2,931 research outputs found
Recommended from our members
Mechanisms Regulating PGC Specification and Epigenomic Reprogramming
No full textInfertility is a broad disorder with numerous causes including physical, genetic and environmental. While techniques are currently in use to address certain causes of infertility, such as in vitro fertilization and hormonal therapies, there is currently no treatment option for those who are either unable to make or no longer possess viable gametes. Recently, advances have been made in the development of in vitro gametogenesis which, if perfected, promises an option for gametes to be derived from a patient’s own tissue. In order to bring this technique to fruition, further research is needed into the mechanics directing the specification and epigenetic reprogramming of the earliest stage of the germline, the primordial germ cells (PGCs).Mammalian PGCs are specified early in embryonic development and give rise to the entire adult germline. Following specification, PGCs undergo epigenetic reprogramming in order to establish a permissive epigenetic landscape for proper gametogenesis prior to differentiation into either oocyte or spermatogonial progenitors. Any errors in either of these processes can result in the complete loss of the germline and infertility. In order to better understand the mechanisms underlying PGC development, we utilized the in vitro PGC-like cell (PGCLC) differentiation to study human PGC specification and PGC-specific conditional knockout mice to assess epigenetic remodeling. In our studies into specification, we further characterized the differences between mouse and human PGC specification mechanisms. Using CRISPR/Cas9 gene editing we identified that EOMES directs human PGCLC specification, whereas in the mouse this role is accomplished by T. Our exploration into epigenetic reprogramming utilized a Cre/lox driven PGC-specific conditional knockout mouse to assess the role of epigenetic regulatory proteins during PGC differentiation. We used two knockouts, the first being UHRF1 which interacts with DNMT1 to promote DNA methylation maintenance and the second being EED, a key component of PRC2 which adds the repressive H3K27me3. Through this we identified that while UHRF1 appears to play no role in regulating the PGC stage of germline development, it is necessary for the viability of the spermatogonial stem cell population within the adult testes. In the case of EED, we identified that PRC2 is essential for regulating the timing of sex-specific differentiation in PGCs as well as a novel role for H3K27me3 in X chromosome decompensation within the embryonic testis. Finally, we identified a dual enrichment of H3K27me3 and DNA methylation within the promoters of gametogenesis genes at the time of PGC specification from the mouse epiblast. This provides an exciting glimpse into the complex interactions between the epigenetic regulatory networks that direct PGC differentiation. Further work will need to be conducted to identify the extent of these epigenetic regulator interactions in human PGCs and to apply these findings into developing better methods to more accurately recapitulate human PGC differentiation in vitro
Recommended from our members
Bone Morphogenetic Protein 4-Responsive Transcription Factor Activating Enhancer Binding Protein 2 Positive Progenitor Cells Undergo Fate Diversification to Give Rise to Human Amnion, Germline, and Mesoderm
No full textThe unilaminar embryonic disc, undergoes complex morphogenesis to create a multi-dimensional human gastrula. This process endows early lineage specification and spatial identities that begin at the posterior end of the disc to create a coordinate system for embryogenesis. By adding Basement Membrane Extract (BME) to the media, we demonstrate that a three-dimensional (3D) extra-cellular matrix (ECM)-driven amnion model enables simultaneous induction of primordial germ cell-like cells (PGCLCs). We further demonstrate that a new two-dimensional (2D) ECM-driven model, Geltrex-incorporated Medium Overlay (GiMO), enables the study of lineage specification and an epithelial to mesenchymal transition (EMT), where early mesodermal cells are forming as a consequence of EMT. Crucially, we show that transcription factor activating enhancer binding protein 2 (TFAP2A) is a progenitor of all three embryonic and extraembryonic lineages that emerge from the posterior peri-implantation epiblast (amnion, germline, and mesoderm). Through the use of lineage tracing via reporter lines, we provide further insight into early human developmental dynamics
Recommended from our members
Transposable Elements and their Epigenetic Regulators Are Necessary to Orchestrate the Transcriptome of the Developing Germline
No full textIn order to maintain organismal fitness, the germline must be able to transmit high-fidelity genomic information from one generation to the next. Transposable Elements (TEs), genomic elements which are capable of mobility within the genome, pose a risk to this process. Within a given species, only a small subset of TEs remain mobile. These mobile elements are often evolutionarily young compared to others in the genome and must be durably silenced during germline development so that their mobility does not impact fitness of the offspring. Conversely, evolutionarily older TEs lose their ability to mobilize due to deterioration or loss of pro-viral regions, which encode viral proteins. In tandem with their deterioration, TEs are often “endogenized” or “domesticated” and become cis-regulatory elements, for instance by harboring binding motifs of transcription factors, repressors, or insulators. As a result, a conserved property of these TEs is their capacity to embed themselves into the cis-regulatory network of tissues, especially in the early embryo. Primordial Germ Cells (PGCs) are embryonic precursors to the adult germline. In mammals, PGCs are specified early in embryonic development ( at embryonic day (E) E6.25 in mice and ~E11-12 in humans). The transcriptional networks that drive and reinforce acquisition of PGC identity have been well interrogated using in vitro models of PGC specification in the mouse (m) and human (h) via generation of PGC-Like Cells (PGCLCs). Here we show that an evolutionarily young TE subfamily, LTR5Hs, is epigenetically remodeled during human PGC specification and bound by critical human PGC factors, supporting the hypothesis these elements act as enhancers. We go on to show that ectopic epigenetic repression of LTR5Hs results in inefficient human PGCLC induction, thus establishing enhancer activity of LTR5Hs as necessary for human PGC specification. These findings demonstrate that LTR5Hs is necessary for specification of the human germline.
After specification, PGCs will migrate while undergoing epigenetic reprogramming including DNA demethylation and imprint erasure. In the mouse, PGCs migrate into the developing genital ridge, undergo rapid mitotic proliferation and differentiate into either testicular germ cells, which become pro-spermatogonia, or into meiotic germ cells which will undergo meiosis and mature into oocytes. To ask how control of TEs impacts a later stages of PGC development, differentiation, we used an in vivo mouse model and employed PGC-specific conditional knockout of TRIM28. TRIM28 is an epigenetic scaffold necessary to repress many TEs, especially those which are evolutionarily young. We found that TRIM28 is regulated in a sex-specific manner and that TRIM28 loss in PGCs during differentiation results in upregulation genes typical of Zygotic Genome Activation, which in the mouse happens during the 2C stage of embryonic development. Upregulation of a 2C-like transcriptome results in reduced mitotic expansion, inefficient fate restriction to the adult germline, and a failure of PGCs to differentiate in males and to properly progress through meiosis in females. Thus, precise control of transposable elements is important not only for germline specification, but also to protect the germline transcriptional program as PGCs differentiate
Recommended from our members
Studying Niche Colonization and the Dynamics of Imprint Erasure During In Vitro Acquisition of Pluripotency in Germ Cells
No full textPrimordial germ cells (PGCs) are the embryonic precursors of sperm and eggs. Scalable in vitro differentiation of PGCs from pluripotent cell types has emerged as a model to study PGC biology. The in vitro PGCs (iPGCs) obtained from ESCs through the embryoid body (EB) differentiation method correspond to a pregonadal stage of PGC development. Thus, extending the applications of this model to the study of more developmentally advanced germ cells requires maturation of iPGCs to different developmental stages. However, the maturation potential of iPGCs is unknown. In order to promote maturation of male iPGCs in vitro and in vivo we use a transplantable niche model where iPGCs are aggregated with male fetal gonadal cells and cultured prior to transplantation to induce de novo formation of the germ cell niche. Initial characterization of this model without iPGCs revealed that despite the different localization of germ and Sertoli cells within gonadal aggregates before transplantation, both of these cell types are found in physical contact inside the reconstructed germ cell niche. Furthermore, we determined that both germ and Sertoli cells mature in the gonadal transplants. Analyzing iPGCs in gonadal aggregates in vitro, we find that unlike the pregonadal PGCs of the E9.5 embryo, iPGCs do not express the gonadal stage marker MVH indicating they do not mature in vitro. Although transplantation of gonadal aggregates did not support niche colonization by either in vitro or embryo-derived exogenous germ cells, unlike iPGCs, PGCs from the embryo do not survive in the transplants. The surviving iPGCs remain in the extratubular space, disrupting the transplant morphology, and express proteins that uncommon in the germline indicating they have acquired a different cellular identity. To further examine similarities and differences between embryonic and ESC-derived PGCs, we analyzed their epigenetic stability at imprinting control centers (ICCs) in a PGC reprogramming assay in vitro using sequencing of bisulfite-treated DNA amplified by PCR. We find that both embryo and ESC-derived PGCs erase cytosine methylation at the Snrpn ICC. However, unlike in the PGCs of the embryo, this demethylation is faster and not stably maintained. Furthermore, we find that ESC-derived PGCs form colonies faster than E9.5 PGCs and this correlates with significantly less expression of Lats2, which indicates a lower barrier to reprogramming. Our results also indicate that, compared to E9.5 PGCs, iPGCs have significantly higher expression of the pluripotency-associated transcription factor Klf4. Interestingly, using this in vitro PGC reprogramming assay we evaluated the dynamics of imprint erasure during reprogramming embryonic PGCs in vitro, which to this date was unknown. We determined that cytosine methylation at ICCs is erased in E9.5 PGCs during the first four days of reprogramming. This is followed by de novo DNA methylation during the last days of PGC reprogramming in vitro. The extent of remethylation is locus-specific resulting in pluripotent EGCs with hypo and hypermethylated ICCs
The relationship of academic performance and admission requirements to teacher effectiveness of practice teachers at Clark College
No full textThe purpose of this study is to determine the relationship between an admission requirement, academic performance, and supervisors' ratings and teaching effectiveness of practice teachers. Nine null hypotheses are tested. Hypothesis one through three indicated that there would be no statisticaly significant relationship between (1) an admission requirement and teaching plans and materials, (2) an admission requirement and classroom procedures, and (3) an admission requirement and interpersonal skills of practice teachers. Null hypotheses four through six indicate that there would be no statistically significant relationship between (4) cumulative grade point average and teaching plans and materials, (5) cumulative grade point average and classroom procedures, and (6) cumulative grade point average and interpersonal skills of practice teachers. Null hypotheses seven through nine indicated that there would be no statistically significant relationship between (7) supervisors' ratings and teaching plans and materials, (8) supervisors' ratings and classroom procedures, and (9) supervisors' ratings and interpersonal skills of practice teachers. The research design for this study was a correlational study with a focus on descriptive research. The sample population consisted of 49 education majors who completed the Teacher Education Program at Clark College during the years 1980-83. Permission to obtain the data necessary for this study was obtained from the appropriate school authorities at Clark College. Grade point averages and scholastic aptitude test scores were obtained from the Registrar's office. Supervisors' ratings and TPAI data were obtained for the Department of Education at Clark College. It is concluded that measures of admission requirement, cumulative grade point average, and supervisor ratings are not good predictors of success in students teaching at Clark College
Recommended from our members
Naive Pluripotency in Human Primordial Germ Cell Development
No full textInfertility is caused by a multitude of genetic and environmental factors, and many who seek treatment are unresponsive to in vitro fertilization. Treatments such as these are costly, as well as physically and emotionally demanding, so it is critical to better understand the individual underlying causes of infertility to improve patient care. Germ cells give rise to the next generation and are responsible for passing along genetic and epigenetic information, and abnormalities in germ cell development can result in infertility. In addition, mutations in certain genes related to germ cell formation can contribute to their improper development, resulting in germ cell tumor formation or other developmental disorders in their future offspring. In these studies, we investigated the earliest cell type in the human germline, known as human Primordial Germ Cells (hPGCs). We developed models and techniques to represent hPGC formation and identified genes of interest to investigate their potential role in healthy hPGC identify and development. Two genes, KLF4 and TFCP2L1, are upregulated in hPGCs and are known to have a role in naive pluripotency. Given this, and other shared characteristics between the hPGCs and naive pluripotency, we hypothesized that KLF4 and TFCP2L1 have a role in hPGC development. We first used CRISPR/Cas9 gene editing technology at the human embryonic stem cell (hESC) stage of our PGC-like cell (hPGCLC) aggregate model, to demonstrate the requirement of certain genes including EOMES in PGCLC induction. Then, we developed an extended culture system to study hPGCLCs in vitro. In extended culture, hPGCLCs begin the process of reprogramming to represent the later hPGC stage, where we can study characteristics such as cell cycle and survival. We knocked out KLF4 and TFCP2L1 separately in hESC lines, and discovered that neither gene is required for PGCLC induction, but each has an anticipated role in re-acquiring naive pluripotency in the hESC state. Despite these functional null mutations, neither gene is required for progression of hPGCLC development in vitro including in progression through S phase as measured by Edu, or for PGCLC colony survival. Given this, we conclude that the naive pluripotent states observed in the pre-implantation and hPGC stages are uniquely controlled, and that these transcription factors may serve alternate roles in hPGC development. Possible roles include protection against germ cell tumors, or facilitation of later and advanced stage hPGC development. Further work will address these possibilities, using animal and stem cell models of gonadal development to study later stage progression
Author response
No full textMethylation of cytosines (5 me C) is a widespread heritable DNA modification. During mammalian development, two global demethylation events are followed by waves of de novo DNA methylation. In vivo mechanisms of DNA methylation establishment are largely uncharacterized. Here, we use Saccharomyces cerevisiae as a system lacking DNA methylation to define the chromatin features influencing the activity of the murine DNMT3B. Our data demonstrate that DNMT3B and H3K4 methylation are mutually exclusive and that DNMT3B is co-localized with H3K36 methylated regions. In support of this observation, DNA methylation analysis in yeast strains without Set1 and Set2 shows an increase of relative 5 me C levels at the transcription start site and a decrease in the gene-body, respectively. We extend our observation to the murine male germline, where H3K4me3 is strongly anti-correlated while H3K36me3 correlates with accelerated DNA methylation. These results show the importance of H3K36 methylation for gene-body DNA methylation in vivo
Recommended from our members
Primordial Germ Cell Differentiation in Vitro: A Model for Understanding Epigenetic Reprogramming and Genome-Wide DNA Demethylation in Mouse Primordial Germ Cells
No full textSperm and oocytes are terminally differentiated, sex-specific germ cells, which, upon fertilization will generate a new embryo and leads to species propagation by sexual reproduction. Though fated only to generate eggs or sperm, germ cells have the unique property to imbue zygotes with totipotent capacity, which facilitates the formation of all tissues the embryo will need to survive to adulthood. These characteristics are facilitated by germ cells' ability to pass on genetic information to the next generation, as well as their capacity to initiate genome-wide reorganization and removal of epigenetic information inherited by germ cells during embryogenesis. It is hypothesized that remodeling of this epigenetic information is essential to drive proper embryo development. While these events are not well understood, it is known that the events that underlie these unique properties are initiated in early development, shortly after the germ line is established as a pool of primordial germ cells (PGCs). Efforts to unravel these mechanisms that underlie totipotency in germ cells have been limited due to the inability to isolate, study, and manipulate PGCs. To overcome this obstacle, we hypothesized that PGCs cells could be differentiated from pluripotent embryonic stem cells, and that these cells would serve as a surrogate cell type for the study of PGC biology.Establishing a new model of lineage differentiation from embryonic stem cells required the development of assays and criteria to rigorously test identity, developmental staging, and epigenetic progression to determine if an in vitro model is able to recapitulate features of endogenous PGCs. To accomplish this, we developed a scalable and transgene-free method to differentiate immature PGCs in vitro using the cell surface markers SSEA1 and cKit that are developmentally and epigenetically reminiscent of immature PGCs. We applied existing assays to validate PGC identity, and devised a new stringent assay based on genetic deletion of a known PGC determinant. We developed a single-cell gene expression methodology to compare gene expression signatures of in vitro derived PGCs and endogenous PGCs, and identified novel criteria to define PGC identity from early endogenous PGCs and in vitro-generated PGCs.We next used in vitro PGC differentiation to investigate genome-wide DNA demethylation, one of the first epigenetic reprogramming events undertaken by early PGCs. By combining the scalability of this differentiation system with next generation methylation sequencing techniques, we generated the first DNA methylation maps of in vitro derived PGCs, and determined with sequence-specific information that DNA demethylation is genome-wide and likely to involve loss of DNA methylation as a consequence of cell division. We also investigated potentiators of active DNA methylation loss, including the Tet proteins, and their roles in early PGC development.Finally, we applied single cell gene expression technology to define developmental progression of human PGCs isolated from the gonads of fetuses from elective terminations. We identified a common progenitor stage of PGC development in the human fetal gonad. Furthermore, we adapted our single cell gene expression approaches to interrogate differentiation strategies in the generation of the common human PGC progenitor in vitro. Together, we have developed a differentiation system to ask questions about epigenetic progression in early germ cells, and have utilized single cell gene expression technology and genomics to characterize seminal events in the epigenetic reprogramming of human and mouse germ cells
Recommended from our members
PRMT5 is an essential survival factor for ground state pluripotency and primordial germ cells: From primordial germ cell differentiation in vitro to mammalian germline in vivo
No full textOur ancestors and our children are linked by a single, special cell lineage called the germline. Germ cells are tasked with the role to accurately pass DNA from one generation to the next. In today's society infertility is an important health concern as it is estimated to affect 10% of the reproductive age population. In many cases infertility can be traced to abnormal germline cell development. The early events of germline formation are difficult to study because of limited materials, especially low germ cell numbers in the process of embryogenesis.To overcome the challenge of limited materials, an in vitro method to derive cell types faithfully reporting in vivo phenotypes needs to be devised to facilitate the research process. The key results from the in vitro model should then be evaluated carefully in vivo to show relevance in development. My thesis first focused on establishing an in vitro model to recapitulate early development of germline, by differentiating embryonic stem cells (ESCs) to form in vitro primordial germ cells (iPGCs), in a dish. This method allows good number of germ cells to be produced for molecular and biochemical studies. Using the in vitro model, key germline modifiers for ESC maintenance and germ cell formation were identified, one of which is protein arginine methyltransferase 5 (PRMT5). PRMT5 is a type II arginine methyltransferase (PRMT) that modifies symmetrical dimethylated arginines (SDMAs) on proteins, which substrates include histone H2A and H4, as well as the Sm proteins involved in RNA splicing. However, the molecular function of PRMT5 in ground state ESCs remains unknown. In our study, for the first time, we generated an inducible knock out of Prmt5 in ESCs cultured in ground state pluripotency (cultured with 2i inhibitors and leukemia inhibitory factor). We characterized PRMT5's role in ESCs through the method of paired-end RNA sequencing to show that PRMT5 affects cell survival, proliferation and chromatin organization etc., by affecting RNA spicing. By generating a conditional knock out to specifically deplete PRMT5 in the germline in vivo, we showed that PRMT5 is necessary for mammalian PGC formation. Taken together, we developed a differentiation system to ask questions about key factors regulating ESC and germline development. One of the key factors we identified is PRMT5. The role of PRMT5 was scrutinized using paired-end RNA sequencing in the inducible knock out ESCs, suggesting that PRMT5 is necessary to ensure normal splicing in "RNA processing", "DNA damage response" and "chromatin modification" for cell to survive. Finally, we utilized in vivo mouse model to validate events that happen in vitro, showing that PRMT5 is indeed a survival factor both for ground state pluripotency and mammalian germline
Recommended from our members
Mechanisms of demethylation in primordial germ cells and the importance of stage-specific demethylation in safeguarding against precocious differentiation
No full textPrimordial germ cells (PGCs) are the cellular precursors for mature gametes which are responsible for giving rise to embryonic development and the next generation of PGCs. During development, proper PGC differentiation results in high quality gametes, which are essential for normal development and future child health. Problems during PGC differentiation can lead to impaired fertility, poor quality germ cells, or developmental defects in the next generation. One of the essential events that occurs during PGC development is whole-genome reprogramming of DNA methylation. The reprogramming of DNA methylation in the context of PGC development is required for appropriate cell lineage differentiation. This process is essential in establishing the correct epigenetic landscape which will impact differentiation, and maturation of PGCs. My goal is to focus on two aspects of genome-wide reprogramming in Primordial Germ Cells (PGCs). First, the molecular mechanisms of DNA demethylation during the gonadal stage of development, as well as the mechanisms involved protecting specific loci from demethylation in order to allow for correct temporal expression of germ cell genesPrimordial germ cells (PGCs) undergo genome-wide demethylation in two distinct stages. Stage 1 consists of global demethylation before embryonic (e) day e9.5 of mouse development. Stage 2 The second phase occurs once PGCs colonize the genital ridge between e10.5-e13.5, and happens in a temporal and locus-specific manner. Results indicate that the second phase is regulated in part by Ten eleven translocation (Tet) protein Tet 1, and conversion of 5-methylcytosine (5mC) to 5-hydroxymethylcytosine (5hmC) at specific loci. The major working model for Tet-dependent DNA demethylation involves replication-coupled loss of methylated cytosines from the genome. However an alternate model would predict active removal of 5hmC at specific loci independent of cell division. In order to address this directly, we have established a new organ culture model involving the growth of dissected aorta/gonad/mesonephros (AGM) tissues isolated from the mouse embryo at e10.5. During three days of organ culture, we show that PGCs divide on average three times. We also show that in the background of global hypomethylation established in phase 1, PGCs isolated from the organ culture undergo locus-specific DNA demethylation, and 5hmC reorganization, and this occurs within three days. Using this model we have targeted the PGC cell cycle using a P-AKT inhibitor, and have determined that imprint erasure can happen in proliferation dependent and independent ways depending on the genomic locusAlternatively, during the removal of DNA methylation in stage 1, some loci are protected from demethylation and the mechanism for this process remains unknown. In the current study we tested the hypothesis that Dnmt1 is responsible for maintaining methylation by being recruited at specific genomic sites during whole genome demethylation. To address this, we created a conditional germline knockout of Dnmt1. Analysis of Dnmt1 conditional knockout (DCKO) PGCs revealed that Dnmt1 is the major methyltransferase that functions during whole genome demethylation to maintain DNA methylation at discreet genomic regions including intracisternal A particle (IAP) transposons, as well as maternal and paternal imprinting control centers. Furthermore, the absence of Dnmt1 results in precocious differentiation that leads to germ cell loss in both male and female embryos. Taken together, we propose a model in which maintenance of cytosine methylation by Dnmt1 is essential to maintain cytosine methylation at discreet regions of the genome during whole genome DNA methylation reprogramming
- …
