Exploring Revolutionary Advances in Embryonic Biology

SDB Ethel Browne Harvey Postdoctoral Seminar Series - Talia Hatkevich and Gerrald Lodewijk

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Summary

In this engaging seminar hosted by the Society for Developmental Biology, two postdoctoral researchers, Talia Hatkevich and Gerrald Lodewijk, elaborated on their groundbreaking research in developmental biology. Hatkevich shared her work on the development of male germ cells using mouse models, focusing on the mechanisms of DNA repair and epigenetic regulation. Meanwhile, Lodewijk presented his innovative approach with synthetic embryo models, utilizing CRISPR technology to explore embryonic cell organization and potential applications in understanding early pregnancy failures. Their presentations spark curiosity about the intricacies of germ line maintenance and embryonic structure formation, while showcasing the forefront of current research developments.

Highlights

Talia Hatkevich discusses her longstanding research on the regulation of male germ cells and the role of DND1 in preserving genetic information. 🐭
Hatkevich's exploration of DNA repair mechanisms is pivotal to understanding heritable genetic integrity in germ cells. 🧬
Gerrald Lodewijk introduces CRISPR-engineered embryo models, illustrating structural and functional similarities to natural embryos. 🏗️
Lodewijk's work emphasizes the potential of synthetic models in studying embryo development and addressing early pregnancy issues. 🤰

Key Takeaways

The seminar unveils cutting-edge research on male germ cell development and embryonic models. 🌱
Talia Hatkevich explores how the integrity of genetic information is maintained in male germ cells through unique DNA repair mechanisms. 🧬
Gerrald Lodewijk demonstrates the use of CRISPR technology to create reproducible embryonic stem cell models, paving the way for deeper insights into early embryonic development. 🔬
The session highlights the importance of meticulously regulated epigenetic and genetic factors in developmental biology. 📊

Overview

Talia Hatkevich's presentation delves into the meticulous process of male germ cell development, particularly focusing on the epigenetic and genetic mechanisms involved. Through her research, she highlights the significance of the protein DND1 in maintaining genetic fidelity across generations and the challenges faced in safeguarding the germ line during development.

Hatkevich also examines the role of DNA repair mechanisms adapted specifically for the germ line, probing into how male germ cells preserve genetic material despite constant epigenetic changes. Her findings have broad implications for understanding genetic inheritance and the potential in addressing fertility issues.

Gerrald Lodewijk shares his exploration of embryonic stem cell models, ingeniously utilizing CRISPR technology to emulate early embryonic development. His technique not only re-creates the structural organization of natural embryos but also opens new avenues for studying cellular interactions and imbalances that might lead to early pregnancy failures. His research proves significant in devising new methodologies for in vitro studies of embryogenesis.

Chapters

00:00 - 00:30: Introduction by Josh York Josh York introduces the Ethel Brown Harvey post-doctoral seminar series, mentioning his role as a moderator alongside Diana Fulmer. They will be highlighting research from post-doctoral members of SDB, featuring speakers Talia Hadkovich from Duke and Gerald Ludovik from UCSC. Each speaker will present for 20 minutes, followed by a 10-minute Q&A session.
00:30 - 01:00: Introduction of Dr. Talia Hatkevich Dr. Talia Hatkevich, with a PhD in genetics from UNCC Chapel Hill, has conducted significant research on meiotic processes using Drosophila. A published scientist in top-tier journals, she is now an NIH-funded postdoc at Duke, researching male germ cell development with mice.
01:00 - 01:30: Dr. Talia Hatkevich's Presentation Introduction The chapter introduces Dr. Talia Hatkevich as she begins her presentation. She is a postdoctoral researcher in Blanch Cable's lab at Duke University. Dr. Hatkevich thanks the audience for joining and expresses gratitude to Josh for the introduction.
01:30 - 02:00: The Purpose of Male Germ Cells The chapter titled 'The Purpose of Male Germ Cells' discusses one of the most fundamental events in biology: the passing of information from one generation to the next. This process allows for evolution, continuation of species, and even just passing on the family name. The information is transmitted via germ cells, which are the sex cells of animals that eventually develop into mature gametes, like sperm.
02:00 - 02:30: Development and Molecular Events in Male Germ Cells The chapter discusses the development and molecular processes involved in male germ cells, focusing on the genetic and epigenetic information that is passed through these cells. It tackles the question of how the integrity of this information is maintained, especially in the context of the mammalian male germ line.
02:30 - 03:00: Key Regulatory Mechanisms in Germ Cell Development The chapter discusses the key regulatory mechanisms in germ cell development, focusing on the male germ cells and their journey to establish the male germ line. It highlights that in male mice, this process begins at embryonic day 6.5 (E6.5) when the primordial germ cell is specified. The germ cell then needs to migrate to achieve its purpose.
03:00 - 04:00: Role of RNA Binding Protein DND1 The chapter discusses the role of the RNA-binding protein DND1 in embryonic development, specifically focusing on the process through which primordial germ cells (PGCs) migrate to the gonad by embryonic day 10 (E10). By E1.5, these cells begin to differentiate into male germ cells, also known as MGCs or prospermatogonia. The chapter emphasizes the process of proliferation and the eventual cell cycle exit of these male germ cells (MGCs).
04:00 - 05:00: Post-Transcriptional Regulation by DND1 The chapter traces the development of spermogonial stem cells (SSCs) starting from embryonic day 14.5, during which they are in a G0/G1 mitotic arrest. This state persists for about 7 days. On post-natal day one (P1), these cells re-enter the cell cycle. SSCs are highlighted as being particularly significant or successful cells.
05:00 - 06:30: Research Findings on Set DB1 Regulation The chapter focuses on Research findings related to Set DB1 regulation and its impact on spermatogonial stem cells (SSC's). SSC's are crucial as they represent the stem cell population within the testis capable of both differentiation and self-renewal. This property allows males to continuously produce the progenitors of sperm throughout their reproductive lifespan, effectively ensuring the passage of genetic material to the next generation. The text marks a sequence where after the establishment of SSC's, mature sperm are produced, hinting at the critical role SSCs play in male fertility.
06:30 - 08:00: Importance of Set DB1 in Germ Cell Development This chapter discusses the critical role of Set DB1 in the development of male germ cells. It outlines the process from specification at embryonic day 6.5 (E6.5) to the establishment of spermatogonial stem cells (SSCs) at postnatal day 2 (P2). During these stages, germ cells undergo unique molecular events, including complete demethylation of the genome around embryonic day 9.5, resulting in a highly hypomethylated genome.
08:00 - 09:30: Second Research Project on DNA Stability in Germ Cells In the chapter titled 'Second Research Project on DNA Stability in Germ Cells', the transcript explains the process of DNA remethylation in germ cells, starting from embryonic day 13 (E13) and proceeding until birth, reaching a stage of hypermethylation. The chapter also discusses unique chromatin rearrangements specific to germ cells and highlights the phenomenon of hypertranscription in germ cells, indicating elevated levels of transcriptional activity during this developmental period.
09:30 - 10:00: Conclusion and Acknowledgments by Dr. Hatkevich Dr. Hatkevich discusses the intricacies of male germ cell development, noting the significantly higher levels of certain elements compared to their sematic counterparts. He emphasizes the unique developmental and molecular characteristics of these cells and the importance of maintaining their epigenetic and genetic integrity. The chapter highlights the need for specific regulatory mechanisms, acknowledging that many remain unknown, setting the stage for further exploration and research in this area.
10:00 - 12:00: Q&A with Dr. Talia Hatkevich In this chapter, Dr. Talia Hatkevich discusses her ongoing research and development work. She first shares a story about a project she's been working on during her postdoc, which she hopes to publish soon. This project focuses on the G0 G1 arrest of male germ cells and its significance in their development into the SSSE fate. Additionally, Dr. Hatkevich talks about a new branch of her program that she is currently developing.
12:00 - 13:00: Introduction of Dr. Gerald Lodewijk The chapter introduces Dr. Gerald Lodewijk and delves into the concept of germ cell identity transformation. It explains the process in which the pluripotent germ cell identity diminishes while the SSC (Spermatogonial Stem Cell) stem cell identity is acquired. A critical component of this transformation is the role of de novo DNA methylation. The transcript highlights that proper de novo DNA methylation is crucial for the establishment of SSCs, and without it, the establishment does not occur. The chapter emphasizes understanding this biological process and its significance.
13:00 - 14:00: Dr. Gerald Lodewijk's Presentation Introduction Dr. Gerald Lodewijk's presentation centers on understanding the regulation of a rapidly changing epigenetic landscape, specifically how DNA methylation influences the histone landscape. The focus is on determining how this regulation ensures the development from MGCs to SSCs, using an RNA binding protein as the lens for investigation.
14:00 - 15:30: Embryo Development and Research Motivation The chapter explores DND1, a vertebrate-specific RNA binding protein crucial for germline development. Researchers have identified DND1's significant expression in G0 G1 MGCs and engineered a mouse line that endogenously tags DND1 with GFP. This advancement allows for direct observation and study of DND1's role and behavior within germline stem cells, providing valuable insights into embryo development and research motivations in this field.
15:30 - 17:00: Gene Editing for Embryo Model Creation This chapter discusses gene editing in the context of embryo model creation, focusing on the expression of DND1 in germ cells at embryonic day 16.5. DND1 is pervasive within the cell, located in the cytoplasm, nucleus, and germ granules. It binds to hundreds of transcripts during the G0ero period, many of which encode for epigenetic regulators. This information was derived from a RIP-seq study conducted using a DND1 GFP line by a former postdoctoral researcher in the lab.
17:00 - 18:30: Success in Creating Structured Embryo Models The chapter discusses the role of epigenetic regulators in the development of structured embryo models. It specifically highlights the interaction between DND1 transcripts and the set DB1 epigenetic regulator, a hysomethyl transferase. The RIPS seek analysis reveals a significant finding; there is no enrichment of set DB1 transcripts at embryonic day 14.5 (E14.5), but notable enrichment occurs at E16.5 and E18.5. This enrichment pattern suggests a regulated temporal expression, which might be crucial for understanding the mechanisms involved in embryonic development.
18:30 - 20:00: Transcriptional and Structural Comparisons with Natural Embryos The chapter begins with the speaker proposing a hypothesis that DND1 regulates the protein expression of its target transcripts, which encode for epigenetic regulators, in a post-transcriptional manner. They plan to use set DB1 as an example to explore this hypothesis. The first question raised is regarding the role of set DB1 in this regulatory process.
20:00 - 21:30: Transcriptional Differences and Improvement Attempts The chapter discusses the challenges of answering questions related to post-transcriptional regulation in their system due to the lack of cell culture for molecular analysis. Despite this limitation, they explore the correlation between transcript levels and protein levels to infer post-transcriptional regulation. The specific protein examined is set DB1, which is analyzed throughout a developmental period indicated as G0ero, with specific time points at 14.5 and 16.5.
21:30 - 23:00: Functional Analysis of Embryo Models The chapter 'Functional Analysis of Embryo Models' discusses the analysis of protein levels across three developmental stages. It highlights a significant decrease in protein levels: a 60% reduction from stage 14 to 16, followed by a 50% decrease from stage 16 to 18. These changes are quantified using FACS analysis, focusing on the fluorescent geometric mean of the Set DB1 protein.
23:00 - 24:00: Conclusion and Future Directions by Dr. Lodewijk This chapter, titled 'Conclusion and Future Directions' by Dr. Lodewijk, focuses on the localization patterns of the protein set DB1 within germ cells. The author discusses how set DB1 is primarily localized within the nucleus at developmental stage E14.5. However, by stage E16.5, there is a notable expression shift to the cytoplasm. By stage E18.5, set DB1 is found in both the cytoplasm and nucleus in a punctate distribution, indicating dynamic localization changes over time.
24:00 - 25:00: Q&A with Dr. Gerald Lodewijk Dr. Gerald Lodewijk discusses the transcriptional levels observed in a study using qPCR, indicating that there is no significant difference in transcript levels between 14.5 and 16.5. However, a notable reduction in protein levels at 16.5 suggests post-transcriptional regulation of the set DB1 transcript, highlighting an interesting aspect of the study related to DND1.
25:00 - 26:00: Seminar Closing Remarks In this chapter titled 'Seminar Closing Remarks', the discussion focuses on the post-transcriptional regulation and transcript localization of set DB-1 protein. Despite a reduction in set DB1 transcripts by 18.5, it's suggested that the transcripts may still be transcribed even in a reduced protein state during G0 phase. The chapter elaborates on these findings by examining where the set DB-1 transcript is localized.

SDB Ethel Browne Harvey Postdoctoral Seminar Series - Talia Hatkevich and Gerrald Lodewijk Transcription

00:00 - 00:30 Hello everyone and welcome to the Ethel Brown Harvey post-docctoral seminar series. My name is Josh York and I'm a posttock at Northwestern University and I'll be moderating today with Diana Fulmer a posttock at UPIN. We are excited to highlight the work of our outstanding post-docctoral members here at SDB. Today Talia Hadkovich from Duke and Gerald Ludovik from UCSC will share their research. Each speaker will give a 20-minute talk followed by 10 minutes of Q&A. Please enter your questions in the Zoom Q&A box. It's my pleasure to
00:30 - 01:00 introduce our first speaker today, Dr. Talia Hatkovich. Dr. Hatkovich earned her PhD in genetics from UNCC Chapel Hill and Jeff Sakielsky's lab where she studied mechanisms regulating various events during meiosis including chromosome pairing and myotic recombination using using Drosophila as a model system. She's been a highly productive scientist in her field publishing in top tier journals such as PNAS current biology and plus genetics. She is currently an NIH funded posttock in Blanch Capable's lab at Duke where she uses mice to investigate the development of male germ cells. Talia,
01:00 - 01:30 thanks for being with us today and I'll hand it over to you. Thank you so much Josh. Let me share my screen here. All right and thank you everyone for tuning in today and as Josh mentioned my name is Talia Hackovich and I am a posttock in Blanch Cable's lab at Duke University. So I just want to start off by talking
01:30 - 02:00 about the one of the most fundamental events in biology and that is the passing of information from one generation to the next indefinitely. And this allows for evolution, the continuation of species and really even just passing on the family name. This information is passed on through the germ cell which is the sex cell of the animal. uh that will eventually give rise to the mature gameamt to the sperm
02:00 - 02:30 or the egg and the information that's passed on indefinitely is both genetically and epigenetically packaged inside this germ cell. So an inherent question that the immortal germ line poses is how is the integrity of information maintained? And you know this question is particularly relevant within the context of the mamleian male germ line
02:30 - 03:00 and I will tell you exactly why. So the male germ cells must undertake a momentous journey to establish the male germ line to eventually pass on their genome. And in male mice, which is the model I use, this day or this starts at embryionic day 6.5 or E 6.5. This is when the primordial germ cell is specified. Then the germ cell has to actually migrate
03:00 - 03:30 through the embryo to uh enter the native gonad and then it gets settled there at E10. And then at E1.5, the primordial germ cell is specified into a male germ cell or MG MGC. Um, I want to point out that this is also referred to as prospermatagonia. However, I'm going to refer to male germ cell as MGC throughout this talk. After days of proliferation, the MGC's will then enter or exit the cell cycle at
03:30 - 04:00 embryionic day 14.5 and they remain in this G0 G1 mitoic arrest for about 7 days where at P1 they re-enter the cell cycle. So post-natal day one and they re-enter enter the cell cycle essentially emerging as spermoggonial stem cells or SSC's. And I like to think of SSC's as the biological cell that has won the most coveted prize. And this is because
04:00 - 04:30 SSC's are the resident stem cell population within the testice that can both differentiate and self-renew. So for the entirety of a male's reproductive lifespan, it is uh able to provide the progenitors of sperm thereby passing on whatever is in their genome to the next generation. Then after the establishment of SSC's, we get to mature sperm. So, not only is
04:30 - 05:00 the male germ line going through all of these developmental steps from when it's specified at E6.5 to when the SSCs are established at P2, but they're also going through unique molecular events that happen only in germ cells. And some of these events that occur during this time is complete uh demethylation of the genome. So this happens at about 9.5 and the genome is hypomethylated by about
05:00 - 05:30 E13 and then dnovo DNA remethylation occurs starting at about 15.5 by birth. We get um hyperthylation finally. So um also facilitating with that DNA methylation and remethylation, we also have a chromatin rearrangements that are unique to germ cells. And we also know that germ cells um undergo hypertranscription. And so this means that germ cells exhibit transcriptional
05:30 - 06:00 levels that are up to 20 times higher than the sematic counterpart. Right? So there's a lot going on developmentally and molecularly that are unique to the male germ cell. And so to maintain the epigenetic and genetic integrity, the germ cell must employ some regulatory mechanisms. However, many of these mechanisms during development are unknown. So today what I'm going to do
06:00 - 06:30 is I'm going to talk to you about two things. First I'm going to tell you a story I've been working on uh during my postoc for the past couple years that I hope to be published soon. Uh and then also a program that um a a branch of my program that I am currently developing. So let's get to it. So this G0 G1 arrest of the male germ cells is actually vital for development into the SSSE fate. Um so
06:30 - 07:00 what's happening is that the pur potent germ cell um identity is actually being faded away and then SSC stem cell identity is really being acquired during this time and what is actually having this required acquired is denovo DNA methylation as I talked about so without the denovo DNA methylation occurring properly the establishment of SSC's does not occur right and then also so helping and
07:00 - 07:30 facilitating with the DNA methylation um is the histone landscape. So this histone landscape is rapidly changing in response to the remethylation of the DNA. So my overall question for this particular project is how is this rapidly changing epigenetic landscape regulated ensuring the development of the MGC's into the SSC's and I'm going to answer this looking through the lens of an RNA binding protein called
07:30 - 08:00 DND1. So DND1 is a vertebrate specific germ cellcific RNA binding protein that is important and vital for the germline stem cell or the germline development. And here we know that DND1 is highly expressed within the G0 G1 MGC's. So we created a mouse line that indogen endogenously tags DND1 with the GFP. And using this we can actually see
08:00 - 08:30 here that germ cells at E16.5 which are all of these cells here are expressing DND1 and it's all over the cell. So it's within the cytoplasm, the nucleus and within these puncta which are germ granules. We also know that DND1 binds hundreds of transcripts throughout the G0ero period and many of these transcripts encode for epigenetic regulators. And we know this because a previous postoc in the lab performed a RIP seek study using our DND1 GFP line.
08:30 - 09:00 And one of the uh epigenetic regulators that binds uh whose transcript binds DND1 is set DB1 which is a hysomethyl transferase. So here is um our RIPS seek and you can see here that compared to input there is no enrichment of the uh RIPS seek at E14.5 of set DB1 transcript but there is at E16.5 and E18.5.
09:00 - 09:30 So uh I am going to answer my question by having posing the hypothesis that DND1 regulates the protein expression of its target transcripts that encode for epigenetic regulators and this happens post-transcriptionally and I'm going to use set DB1 as an example today. So the first question that I want to pose is is set DB1 set DB1 actually
09:30 - 10:00 subject to post-T transanscriptional regulation and this is actually a really hard question to answer in in our system um because we can't do a lot of molecular analysis because we don't have any cell culture or anything. But what we can do is we can look at the correlation between transcript transcript levels and protein levels to then infer something about post-transcriptional regulation. So by looking at set DB1 protein throughout G0ero i.e. 14.5, 16.5 and
10:00 - 10:30 18.5 we actually see that the protein decreases significantly throughout the three stages. So it decreases about 60% from 14 to 16 and then about 50% from 16 to 18. Um this is quantified using uh fax analysis and looking at the um the fluorescent geometric mean of set DB1 protein. Right? So this is also seen
10:30 - 11:00 here when looking at the set DB1 localization uh within germ cells. Um so here I just want you to look at the set you can just look at the set DB1 panel. So at E14.5 we see that set DB1 is localized specifically in the nucleus. And then at 16.5 we actually see this really interesting pattern where set DB1 starts to actually uh be expressed within the cytoplasm. And then at 18.5 set DB1 is seen in a punctate manner both within the cytoplasm and
11:00 - 11:30 within the nucleus. All right. So what about the transcript levels? Well using qPCR we see that there is no difference between transcript levels between 14.5 and 16.5. This is interesting simply because at 16.5 we see a strong reduction of post of protein levels. So this in itself is suggesting post- transanscriptional regulation of the set DB1 transcript. Importantly at 16.5 this is when DND1 is
11:30 - 12:00 binding set DB1. We also but we see a a strong reduction of set DB1 transcripts i.e. 18.5. However, post-transcriptional regulation of set DB-1 to regulate protein expression um would suggest perhaps that maybe even though the protein is being reduced during G0 that the transcript is actually still being transcribed. So we actually just looked at this by looking at transcript localization of set DB-1
12:00 - 12:30 in germ cells throughout um G0 and we see that set and we did this using single molecule fish and we see that set DB1 is localized to the nucleus at all stages because M mRNAs are not um sequestered or stored in the nucleus. uh this indicates that set DB1 is being actively transcribed at all three stages. And when you look at the percentage of set DB-1 localization per
12:30 - 13:00 cell, it's it's within the it doesn't change uh the localization within the nucleus at all. So we uh this suggests to us that set CB1 is actually being transcribed all throughout G0 even though the protein is being decreased. So this suggests to us that yes sight CB-1 is in fact being regulated post-transcriptionally but is it actually good um being regulated through DND1. So to answer this question um we
13:00 - 13:30 collaborated with AUSC Suzuki who has a DND1 flockbox mouse and so we created this DND1 mutant mouse by um mating these two together with an O4 CRE ER we tomoxifened at 13.5 and then at 16.5 um I was sent the the samples and I analyzed set DDB1 proteins via if um at 16.5 and this is from a paper that Tishi previously published showing that if you
13:30 - 14:00 tmoxifin at 13.5 you get a strong reduction of DMD1 at 16.5. So I just want to say when looking at the mean fluoresence um of set DB1 via um if quantitation we actually see a significant decrease of set DB1 protein in this mutant. Um this was actually really surprising to me because uh my hypothesis was the complete opposite that D&D1 is promoting the degradation
14:00 - 14:30 of the transcript. But because of this it actually suggests that set DB1 is um playing a protective role of the transcript. So let me just show you how I'm actually interpreting this. So set DB1 at E14.5 it's not bound at all. And then we get high transcript translation of the transcripts 16.5. How I see this is that maybe perhaps some of the transcripts are being degraded but DND1 is binding to
14:30 - 15:00 some preventing that degradation and that's resulting in some protein and we also see this happening at E18.5. So the mutant you would get rid of DND1 and therefore you would have a reduction of the set DB1 protein right okay lovely but if set DB-1 is reduced too early in MGC development then what is the consequence this I'm actually asking is this protective role of DND1 really
15:00 - 15:30 important so to answer this question what I did is we created um a knockout mouse um of site DB1 one specifically in the germ cell using a creer system in germ cells specifically uh with the germ cell specific driver similarly to the dnd1 knockout mouse it tomoxifind at 13.5 and analyzed at e6.5 now this led to a mosaic knockdown which is pretty common but through analysis we see that about 50% of the
15:30 - 16:00 germ cells are affected so um just gross morphology analysis here at 16.5 this is our wild type control we can see that um nice testus cords and germ cells are marked with this tra 98 so um it's a nice round um testice with nicely organized chords now the septi b1 mutant morphologically at the tissue level does not look very different however let's
16:00 - 16:30 look a little bit closer so here we're looking at wild type we're looking at um uh testus cords marked by TR98 with which are marking the germ cells um and you can see that the nucleus of the germ cells are beautifully round and it's very consistent. Our set DB-1 mutant looks completely different. The TR98 which is a germ cell marker is not marking nearly as well indicating some unhealthy cells there. And you see that
16:30 - 17:00 the nucleus of the nuclei of the um sitb1 mutants are actually very abnormal and not round at all. To try and quantify this, what I did is I took the nucleus of all the diameters and I took the largest um nucleus of each cell to um and then compared between wild type and set DB1 mutants and we see a significant difference and we see a new population that's really large. Um but then we also see some germ cells that are pretty small and you can see
17:00 - 17:30 here. So um I also wanted to ask well if these cells are unhealthy are they undergoing apoptosis and a marker for apoptosis is gamma H2AX which marks double strand breaks but it would have to be completely nuclear wide um gamma H2AX positive for it to be a dying cell. Uh so uh we quantified the percent nuclearwide gamma H2AX germ cells per chord analyzed and we see a significant
17:30 - 18:00 increase in set DB-1 mutants. So this is indicating to us that these cells are most likely dying. Um but a question is is so this is at the MGC um level that we see this hap this um defect happening but can SSE actually be established. So to ask this question um we looked at um testes at P4 in wild type and set DB1
18:00 - 18:30 and you can see here this is the cross-section of a testice uh and we're again using TR98 to mark our germ cells and our set DB1 mutant we see no germ cells in the left testice this indicates that SSES are not established but what's really weird is that in the right pestos we get plenty of germ cells this actually looks normal morph morphology wise um and even at 6
18:30 - 19:00 weeks uh this phenotype um persists so at six week old pups in our wild type we have left right testice in our mutants we really see the defect only in the left testice um we have no idea for this I would love to talk about it um in Q&As's and if anyone has any ideas I would love to too. So overall with this story um so what we see is that set DB1 is most
19:00 - 19:30 likely being um actively translated at 14.5. At 16.5 it's time for the protein to be reduced but not too much and not too quickly. So we see DND1 coming in binding set DB1 um and ensuring that there's still some protein that's being made thereby throughout 14 to 18 um we're seeing just a gradual reduction of the protein and this in its own right is setting up the a proper genetic
19:30 - 20:00 landscape to ensure that MGC's develop into SSC's. So the next thing I I want to talk about is a what I've been um working on developing for another arm of my research program and that is stability in MGC's during this G0 G1 developmental time. So DNA is constantly exposed to sources
20:00 - 20:30 both endogenously and exogenously that damage your DNA. It creates uh you the average is about 200,000 lesions per day and the exogenous sources are UVIR and then also just chemicals that you come in contact with. Endogenous damage includes reactive oxygen species things simple as um transcription replication and even removing and um adding um methylation to DNA. one minute will uh
20:30 - 21:00 create double strand breaks or damage and double strand breaks um or DSBs are the most deleterious form of the DNA lesions and so there are double strand break mechanisms that are able to be uh to come in and repair those double strand breaks and the two canonical pathways are uh homologous homologous recombination or HR and non-homologous endjoining or NHG
21:00 - 21:30 J. So NJ is thought to be like uh the quick and dirty repair mechanism for double strand brakes. And this is because a complex will come and just kind of clamp on the ends of the double strand brake and then just liate them together. So smush them in. And here we get um genetic information lost. However, this is able to repair quickly. So there's a give and take with using NHJ. Whereas with homologous re combination,
21:30 - 22:00 it's a lot more complicated and involves a lot more proteins. However, no genetic information is lost because you are synthesizing off of a homologous template um to to get the information back that's lost during the double strand break. Now, the pathway in mammals that is is used NHJ versus HR is largely dictated by the cell cycle. So in the cell cycle in G1 right you you
22:00 - 22:30 are not replicated your chromosome is not replicated you do not have a sister chromatid however in sphase you um are getting synthesis in G2 you have a sister chromatid right so this means that in G1 you don't have a homologous template readily available so the cell will usually use NHJ to repair a pair and then uh the cell will use the homologous re combination when they have
22:30 - 23:00 their sister chromosome or sister chromatid nearby. Right? So this sets up a paradox in the germ line. Using NHJ to actually repair the germ cell in the genome will lead to genetic deletions which can be then codified into the heritable germ line. Um and this is this is really interesting because during the um the G the arrest there is a G0ero meaning that there is no sister chromatid nearby and
23:00 - 23:30 during this time there's hypertranscription DNA methylation and nuclear arrangement all which across the DNA and causing double strand breaks. So the question is is what is the mechanism by which the G0 G1 MGC's are actually repairing double strand brakes. So this is something I'm developing and I have preliminary data. I just don't have time to talk to you about it but um we can talk about it
23:30 - 24:00 later in Q&A. So going back how is the integrity of the information within the mortal germ line actually maintained? While genetically my preliminary data suggests that it does it by using unique DNA repair mechanisms and epigenetically it is through highly regulated expression of epigenetic factors and this is facilitated by proteins such as RNA binding proteins and
24:00 - 24:30 DND1. And with that there's a lot of people I would like to acknowledge um Blanch for being a lovely mentor. I I appreciate everything that she's done for me. Um Kayla is an undergrad from University of Richmond who has quantified a lot of the microscopy data. Um and then also Atushi um who is gracious enough to share the DND1 uh conditional knockout mouse. And with that uh I'm happy to take some questions.
24:30 - 25:00 All right. Thank you Talia. That was a fantastic talk. So just a reminder for everyone if you have questions you can input them in the Zoom Q&A box and I will uh give those to Talia and then any unanswered questions we can um you can answer uh we can answer later in the Q&A box. Uh I'd like to begin actually. So Talia I had two general questions. So one um have you considered genes the asymmetric test phenotype um genes that play a role in left right organogenesis? I was just
25:00 - 25:30 curious like have you considered like pitex or lefty or nodal there genes like place heart in different sides of the body and you know are you are you thinking those genes are involved in this as asymmetry um you know I I honestly I don't know um we don't know the reason for the asymmetry but lefty is actually an interesting gene to think about because it's highly expressed in the developing germ line so um I should perhaps check that out
25:30 - 26:00 Um I will say that one one thing that is different between left and right is that there's been a paper is in that there's more territoas in the left testice than in the right testice and Blanch actually showed uh a couple years ago that um this correlates with differences in vascular architecture. So my hypothesis right now um is that
26:00 - 26:30 perhaps the left testice cannot recover as as greatly as the right testice simply because there's too low of oxygen or energy. Um but who knows but thanks for the question. Yeah one one more followup just a general question. So um for PGC's NGC's is there evidence that the gene regulatory networks involved in those how how similar are those to other types of of potent cells in the embryo
26:30 - 27:00 are they very different? Are they sort of similar in many ways evolutionarily conserved? What's what's your take on that? Um I I honestly don't don't know. Um I will say that the germ the germ cell is very unique in that it can turn in at at that stage going from PGC's to MGC's it's highly pur potent so it has to shut down a lot of other um pathways or all
27:00 - 27:30 pathways um to be able to develop into the germ cells. So I would suspect that it's probably different. there's a lot of uh germ cell specific genes that are expressed during this stage. Um so I would say it's probably specific to the germ cell. That's all for me. We do have a question here from Teresa Gross Theing. uh she asks what exactly is the function of set DB1
27:30 - 28:00 and how is its function affected by its dosage that is reduced but not eliminated by DND1. Um great question. So set DB1 what it does so it's a hysomethyl transferase and it deposits H3K9 um well it deposits trimethyl onto H3K9. So what that does is that is supposed to um uh reduce uh the expression of genes
28:00 - 28:30 and create heterocchromatin. So it's a heterero chromatin modify or promoting mark. Um how set DB1 is actually being affected um how this mark is being affected by set DB1 during this stage in germ cell development is unknown. One of the things I would like to do is to do a chip seek for the mark that set DB1 lays down um to see the developmental
28:30 - 29:00 progression of that. But that field is is rather crowded and I think someone's probably going to get to it before I do. Um now why would it need to be reduced? Um and how would that change the function of it? I'm guessing perhaps maybe to maintain that mark um after or while the um the DNA is being remethylated, but again this is all speculation. Uh we have another question here from
29:00 - 29:30 Michael Leaden who asks, "Do you see the same phenotypes for RNA and protein levels for genes other than set DB1?" Yeah. END1 always protective. Um so we do see a similar well okay so the transcripts that DND1 um binds to it can be it can be protective or it can actually promote um the degradation of
29:30 - 30:00 so said DB-1 is known to have both of these roles. um the genes that we see or the transcripts that we see uh a lot of them do not have the same patterning that set DB1 does have some will um increase indicating that DND1 is um promoting translation of um and these are more analyses that I have done that will hopefully be uh in the manuscript that I publish. Looks like we have time for one more
30:00 - 30:30 question we have from Hector Perez who says, "Hi, awesome work. Did you find aberrant expression of gonatal somatic cell markers in the left testice? Is the lack of GC's modifying the developmental morphology of the testice or is this modification only attributable to mutation?" Um, I think that the lack of GC's is what's really um leading to the morphological defect of the testice. Um and in the left testice uh that has no GCS in the set DD1 mutant
30:30 - 31:00 we have not done any RNA sequencing or anything with that. That again is a future direction that I would love to study. Um but that can provide some insight onto perhaps what is really going wrong on on the transcriptional level. Excellent. Okay, I think that wraps up time in our end. Again, thank you Talia. Fantastic talk. and I'll hand it over for the second moderator. Thank you, Josh. And hi,
31:00 - 31:30 everyone. Uh, again, I am Diana Fulmer from the University of Pennsylvania. And I now have the pleasure of introducing Gerald Lorovik, a post-doal researcher from the University of California, San Santa Cruz. Gerald joined the Ali Shared Lab uh in 2020. So, he is a pandemic postoc much like uh myself and many of us. um and he joined his uh current lab after earning his PhD from the University of Amsterdam under the supervision of Frank Jacobs. Uh Gerald
31:30 - 32:00 has won numerous awards including a recent GAN mentor uh mentor fellowship from Gene and today he's going to share his latest work on the self-organization of embryionic stem cells into reproducible embryo models. So take it away Gerald. All right, thank you for the introduction. Um, and yeah, been a postto here for a few years, started in the pandemic as you mentioned and um, yeah, still still going. Uh, so I want
32:00 - 32:30 to talk about that recently got published and highlight a couple aspects of that in the time that has been given by the organizers. So thank you for inviting me and given the opportunity to talk here. Um so how about engineering pro cell to generate uh embryo models in vitro and just to segway a little bit of the previous talk. Um so life starts from a humble beginning which is one cell and this is the sperm fertilized egg to create a
32:30 - 33:00 zygote. This is the one cell which in in in mammals placental mammals will divide to generate more advanced stage of the embryo. So coming from this one cell to find to make two cell, four cell, eight cell, so on and so forth. But eventually uh you create a structure called the blastoy. Um is highlighted by the formation of embionic cavity and differentiation into a few different cell. Okay. So first there is the blast
33:00 - 33:30 cells which generate the placenta which helps to implant into the maternal uterine wall. Um there's the hyperbolast of endoderm um which generate the yolk like amnons. So another supporting tissue and the inner cell mass or epiblast actually forms the tissues of the adults body later on. So so all the tissue as we are you know sitting and listening here and so this pathways are gen like quite the same between species but you can
33:30 - 34:00 also see that there are difference in how these cells are organized. The human you see this more like disc shaped structures of the cell types whereas in the mouse you get this sort of like cylinder structure even though the cell types are similar they organize in different ways and for this talk I'm mostly focusing on this mouse development and especially this sort of like early implantation stage uh between sort of day five and day six as part of this initial study. Uh another interest in this to find out
34:00 - 34:30 like well how does one cell know how to form an a whole embryo. So you have this one cell that form different cell types cells then collectively like migrate or divide. So how is this from a fundamental aspect? How does this sort of collective and and individual behavior reproducibly lead to this development of an embryo like mouse or human or other species and also from I think a more of a clinical perspective where we know that in human about 30% of
34:30 - 35:00 every sort of early embryo fails to implant leading to early pregnancy termination and we don't really know why there's not really a lot of information on what's going on but it seems that troph blood cells would have some kind of role in this because they are the ones that implanting facilitate the implantation. Uh so that led to my interest in like developing some of these tools and models to make uh sort of embryo like structures in vitro model some of these aspects from a
35:00 - 35:30 fundamental clinical angle at some when you compare development across species I think in develop developmental biology uh there's not just work done in mouse and human but also in frogs and fish and other species and especially species that do develop in egg like in this case the zebra fish in the middle here. The advantage is you can take the egg and sort of image the whole process of development um relatively easily whereas you deal
35:30 - 36:00 with a mouse or a human there's like all kind of ethical restrictions but also the embryo is actually deep inside of the maternal organism. Uh so you can access it very easily. So I think that's another reason to use some of these embryo models to sort of scale up some of the developmental studies we're doing in both a mouse and human context. Um um and so yeah just to coming back to some actual like work um so basically in a mouse
36:00 - 36:30 embryo there's this structure of the mouse accelerator the trophoblast extra embriionic enderm and epiblast that basically we're trying to rec recapitulate to sort of get this early embryo model in this case and I aim to do this with epiggome editors so these are cast 9 based tools that bind to the DNA they don't cut it, but instead you fuse them to transcriptional or epigenomic modifiers. So you can actually
36:30 - 37:00 locally activate or repress elements of of the DNA and do like functional uh DNA modifications. Um part of the main reason for that is that we only use sort of the endogenous cell machinery to regulate. So indogenous vector elements to to induce different cell types and really have like a cell intrinsic based method to make cells of the early embryo. Um which has an advantage that we don't we hope we didn't need to use
37:00 - 37:30 any sort of media supplements to guide the cells into a specific state. And of course naturally because it's a cris based system we can also uh use this to do additional pertubations like function modifications to follow up with functional experiments. So it's like a programmable device use either pertubations or larger scale larger scale screens to study these embryo models in vitro. Basically this is what I what I set up. So I have my embryionic stem cells on the bottom left here. I
37:30 - 38:00 integrate uh transgenic gest. So it's a transcriptional activator cassette that is a doxy inducible system. Um so we can control the expression and the second part is the entifier foricette. So we can actually direct the GKS9 to our target. Uh and again this showing that without dogs there's no activation plus docs we get activation. So we can choose when do we do this activation start at the end. We are
38:00 - 38:30 flexible in that. Um and this is basically going back to like well we know that are three main cell types in the early embryo that we need to produce embryionic stem cells we can look at them for free because that's the cells that we start with these sort form those epiblast or inner cell me like compartment so the cells actually make the tissues and the embryo and cells that we have to make specifically from these embryo samples are the tropha
38:30 - 39:00 cells and extra embryo endoderm cells and we know from previous studies that CDX2 and GA6 are important for inducing those sulfates in the embryo. So we started out by inducing those factors and and see do we actually see changes that that lead to those kind of cell types. Uh this is some very simple validation. Left you have RNA. On the right we have CDX2NA. We see that we get CDX2
39:00 - 39:30 activation. uh no activation the control and also the activation of the downstream gel called T effect. So we do the crisper activation we not only induce CXQ but also downstream factors are getting activated indicating there's sort of like a self uh activated program happening and the same when we do our extra embriionic induction uh extra ambionic under induction with ga 6 like no ga 6 in the control but we see high levels of ga 6 with the case of guinea
39:30 - 40:00 and also with downstream foxy one foxy 2 activation this is very promising structures per activation seem to work quite efficiently. Um but then of course the question is do these cells organized into embryo like structures? So have they formed sufficient unique features that they actually are able to form organized structures that mimic the embryo. Um, and that's basically what we did
40:00 - 40:30 here is that we put these cells into agar well plates and these are multi-well plates that again have hundreds of microwwells within each well and the cells will sort of land in these micro wells and they start to form little balls of cells. So you can have like you know 30 cells approximately in each of these microws. Then the cells will start growing and interacting with each other. And we see the cells at time zero. We add dogs at time zero. All the cells are the same.
40:30 - 41:00 We add dogs to the differentiation starts. Three days later collect the tissue and we did immunofesscent staining to see what what happens. Um it is kind of what we get. Uh so we label all of our nuclei. CDX2 for trophoblast cells, oxy12 for the extra endoderm or primitive endoderm and oxyor 51 for the epiblast merge combination. Uh so we see that similar like the natural embryo this fox a12 is
41:00 - 41:30 on the outside c2 is on one end of the embryo fox for the other side of the but a little bit more about the structure later in the talk but we try to quantify some of these structures actually get some measures like well how many cells do we get so we can measure the number of cells per organoid by segmenting the the full like zstack of these 3D structures um to get a sense of which kind of cells do we get and how many uh but also some
41:30 - 42:00 like more complex features like do we get formation of the embryionic cavity by measuring the signal of cells in the cross-section of these tissues and princely using that we find that 80% of our structures farmer's embionic cavity which is an essential step in embryogenesis and sort of summarizing that is that we have this simple crisper based method We use epigenome editing to induce this a cylinder structure without
42:00 - 42:30 using any morphagens that we add to media. It's a completely cell intrinsic induction. We only need two elements. So we just need the gator 6 and cdx2 elements to basically induce this cell constructing pattern. And there's some other examples here just to show for you know for fun um like another cross-section here and sort of 3D representation on the bottom right here of what it looks like in comparison with the schematic of the natural end top.
42:30 - 43:00 That's one aspect is a comparing like the structure. Um but another question is like well are these cells actually assembling on a molecular level the the embryionic cells? So do these for instance show transcriptional similarities to the natural mouse embryo and for that we use single cell R sequencing to find out are the cells that we have in our crisper programmed embryo models or CPAMS are they the same similar to the natural mass
43:00 - 43:30 embryo single different thing we find distinct clusters like extranic emodermoblast cells and epiblas cells in model which are sort of highlighted by canonical markers like ga6 for extra and endoderm celix 2 for trophoblast and nano for the for the epiglast. Um and I want to zoom in a little bit more on the comparison between the natural embryo. The left here are a set of marker genes shown in
43:30 - 44:00 our model and on the right same genes in the E5125 mouse embryo. So actually like most embryo data and we find like highly similar expression profiles of the marker genes for instance epiblast here in the purple highlighted box uh for extra extra embriionic endoderm. However, we doc also like some subtypes of of cells forming that for instance have one expression or LHX1 expression
44:00 - 44:30 that is higher in cluster one to cluster two and we see that same kind of expression pattern in the natural embryo show that these groups are quite similar and finally the same for trophobus channels where we see our CDX2 population upregulating several markers related to troph development. such as T52 C and PGFA similar to the nature mouse embryo. So combined this shows that this model
44:30 - 45:00 both has transcriptional similarity and structure similarity to the mouse embryo. Uh there was like a great initial finding that we could like pretty easily induce these cells uh mix them together and they s organize into this accidental like structures. Um, one thing we did observe from this data is that there seem to be uh some level of of epiblast markers left in actual blast compartment in the cams compared to the natural mass embryo. I
45:00 - 45:30 thought like well if we use this data to perhaps enhance some aspects of our model like we improve the trophoblast compartment would it our efficiency of the overall model formation? Uh there could be like two reasons why we see this sort of little bit of blast expression there. Is this um here? Okay.
45:30 - 46:00 Um so do we see um improve compartment by either increasing differentiation time? So do we just need to add induce CX2 for longer uh to induce uh like proper or like more differentiated troph cells or we the other way we activate additional factors by multipplexing our crisper system to induce additional trophoblast cell factors that would enhance
46:00 - 46:30 differentiation in those sense so we tried to do both um so doing a pre-induction and a multiflexction approach Uh so a control just like using our initial system with the CX2 adding do set.0 zero when we add the cell to the agar well plate and the second condition we pre-induce this for two days with dogs before feeding the cell into the egg well and then another
46:30 - 47:00 condition is basically doing the same but in this case we also add a L5 gna in the cex cells now these cells are dual cexus L5 double activating system and the reason why I choose L5 5 is because L5 is known to be a gatekeeper for [Music] trophenic repression of L5 in the embryionic cells basically preventing preventing this troph differentiation.
47:00 - 47:30 So I think this would be like a perfect candidate for us to study because we can retro activation tool to the L5 locus to activate or like remove this epigenic barrier and and hypothesize that this would improve our differentiation of the trophy model that we used before and basically this is what we get. So just looking at this pre-induction I'm here just looking at the number of uh CPMs number of tissues
47:30 - 48:00 that have a significant role compartment and we found that if we just do the pre-induction with CDX2 uh go from like a 25% to like 40% of doubling our our number of of CPMs that has like a proper TS number. Um, surprisingly when we did the activation with CX2 and L5 sort of at the 10.0, we didn't see any changes. Um,
48:00 - 48:30 but doing this combination of the pre-induction versus the dual induction really like boosted that efficiency to 75%. So the combination of sort of the longer induction moving that alpha barrier really seem to significantly enhance the troph cells we get. highlighting that you can use this crisper multiplexing method to like teeth out how these cells are formed in vitro and using them in this kind of
48:30 - 49:00 models. Another question is like well just having the cell numbers is important but are they actually localized into the right compartment? Um and to measure that basically I I traced the signal in the X cylinder for both the O4 and the CDX2 staining and plotted that sort of in this graph and clustered it based on the on that staining pattern in three different clusters. So cluster one is
49:00 - 49:30 basically kind of random. You have both like the CDX2 and R4 signal sort of intermixed indicating there's no patterning. Cluster two there's like small domains of CDX2 positive cells green peak here and cluster three is sort of like the real like proper separation of like a distinct CDX2 and a distinct box for domain kind of shown in the example here and we find that um when you do this pre-induction uh the majority of of actually get this like highly organized
49:30 - 50:00 structure in separate domains where like the one end is CX2 trouble blood cells and the other end is for fbl blast cells. Uh so this combination again is like really helping to uh properly organize our model in like the proper uh access information that we see nature and again to summarize this is that able to uh show distinct domains of CX2 and and opt for trop and epiplast
50:00 - 50:30 compartments and we could enhance this efficiency efficiency of this summation by doing the pre-induction and also like using multiplicing capacity to to to get the numbers and the organization matching the nature embryo formation. Uh and the final aspect I want to highlight of this is can we use this for any functional aspect. So I think that is maybe the powerful tool that we can
50:30 - 51:00 hopefully do in the long term is actually do more functional experiments like observing certain pathways certain genes certain like risk factors in development and and see how does this affect the development of our model and linking that to actual real natural embryo development. One minute. Thank you. Um so we chose to work on the basement membrane for this. The basement membrane is a structure that's in between the extra endoderm and the other
51:00 - 51:30 cell types. It's sort of this blue like line that is sort of like a a layer of extra matrix that is separating the different cell types. Um and this is our natural embryo. And again we find this in our model. We quantify this where's a green graph. uh it's like in between uh the the ga 6 on one end the oxorn on one end lemon is like nicely wedged in the middle sort of when you take this cross
51:30 - 52:00 tension of the embryo measure them and we also know that this this lemonine is highly produced by this extra estro amiotic endoderm cells the outside cells they pump a lot of lemonine into into their environment um and we also know that it's epibblast cells they sort of try to counteract that at some point they will like break down this matrix because they need to sort of make space to expand and and cells to migrate for gastulation. So there's like a continuous sort
52:00 - 52:30 of dynamic like establishing an ECM to maintain a structure but also like degrading locally some of this ECM structure so like cells can migrate and move to their proper location. It's quite a dynamic process and interesting uh to study. Uh so we thought like well we can advantage of our ESLs that currently have a control guide RNA only um and we can use crisper A to either overexpress lemonine by targeting a lemon alpha one gene sort of let the
52:30 - 53:00 cell pump out more uh lemonine that they're supposed to or do the the opposite where we let the cells overexpresses that degrade this based on memory matrix. These are metallo matrix proteasis for MMPs. And because we guaras in our ES cells, they're only sort of being activated in this sort of purple compartment of the cell. Basically, only one half of our model is like making these additional
53:00 - 53:30 factors like creating a localized effect. Uh and again, we do lamining to quantify this. And what we find is that when we do laminate activation, we get increased integrity of this basil membrane. So there's like less gap uh in this sort of continuous matrix surrounding those those cells. Uh and it was not affected when we did the the MMP overexpression. Uh but instead when we did the MMP over expression the basement membrane was thinner. So it seemed to
53:30 - 54:00 have had the lemon over expression or MP activation. They seem to have kind of reciprocal effects where you know the one is sort of strengthening it, the one sort of weakening it but they use sort of different different uh slightly different outcomes. And probably one exciting thing about that is that we actually saw a phenotype in a structure of those embryo structures where when we strengthened the the bas membrane, our embryo models had a more round structure
54:00 - 54:30 by measuring the difference between a long and a short axis of of the overall shape. Uh suggesting they have some kind of like block in their their elongation uh in the axon there. Whereas if weakened the basement membrane uh this allocation seem to be uh promoted. So the actual embryos seem to get a little bit sort of like longer shaped probe because they didn't have this barrier that is like preventing their growth as much. I guess the summary of this of
54:30 - 55:00 this crisper pertubation experiment. Uh just to quickly sum up in future work in comparing this both in human and a mouse system to see what are some human specific aspects of how these cells are develop developing and how this really implantation defects. We can use like tools to really like dig into the specific pathways that are regulated uh in this mechanism uh by
55:00 - 55:30 also introducing for inance like orthogonal crisper systems. So you're going to combine a crisper activation with a different crisper repressor system. So really have the flexibility uh to to independently target different pathways within specific cell types and see how that relates to their development and interacting with their environment. And with that I want to thank all of my lab members and the contributors especially
55:30 - 56:00 Saiaka who's an undergrad who did a lot of work on this paper and also co-authoring the paper and of course the funding sources from both a firm and a post fellowship and wubicon and I'm happy to take any questions. Thank you so much, Gerald. That was an excellent talk. Um, everyone, please put your questions in the Q&A box. And I'm going to go ahead and start us off. Um,
56:00 - 56:30 I was really excited about these, um, basically mini embryos that you could make. And I was kind of curious, um, how functional, you kind of touched on this a little bit with the MMPs and the basement membrane, but I was wondering how functional they were. for for example, do they try and embed into the matrix uh that you're you're actually plating them on? Um could you kind of use this as a proxy to study sort of the black box, I guess, if you will. Yes, I think that is uh so I'm not the
56:30 - 57:00 only person who work on this. Other labs have tried some of these things where they try to like put these sort of embryo models in like a pseudo pregnant mouse um um and they find that they can implant but then they don't really develop further. So there's something that is missing. We know there's some of the extra ambionic cell type that are missing which probably lead to some like developmental block. So I think that is something that is currently unknown like what do
57:00 - 57:30 we need to do to sort of make them develop further uh with especially with high efficiency. That's really cool. Um let's see we have a question here from Michael Leaden. Extremely cool. The transcriptional profiles are similar but not identical. In particular, all cells seem to have higher expression of all the genes you showed. How do you interpret the differences between embryos and engineered systems? And do the differences allow you to predict what
57:30 - 58:00 the limits are for how the engineered systems can be used to infer the biology of the embryos? Yes, I think that's a great question because we have this natural data to compare it to. Um, so in our case, we did use the L5 as one example to really improve our model because the L5 was like lower compared the L5 in our model was lower compared to the natural embryos. And well, this is really something that is potentially lacking in the engineered
58:00 - 58:30 cells. Um and yeah, I think the levels are important and I think uh yeah, having more control over those levels could indeed lead to more robust formation of these different cells and also the timing of it. So I think it is known that some of these transcripts are turned on a certain time then they are repressed at a certain time. Um so having control over that would definitely be helpful to sort of
58:30 - 59:00 uh more like specifically induce the cells especially if you take like time course data into consideration. So you can take like the the day four, day five, day six, day seven mouse embryo and really like figure out okay like this factory needs to be on for like one or two days. So then we need to shut it off. Uh so you can really like engineer the circuits uh to to sort of mimic that temporal and and level aspects in vitro by real leveraging that that natural data that we have now for both I think mouse and
59:00 - 59:30 also for human data. There is actually some data out there that that we can use for them. Thanks. Um and I actually had just another question um about um culturing the embryo the synthetic embryos. How far I know you said that um some people have actually tried to implant them in a pseudo pregnant mouse. How act how far are you able to take them while they're actually in culture? What embionic stage would you say you could get them to? Uh so this it's like yeah I think we get
59:30 - 60:00 most similar to this E5.25 mass embryo. We do see like some excess formation in the sense that we see in about like 25% of our structures we see for instance local expression of left one TK1 and sir one which are apparent in factors that that will start to induce like another access formation in the mouse embryo and start distillation around like E5 and a half to E6. So I haven't quite we haven't gotten there quite yet.
60:00 - 60:30 So all this stuff has been on sort of E5.25 25 uh timing and I think what other labs have shown is that if you want to go further there's definitely the sort of like bottleneck in efficiency u um where it's it gets harder to sort of maintain this like proper access formation into like the more complex structures of both the guest and like further
60:30 - 61:00 um further advanced structures and I think the latest the latest structure a happy mate in vitro from mouse or think about they've grown them think for eight for about eight days and they do see like some like organs forming like brain like other organs in in these structures. Um but I think the efficiency is something that is uh probably like the main thing is like well how do you make this differentiation robust where we start
61:00 - 61:30 out with maybe like some factors we can't control that at some point lead to maybe like an aberant development. So how can we control these factors doing different parts of differentiation so we can keep sort of the differentiation in the right trajectory. I think that is the challenge in that the challenge and the excitement. Well thank you so much and if there are no other questions I just want to thank one more time Dr. Zahekovich and Dr. uh Lovik for your excellent talks. I did want to let everyone know that this seminar uh is being recorded and will be
61:30 - 62:00 available on the SDB website next week. Please join us next uh month for our seminar on Friday, May 9th, when Taylor Medwig Kenny from the University of North Carolina at Chapel Hill and Ru Yen from Harvard Medical School will present. Thank you all for attending.