatt really got me turned on to reproductive biology the science and then I went to Michigan State where I spent some time working on fertilization and the cryopreservation in non-human primates and hamsters and then I joined the laboratory of David Bullock in the 1983 and the goal was to study the hormonal regulation of a hormonal regulation of a million genes. So the 80s were hormonal regulation of a million gene. 90s was knocking out progesterone receptor. 2000s was what does PR do? Identify a system to look at uterine gene expression, map the progesterone related pathways, identify the roles of the receptor and uterine receptivity and then identification of the role of PR and parturition. So we moved through the uterus with PR as our major a major tool. So when I joined the laboratory of David Bullock back in 83 there was really no good cell line to study hormone regulation of a mammalian gene. Most of the work was done by a lot of people on ovalbumin and so we wanted to set up a way to look at the role of uteroglobin's regulation by progesterone in vivo and at that time there was very few labs doing transgenic work and I think I came as a postdoc. I gave me a couple of sheets of yellow paper telling me well this is the protocol we have set it up. So I did that and it was actually a lot of gray hairs but it was a lot of fun when it finally worked. So uteroglobin is abundant protein in the rabbit uterus. It's expressed at the time of receptivity. It was originally called blastokinin. It's got a ton of names over the year before they before they actually clone the gene. It's regulated by progesterone. It's also regulated by glucocorticoids in the lung and our goal was to identify the elements responsible for hormonal regulation of the rabbit uteroglobin gene in the uterus and we're going to do this by establishing a transgenic mouse approach. So this is this is how old this is. This is from a Malindo paper and it's overexposed because we could barely see expression of the rabbit uteroglobin gene in the uterus. We see it mostly in the lung but with that one mouse that expressed in the uterus we could show that it was hormonally regulated. So that was a victory but what it told us was is we really couldn't do anything because this was you know two lines of mice and they didn't express it very well and it turns out that uteroglobin is actually you know I was in an exile from uterus because uteroglobin is actually a cell-specific gene in the lung. It's expressed specifically in club cells. So I went into the desert and studied lung biology for about 10 years looking at the you know cell differentiation along lung cancer models and such and then we've got back to the uterine biology in collaboration with John Lyde and Orla Connealy and Bert O'Malley when since I was doing most of the I graduated or you know got demoted to running a genetically engineered mouse core and we will knocked out the progesterone receptor. So we all know what progesterone receptor is. It was the receptor with you know it binds progesterone. There are two isoforms the B isoform and the A isoform. It was you know the chicken progesterone receptor was cloned by Shambone and O'Malley within a month of each other was published and then the gene we knocked out the gene. So historically this was the targeting construct to knock out the progesterone receptor. We had gotten a BALB-C clone from Shyamala and I think she's a member of the society and she's actually the mother you know was the mother of our vice president. She gave us a BALB-C clone which really was difficult to target so John went and cloned the 129 clone and we knocked out the progesterone receptor and we found that you know every aspect of female reproduction was impaired by progesterone but with respect to the uterus there was no implantation, no decision response, and the ability to inhibit estrogens regulation of growth was impaired. And then we went into the desert again because what do you do with this? So I think Orla's lab since there's two isoforms she mutated the methionine for the B or the methionine for the A and made a PRB only or a PRA only mouse and basically in the mouse PRA uterus explains everything. It's all PRA and if you look at the expression pattern PRA is the dominant isoform in the mouse uterus unlike the human uterus. So you know we still where do you go with this study? So the goal was then well you know we knew that there was some PR targets but we really wanted to know what the PR targets were. So again this was back in the day when you did a spotted array. So I found a thousand dollars and I went and I got a spotted array and I probed it with labeled RNA from the perco and the you know petri-related perco and wild type and I found this one there aren't many different genes but I found this one spot which turned out to be Indian hedgehog. So that was exciting and a hedgehog was expressed in the uterine epithelium at both the RNA level and the protein level and this is work that was done by Nuryo Takamoto in collaboration you know he was in a lab which was right next door to mine. And so we looked at what are downstream targets of hedgehog and patched hip and coop TF2 were all expressed in the stroma. So we had known from being very close labs with the psi lab that in the spinal cord sonic hedgehog regulated coop TF2 from epithelial to stroma and we hypothesized that Indian hedgehog regulated coop in the uterine stroma in a similar way. And so we came up with this pathway that progesterone receptor regulates hedgehog and then you get activation of coop TF2 in the stroma. So again what do you do with this data it's all descriptive it means nothing it's just you know describing it and there were no good uterine models to knock genes out. In fact the uterine field was lagged behind the mammary field and a lot of other reproductive fields because you could not go and do conditional knockouts in the uterus. So and this is work by Selma Soil, Atish Mukherjee, Rodrigo Valdivia and all my work is done in collaboration with John Light. So John and I sat around so well let's see if we can what if we target something to the PR locus can we see something. So initially we knocked in LACC into the progesterone receptor locus and wow the uterus turned really blue. So he said we got something. So the next thing we did was we knocked CRE into the progesterone receptor locus and you can see that it went across to this reporter mice we got really good ablation in the uterus. We also got ablation and other reproductive tissues but being creative we could actually you know you make a lot of mileage by isolating the uterus and studying the effect in the uterus and a lot of people have gone and used the PR CRE since then. So then we want to map receptor pathways used to transcriptomics knockouts in mice, uterine specific knockouts or uterine knockouts and knockdowns in human embryonic not human endometrial stromal cells. And the critical thing for our research was not to only work in the mouse but to be able to get a hold of human samples, primary tissue, primary cell lines and then actually ask what are the pathways that we find in the mice are they the same in the human and only focus on clinically relevant pathways. I think that was the key. And so we came up with this pathway that hedgehog binds its receptor in the stroma, activates COOP and then it triggers decidualization. The Bajis who actually got their origin in post-docs at Baylor College of Medicine found that a PR target was HAN2 and HAN2 regulated FGFs which inhibited the proliferation of the epithelium. So that pathway was mapped and then so our you know nice little animation pathway we have progesterone from the blood binds its receptor, we get activation of hedgehog, it binds its receptor activates smooth in, we get activation of COOP which then turns activates EGFR, we get open up receptivity the embryo implants and then we get activation of BMP2, WN4 and decidualization in stromal cells. So this was great. So now we want to you know most people thought that you know progesterone binds its response I'm going to regulate gene expression but we thought there has to be more than that. So we went and we conducted a lot of knockouts and chip-seek and we found that two transcription factors which were targeted progesterone receptor GATA2 and SOX17 bound all in a peak about 19 KB 5' to the hedgehog gene. And basically when we looked at what other factors may potentially bind in this peak it was basically the red carpet of uterine specific transcription, uterine transcription factors. So we thought this peak was something so how do we ask this question. So we used CRISPR to knock out this peak and when we knocked out this peak we showed we got reduction in the hedgehog and severely reduced fertility in the mice. So that was a win. So then so we now have you know this enhancement may give some light to an epithelial code. We can use this to target this type of enhancer to target gene therapy for uterus and maybe be able to start working on making uterine cells from iPS cells by activating these transcription factors. So the isoforms was really depressing because we knocked out A and it was a perco, we knocked out B, it didn't do anything in the uterus. But it was known that from Bruce Leslie that loss of progesterone receptor at the window of receptivity was critical but nobody proved that that was the case. And so we asked could we express progesterone receptor through the window. So a very talented graduate student Margo Wendorf went and we made a pre-activatable PRA or PRB you know it knocked into the Rosa locus and when we overexpressed PRA or PRB in the uterine epithelium through the window of receptivity the mice was sterile. And so we came and do a lot of molecular biology I'm not going to bore you with we came up with a new model of receptivity. You have FOXO1 in the cytoplasm progesterone receptor in the epithelium. Again we get hedgehog it comes down activates the stroma but then PR goes away from the epithelium and FOXO1 goes into the epithelium. So there's a mutual exclusivity between PR and FOXO1 in the uterine epithelial nuclei at the window of receptivity. And then the embryo can implant and you get the sigilization. So and then we looked at human samples and we found that at receptivity in humans FOXO1 goes into the nucleus and PR is gone. So this is a very simple marker for receptivity in humans and it'd be really curious to see if anybody picks up on this as a diagnostic tool. But we came up with this yin and yang of progesterone receptor and FOXO1 being mutually exclusive for you in receptivity. Serendipitously we found that when the mice expressed PR in the corpus luteum we ended up getting ovarian cancer and this was regulated through the AKT pathway and that was a sighting showing that PR can actually promote cancer development in an ovarian lineage. So finally Sam, well second to last, Sam Asciano showed that in humans there's a difference between PRA and PRB and that at labor you get an increase in PR, PRA over PRB. And he did a lot of work showing that PRAs tends to promote parturition and PRB tends to inhibit parturition. So we overexpressed PRA and PRB in the myometrium and we found that PRA increased contractility, PRB decreased contractility and that when you express PRB in the myometrium you end up getting a delay in parturition, uterine stenosis and decreased motility of that. So that was actually a cool finding looking at the role of PRB in identifying specific targets. So finally I think anybody went to Carmen's talk has already heard this. The final thing is you know we've done RNA-seq, we've done single-cell-seq, but the future of uterine biology is to do spatial transcriptomics where you have slides with individual dots at about 55 microns that will actually barcode the RNA from 5 to 10 cells. And we look at this as tagging a uterine micro-environment. You know it's not a single-cell thing, it's a micro-environment thing. So we did the day 3.5, our favorite time point, the pre-receptive thing where you have lumal epithelium, glandular epithelium, and myometrium. And this approach was totally not sensitive enough to detect differences between the, I mean basically told us myometrium, it told us endometrium, and you know basically not good. But we then looked at day 6.5 pregnancy and day 6.5 pregnancy of the embryo, you have the primary decidual zone, secondary decidual zone, you have glands, you have myometrium, you've got you know vascular sinus, you've got a mesometrial decidua, and isn't that nice? That's what the single-cell, that's what the spatial transcript told us. It identified all of these including a transition zone between the primary and secondary decidual zone. So using this approach when you have enough of a system you can identify the micro-environments. And what's nice about this is using cell chat you can identify potential communications between the different compartments. So the future I think is to improve the tools to investigate hormonal regulation of human biology. I mean there's organoids out there, we've just published a paper in cells that shows that organoids are not really good for looking at progesterone regulation and gene expression. I mean they'll say they respond to progesterone but these are target genes that really I don't know what the physiological significance is. So we've done that. You know engineer or engineer or and create tools where you can exploit gene editing tools to do that. You always must integrate mouse models with primary culture patient data to better define the mechanisms. I think something really exciting now is you can make myometrial stromal cells and epithelial organoids for menstrual tissue. So it makes collecting those samples much easier. And overall you want to understand the impact on you know especially now the impact of uterine biology and hormone regulation on health disparities which uterine biology is a lot of health disparities and also look at the effect of climate change on urine function. So I think the moral of my career is you know you have sometimes you have to you know you can't just wait for somebody to develop the tools. You have to go and develop the tools yourself. I think science nowadays is too kit oriented and it really reduces creativity. The best creativity is when you identify a problem and you come up with a way to develop the tools to do that. You know I've done that by generating transgenic models, by generating knockout models, knock-in models. So we're really happy with the things are going. So I think uterine biology is an exciting field. I think uterine biology is uterus is the one organ where stromal epithelial communication is key. I think more than any other organ system in the body. So I think for all you that want all you all that want to study uterine biology I think it would be a great a great field to go into. That's it for me. So we'll open up the interactive part of this afternoon's session and so Dr. DeMaio is here to answer any and all questions that you have about science, career development, lab, that sort of thing. We also have an online audience and you're welcome to type in type in questions. Maybe I'll get things kicked off. So you're you're at a government agency now but you used to be in a university lab. Can you comment to the audience about how your job has changed? What's similar different working in the NIH environment versus the Baylor environment? So I think they're both extremely good. So I keep the people at Baylor and keep the people at NIH happy by saying that. But I think that the execution of research is pretty similar in both. It's just that in academia you have to scramble to keep everything funded and you end up doing multiple. For a while there I had a cancer grant, a uterine grant, liver grant, a lung grant just to keep it funded. You end up robbing Peter to save Paul to keep things going. Moving to the government allowed me to focus specifically on the uterus which was a real pleasure for me. The nice thing about Baylor was a highly competitive institution. A lot of great ideas, a lot of people doing different things. So I think that environment was a little bit more challenging than the government environment. But now with Zoom we've been able to keep that communication going. And the one thing I've done over the pandemic is we've actually established a uterine workshop. It runs from September till May. Everybody is invited to send me an email. I'll send them a link. We have about 77 people throughout the country participating with different speakers. And the whole point of that workshop is to have trainees present for 40 minutes max and then open it up for 20 minutes of questions. And sometimes the questions mutate to just old-timers talking about the problems they've had or trainees talking about techniques that didn't work or what worked. And it's been really productive. So anybody that wants to do that. And we kept on shooting for a hundred but we haven't had a fixed time with people jumping on and off. We haven't been able to do that. But we'll probably start it up in September again and try to get people doing that. The nice thing about it being NIH is we have a reproductive group that I think is outstanding. We have Carmen Williams Humphrey, Carlos Guardi and Marcos Morgan. I ran a center. I was a co-director of a center at Baylor for quite a while. And really they kept on changing the rules in the center and focusing the research. And the CIPA centers kept on mutating and it kept on getting harder and harder to get it funded. And if you look at the reproductive group at Baylor and stuff, it's pretty much been scattered. And I think Marty Matsyk is doing a really good job of trying to rebuild it. But now at the NIHS, I have a really good reproductive group that doesn't do the same thing, which the centers end up being a program project grants, which was terrible. They end up being diverse with different mechanisms, different tissues. So we really get a lot from it. So the message I want to take to people is, how many people here want to study the uterus? Or you just hear from my jokes? I mean for those people, whatever tissue you're studying, don't just know everything about your tissue. Because you're not going to learn anything new. Learn everything about everything and adapt from other systems to your tissue. I mean hedgehog, how do we come up with hedgehog? Well not because we're geniuses, but because the science had already shown that hedgehog regulated coops. So we knew from that and we just looked at that. How do we know about all these other transcription factors and what they do? What are the mechanisms by understanding other tissues? I mean when I was on graduate student committees that was studying something in the mammogram or something, we'd ask them what else is known about this? And they would only know their specific tissue. And I think that's, you end up discovering something that somebody already knows in another tissue. So I really think it's critical for you all to really not just limit yourself to the organ you're studying, but try to be a renaissance investigator and know what the biology is in general about what you're studying and then apply it to the uterus. And so we have an online question that kind of is in a similar vein and that is, did you start out wanting to study uterine biology and if not what made you passionate about this subject? Well, I started wanting to study uterine biology by doing a postdoc on uteroglobin in the uterus. I was not a uterine biologist, but I thought that would be a good starting point, but we ran into so many milestones. First of all, it took us a couple of years to set up the transgenic system, because back at the time, we had like seven labs doing it, and it was like a top secret, and you basically had to figure it out for yourself. And then we finally got it going. It was depressing. Uteroglobin is actually a claricell-10-gated protein or whatever. I was a reproductive biologist at heart, and I found it really intriguing, but not really what I wanted to do in studying the lung. And then we jumped on a collaboration with BERT to knock out the progesterone receptor, and that really opened it up. But again, it wasn't like the floodgates opened once we knocked out progesterone receptor. We had to keep on taking chances. I mean, we had to come up with 1,000 bucks for that 1.1 kb array, and that wasn't easy at the time. And then we got this gene, but what do you do? How do you knock it out? There were no good knockout models, and then we said, well, let's take a chance, and let's knock something. And the amazing thing is, you write a grant to say you're going to knock CRE into the progesterone receptor locus, and all the people on the study section, or I don't know who it was, but people on the study said, well, this is, you're going to knock out progesterone. It doesn't work. It's not going to work. It's not going to work. It's going to be expressed everywhere, whatever. Now, I wonder how many people that criticized those grants back then have actually requested the PR CRE mouse since then. It would be really nice to know that. So, the other secret is, is when you make a tool, people are going to tell you it's not going to work, but the minute it works, everybody wants it, and that's reassuring. So, and then always don't be satisfied with the tools you make, because they get overexposed. They get to be, you know, people, you really keep on making new tools. Keep on moving forward. I mean, the thing about making tools in mice is we made a lot of, in fact, this year, we made three tools that did not work, so we're very sad, but if we didn't try, we wouldn't know. So, you have to be creative, and, you know, what science used to be like. I mean, that's what turned me on about biology in the 70s was there was no, most of the factors weren't even isolated proteins. They were factors, and that meant that the proteins were unisolated, and there were all these bioassays, and people were coming up with creative ways to assay what it was. I mean, I remember when hibin was an estrogen contaminant of a follicular fluid, and then it turned out to be a clone protein. So, you have to be able to, you know, take chances, be inquisitive, and that's what turned me on about reproductive biology. Plus, it's the most important science we have. If it wasn't for reproductive biology, we wouldn't be here, and, you know, a lot of the health, women's health issues, you know, are centered around reproductive issues, and a lot of men's health issues, especially as they get older, are centered around their reproductive tract, too. I think we have a shy group. Come on up to the microphone if you have even a half-baked question. We can flesh it out. We have time. Please state your name and affiliation. Okay, my name is Asma Alknawi. I'm from Moffitt Cancer Center. I would like to ask about a barrier that have you faced during your career, and how you solved it. Thank you. A lot of the barriers were not having the tools, or not having the money to try to make the tools, and, you know, beg, borrow, and steal, take chances. I mean, that was the major barrier. Trying to get people interested in what you're doing, you know, to get support for doing it, and a lot of times, people are very interested in what they're studying, and really don't wanna take a chance, and that's when you go from begging, borrowing, to stealing to get things done. There's a lot of stories about how we made the, how John and I made the PRCree on the, you know, basically, SubRosa, but we got it made, and then, you know, getting some data to get it funded. I mean, that was the major obstacles we had. And plus, you know, people, you would hope that scientists would be open-minded, but sometimes scientists are closed-minded. If they think something's not gonna work, they will tell you it's not gonna work, and they will try to discourage you. I remember one faculty at Baylor told me, what do you wanna study, what do you wanna knock out progesterone receptor for? Just over-rectomize the mouse, and you don't have progesterone, and that'll tell you what progesterone receptor does. And I said, well, it'd be really hard to prove that progesterone receptor regulates ovulation if you over-rectomize the mouse, number one, and number two, it turned out that, yeah, we learned a lot from the Perco mouse, that, you know, and this person was very influential, you know, and it really delayed us being able to attack that for a couple of years and stuff, because this person kept on saying, no, no, it's not gonna work. And it wasn't any of the authors on the paper. It was another faculty whose opinions were very well-respected at Baylor. And then, you know, just taking chances. I mean, you know, I could be, you know, at Chick-fil-A right now making, you know, chicken clubs if some of these things didn't work, but it just worked out, so, but, you know, knowing biology and, you know, stacking the experiments in your favor by, you know, going at it, I mean, I think, what, I mean, the Battle of Waterloo was won on the playing fields of Eaton, you know, you don't go into it, the battle is won before, before, you know, you even pick up a weapon, and I think that's because you really have to do your homework before you take a chance to make sure everything is hedged in favor. And actually, the PR Lax-Z, which was never used except for the one paper, actually gave us the go-ahead to make the PR Cree, because it gave us the confidence that it would work. If we had made the PR Cree directly, that would have been a year or two to see if it worked, and we'd have been very nervous. I mean, I give Selma Soil credit, because when she was making, you know, in the old days before CRISPR, targeting constructs took about a year to make, they were very difficult. And we'd go down to Selma every day and go, how's it going, Selma, are you getting close? How many more steps you got? Are we ready to target and stuff? And so, it was, you know, you look back, I was actually at a dinner with Dory a while back, and O'Malley, and I said, wow, John and I look back on those days as some of the best days of our scientific career, because there's a lot of pressure, and we were working to do it, and it was really great. And I told my son, I said that, and he goes, Dad, back in those days, you weren't a happy person. So, in hindsight, it all looks like, you know, it was easy on a go, and maybe that's the beauty of memory, but it wasn't at the time. So, a lot of these tools and systems that we develop in our labs are developed by our trainees, and some projects that we can give them can be higher risk than others, and some of them can be lower risk than others. So, for some of these models where you know that there's a degree of risk involved, how do you decide to work with your trainees to assign those projects, and how has that changed or not as you've transitioned from a university environment to government? I mean, so in both cases, I vowed, after my first graduate student was working on a mouse model, never to have a graduate student work on making a mouse model. It's too high risk. I mean, for a postdoc that wants to make their name, that's the job. Except Margo Wendorf was a graduate student, and after I'd swore nobody would make a mouse model, Jay Wook Chung and I were sitting in the hall talking about, you know, we'd really like to make this lock, stop, locks thing, but we need somebody that knows how to sub-clone. And then Margo came walking down the hall, and she goes, Margo, do you know how to sub-clone? He goes, yeah, but I don't tell anybody about it because I'll be doing it for everybody. And Margo, you have a new thesis project, and she got quite a few papers, made the cover of Science Signaling and stuff with her mouse and stuff, so we're really happy about that. But, you know, making a new model, I'm at the age now where, you know, it's like a postdoc has to have a side project that pays off, and the mouse model has to be big. But remember, in the old days, to make a targeting vector, it took a full-time postdoc doing a lot of mini-preps and sub-cloning, and maybe even screening a library to do that. Nowadays, with CRISPR targeting, I mean, you know, we have a vitamin D project where we said, let's make a lock, stop, locks vitamin D project, and that idea came up in February, and now we're injecting ESLs now. So it's much faster, so you can make new tools now, given, and we have a great core, we have a gene editing and mouse models core with Artyom Gruzdev there, that really can, you just, Artyom, make me this mouse, and he will take out the CRISPR tools and come up with a targeting thing. So that really helped out. We had a great core at Baylor, which I ran, so that's why it was great, but it was only a mouse core, and we didn't have this genetic engineering core, so that's what makes it so great now. I mean, the bottom line for everybody is to take chances, and I mean, and put the us in uterus. So it was largely questions that you had driving the tools that you developed to answer those questions, so, and then you also touched on new technologies and how that's enabled you to, you know, maybe address some of those longstanding questions. What questions do you still have burning that you may have had burning from an early part of your career for which we still don't have the tools or may still not have the technologies? I mean, so we still don't have a good, in vitro, so there's so much you can do in vitro. You can use CRISPR-A, you can use CRISPR-I to really study regulation of genes, and with CRISPR, you know, capture, you can actually try to capture transcriptional complexes from the thing, but that depends upon a non-primary good endometrial cell line, and then we got, so there are stromal cell lines, but trust me, the stromal cell lines aren't the greatest in the world. Then there's, you know, the epithelial organoids, which aren't the greatest in the world, and so the biggest issue right now is an in vitro model to study hormone regulation of uterine epithelium and stroma that mimics the in vivo situation, and that's gonna require co-culture. I think Julie Kim at Northwestern is doing a great job with trying to establish co-culture models and microfluidics to do that, but if you look at the, and I think, because we want to look at how does a hormone or a bunch of transcription factors regulate gene expression, and how does the environment impact those complexes, so we've done, I mean, Ken's lab has done Hi-C on whole uterus, and we'd really, it lacks the resolution to look at chromatin structure in an individual cell type, so, you know, we've been trying to do Hi-C and look at, you know, knock out some things that regulate chromatin looping and stuff to really get a feel for how they regulate gene expression, and that's the biggest challenge right now is an in vitro model, and, because if we could do it, because the beauty now with collecting endometrial samples is you can collect them from menstrual tissue. In the old days, we had to get, you recruit patients for a biopsy and either isolate epithelium or stroma, but now you can recruit patients for, you know, and actually it's discarded tissue, so the IRB process is a little bit easier, where you collect menstrual fluid from them, you can then culture out epithelium and stroma, and our goal will be to culture out epithelium and stroma from the same patient, because they respond differently patient to patient, so when you want to put the co-culture together, you can have a patient that should be, have the similar background. Plus, the other thing is, is instead, you know, we can try to collect samples from an ethnically diverse group of patients, and then ask what are the, you know, there are a lot of health disparities in human biology, and try to come up with some mechanisms that way. So I think that is the biggest challenge. I mean, like any field, the mouse ruled the 90s and 2000s, but even if you look at cancer now, people are moving away from mice and going more to the human tissues, right? And in humans, you know, in cancer, it's patient-derived xenografts. I don't think we could do that in the uterus, and because they're not cancer, so it's not gonna grow as well as the xenograft thing. We want to study normal uterus. We don't want to study uterine cancer. So I think that is the major milestone we have to overcome, and I think it'll be overcome relatively shortly, because a lot of people are working on organoids. You will hear Tom Spencer talk about organoids, and I think for those people hanging around, the Tuesday morning sessions should be really enlightening about what's going on in uterine biology. So when you talk about this co-culture and use the word stroma, you know, that means many different things. So what would be your vision for the stroma in such a co-culture system? Well, I mean, so even if you look at the telomerase transform cell lines, we actually have a paper that's submitted now that compares the single cell of the primaries to the single cell of the transform, and they're different, but they still have the similar cell types, but different. So the secret is, you know, what is the stromal cell, you know, I mean, ideally, can we make uterine stromal cells from iPS cells, uterine epithelial cells from iPS cells? That was the whole point of doing the epithelial promoter analysis. So start from a stem cell and go forward, and so I think that that would be the way to go, because we have a paper we're getting ready to submit where we've identified a transcription factor where we knock it out and all the progesterone-expressing cells in the stroma are gone, but all the non, it's repopulated by a group of non-expressing progesterone receptor cells that still express the transcription factor, but you know, when you express, knock this gene out, PR cells die, and it's a whole different cell population. So I think, so what I meant to people is, especially junior people that are writing grants, I just, a former trainee gave me his grant to look at that didn't do well, well, it did, it got a score in study section, and basically it was overambitious and to lack of focus, and basically what the trainee did was took every cool tool, I called it a kitchen sink grant. He took every cool tool that he could think of. He had single cell, he had single cell ATAC-Seq, single cell RNA-Seq, he had spatial transcriptomics, and that was just the icing on the cake, and I told him, you know, these techniques are really cool, and I think, you know, in places that have a good, you know, genomics core, it's really easy, and with the government, it's really doable for us, but it's expensive, you have a lot of data, and if you really look at the single cell data, I think Carmen did a good job of identifying new stuff, but if you look at a lot of single cell data, they've identified the populations they already knew about, so what's the point, right? And the sequencing depth is so low that you're really not gonna get anything new, and I'd be very careful about making conclusions about cell specificity from single cell. I'm gonna, another story we had where we were looking at, somebody published a paper on single cell and identified a cell-specific thing, they knocked it out, the mouse was sterile, so he said, let's knock CRE into that locus, so we knocked CRE into that locus, and it was not just expressed in that cell type, it was expressed in others. They were just relying on the depth of the single cell, so caveat emptied with single cell, and so it's not so much to use every new fancy tool and be cool, the secret is to use the tool that's gonna ask the question you want, and don't go beyond that. You know, eventually, if you wanna use a new cool tool, eventually you'll come up with a question that will make you use spatial transcriptomics, but spatial transcriptomics is not that good for some things, right? You look at the small uterus, it's, you know, and then so I think last year when we had this result, we heard that, well, you know, 10X is gonna come out with a five micron bead thing, right? And that's sort of like waiting for the Yankees to win the World Series again. You know, they might do it this year, they might not. I mean, you know, it's been 10 years since they won the last one, more than 10 years, right? So you can't count on a company developing what you want, because, you know, maybe there's problems with that to begin with. We have about five minutes left if there are any other questions for Dr. DeMeo. I see Dory moving. I can say it from here. Yeah, I'll repeat your question so we can repeat it. So, you know, you've been wonderfully successful when you were at Baylor at getting funded, and so I'm wondering if you could give some advice on, you know, sort of the keys to doing that, and I'm talking on a global scale, you know, what elements you really think that the key ones to get grants funded. Okay, yeah, so Dory asked, you know, I've been successful when I was in the extramural world of getting funded, what was the keys? The key was, first, you know, going on a study section and finding out exactly what criteria the study section uses, because whatever you think goes on, unless you've been on a study section, you have no idea what, you know, what questions are they asking, what they look for. I think now the NICHD at least has a program where trainees can go, where junior faculty can go on and learn it. The secret to success was coming up with novel ideas that meet the need, and most importantly, get the grant. I mean, O'Malley, I asked O'Malley for his big, O'Malley used to have this top 10 list of how to get a grant, and I actually wrote him and said, could I get a copy of that, and it was a paper thing that's gone forever. But, you know, one of it was, you know, I know there's a deadline, but don't shoot for the deadline, get it done way early before the deadline. Get it sent out to people that don't know what you're doing. Do not give it to the people in your lab, don't give it to the people who are in your department. Get somebody that doesn't even know what you're studying to read it, because one thing I learned from being on a study section is you can have two people from two ends of the country that don't talk come up with the exact same flaws that sink a grant, which meant that if this was so obvious that two people caught it, the person writing it had they got it done in time and sent it out. So, you know, novel idea, health relevance, but the big thing was to make sure that it was done on time. The grants I never got renewed or had to send in a second time, I was, you know, basically the guy diffusing the bomb with the clock going off and getting ready to cut the wire. And then when I had to resubmit them or put in a grant where I had it done well in advance, I had people, critical people. So, if somebody, the first grant I ever wrote, I gave to somebody, I'm not going to mention his name, I gave to somebody to read and basically, oh, this is great, no problem, it's the worst score I ever got in my life, okay? When I gave it to another person who I cringed about giving it to, and basically, I mean, it was Sophia Tsai, basically just criticized it and I felt this big after I wrote it, I thought it was never going to get funded, I actually went back and addressed every question and I got it funded. So, you know, people that tell you good job, good job, you know, this is great, it should get funded, they're not helping you. The people that tell you, you know, you got a problem here, you got a problem there, you really have to address this. And then once you start explaining, when they ask you questions, you start explaining to them, you're wasting their time because they don't want you to explain it to them, they want you to explain it in the grant. So, whenever you write a grant, you know, you get a pink sheet and you have all these issues and you go, oh, wow, this study section was terrible, they just didn't get it, reviewer three didn't get it or whatever, and you blame the thing and you go away mad and you come back a couple years later, you look at the pink sheet, you look at what you wrote and you went, man, I should have never sent this in, you know, because I look at grant writing like Stockholm Syndrome, it's like you're captive to this deadline, you're there working on it, you're sweating on it, nothing's, you know, it's painful and then all of a sudden you were in love with it, it's like you're a master, this grant is the best thing I've ever written, every grant you submit, it's the best thing I've ever written, right? Because you're Stockholm Syndrome, you're under stress and you think about it. When you walk away from it and come back, you realize, you know, the strengths and the, even goes with papers, you may write a paper and think, oh, why didn't they accept this paper? And then you go back and you go, wow, I really didn't explain it that well, or, you know, your reviewers aren't out to get you, reviewers are out to review the science and the problem may not be with the reviewer not understanding, but with your ability to communicate what you want to the reviewer. So you really, I really have to say, again, the most important thing you all can work on is your writing skills, that is critical. And I don't mean English grammar, I mean that you give to an editor to do the grammar corrections. What I mean is, you have to be able to communicate your idea in a linear faction. You want to go from point A to point B, or to point Z, B, C, D, E, and F. If you start writing and you're going backwards to A, B, O, B again, and then C, and I'm going to jump ahead and go back, you're going to just confuse the reviewer. Remember, study section reviewers, they got other jobs to do, and they're going to read your grant, hopefully, before the deadline, but they got a whole bunch of grants to review, and if they can't understand what you're writing or can't make it clear, they're just not going to buy into it. So, I mean, I think that was the success. I mean, getting uterus and funding is tough. Trust me, it's not diabetes, it's not cancer. In fact, getting endometrial cancer funded for a while there was difficult because basically the study section people say, well, you know, nobody's ever died, very few people die of endometrial cancer, you know, just remove your uterus and you're cured. And then now with obesity and a whole bunch of other issues and saying stuff like that is not right, it's now a fundable field. So, you know, but uterine biology is extremely important for multiple reasons. I mean, the quality of a woman's life depends upon, in large part, the health of her uterus. I think I'm not going to get in trouble for saying that. Okay.
Video Summary
Dr. Francesco DeMeo, a senior principal investigator and the chief of the Reproductive and Developmental Biology Lab at the National Institute of Health Sciences in Research Triangle Park in North Carolina, presented on his career in uterine biology. He discussed his path to studying uterine development and function, starting with his undergraduate studies at Cornell University and then his time at Michigan State University where he worked on fertilization and cryopreservation. DeMeo went on to join the lab of David Bullock and focused on studying the hormonal regulation of genes in the uterus using transgenic mouse models. His research involved mapping progesterone-related pathways and identifying the roles of progesterone receptors in uterine receptivity and parturition. DeMeo also discussed his work with the Indian hedgehog protein and its role in uterine stromal development. He highlighted the challenges of working with limited cell lines and the need for better in vitro models of uterine biology. He emphasized the importance of creativity and taking chances in scientific research and encouraged researchers to look beyond their specific tissue of interest and consider the broader biology. DeMeo also emphasized the need for effective communication in grant writing and the importance of addressing critiques and seeking feedback from diverse perspectives.
Keywords
uterine biology
transgenic mouse models
progesterone receptors
Indian hedgehog protein
in vitro models
scientific research
grant writing
diverse perspectives
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