Regulation of human germline commitment by DMRT1 [CUT&RUN]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE223035 | GEO | 2023/07/18
REPOSITORIES: GEO
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Project description:Nascent human primordial germ cell-like cells (PGCLCs) can be specified with BMP2 or BMP4 together with SCF and EGF from germline competent embryonic stem cells which are maintained in the presence of inhibitors for GSK3, MEK, p38 and JNK together with FGF2, TGFβ and LIF (4i ESC). Here we discovered that Activin A and retinoic acid treatment after PGC specification induced genes expressed in migrating and gonadal PGCs in vivo, including DMRT1 and CDH5. Ectopic expression of DMRT1 and SOX17 resulted in repression of pluripotent genes as well as induction of DAZL and a subset of mitotic arrest PGC genes. Our findings provide novel insights on the roles of DMRT1 for the transition from nascent PGCs towards germline determination and molecular mechanisms of early human germline development.
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Project description:Nascent human primordial germ cell-like cells (PGCLCs) can be specified with BMP2 or BMP4 together with SCF and EGF from germline competent embryonic stem cells which are maintained in the presence of inhibitors for GSK3, MEK, p38 and JNK together with FGF2, TGFβ and LIF (4i ESC). Here we discovered that Activin A and retinoic acid treatment after PGC specification induced genes expressed in migrating and gonadal PGCs in vivo, including DMRT1 and CDH5. Ectopic expression of DMRT1 and SOX17 resulted in repression of pluripotent genes as well as induction of DAZL and a subset of mitotic arrest PGC genes. Our findings provide novel insights on the roles of DMRT1 for the transition from nascent PGCs towards germline determination and molecular mechanisms of early human germline development.
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Project description:Nascent human primordial germ cell-like cells (PGCLCs) can be specified with BMP2 or BMP4 together with SCF and EGF from germline competent embryonic stem cells which are maintained in the presence of inhibitors for GSK3, MEK, p38 and JNK together with FGF2, TGFβ and LIF (4i ESC). Here we discovered that Activin A and retinoic acid treatment after PGC specification induced genes expressed in migrating and gonadal PGCs in vivo, including DMRT1 and CDH5. Ectopic expression of DMRT1 and SOX17 resulted in repression of pluripotent genes as well as induction of DAZL and a subset of mitotic arrest PGC genes. Our findings provide novel insights on the roles of DMRT1 for the transition from nascent PGCs towards germline determination and molecular mechanisms of early human germline development.
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Project description:The DND microRNA-mediated repression inhibitor 1 (DND1) is a conserved RNA binding protein (RBP) and plays an important role in survival and maintenance of primordial germ cells (PGCs) and the development of the male germline in zebrafish and mice. It was shown to be expressed in human pluripotent stem cells (PSCs), PGCs, and spermatogonia, but little is known about its specific role in pluripotency and human germline development. Here we use CRISPR/Cas mediated knockout and PGC-like cell (PGCLC) differentiation in human iPSCs to analyse if DND1 (1) plays a role in maintaining pluripotency and (2) in specification of PGCLCs. We generated several clonal lines with biallelic loss of function mutations and analysed their potential to differentiate towards PGCLCs and their gene expression on RNA and protein level via bulk RNA sequencing and mass spectrometry. The generated knockout iPSCs showed no differences in pluripotency gene expression, proliferation nor trilineage differentiation potential, but yielded reduced numbers o PGCLCs compared to their parental iPSCs. RNAseq analysis in PGCLCs showed significantly reduced expression of genes associated with cellular developmental processes and cell differentiation in knockout cells, including known markers for PGCs (NANOS3, SOX17, PRDM1, EPCAM) and naïve pluripotency (TFCP2L, DNMT3L).
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Project description:In vivo reprogramming provokes a wide range of cell fate conversion. Here, we discover that in vivo induction of higher levels of OSKM in mouse somatic cells leads to increased expression of primordial germ cell (PGC)-related genes and provokes genome-wide erasure of genomic imprinting, which takes place exclusively in PGCs. Moreover, the in vivo OSKM reprogramming results in development of cancer that resembles human germ cell tumors. Like a subgroup of germ cell tumors, propagated tumor cells differentiate into trophoblasts. Moreover, these tumor cells give rise to induced pluripotent stem cells (iPSCs) with expanded differentiation potential into trophoblasts. Remarkably, the tumor-derived iPSCs are able to contribute to non-neoplastic somatic cells in adult mice. Mechanistically, DMRT1, which is expressed in PGCs, drives the reprogramming and propagation of the tumor cells in vivo. Furthermore, the DMRT1-related epigenetic landscape is associated with trophoblast competence of the reprogrammed cells and provides a therapeutic target for germ cell tumors. These results reveal an unappreciated route for somatic cell reprogramming and underscore the impact of reprogramming in development of germ cell tumors.
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Project description:Primordial Germ Cells (PGCs) are a rare population of cells specified early in embryonic development that give rise to gametes - sperm and egg cells. Current in vitro systems to derive PGC-like cells (PGCLCs) from pluripotent cells use large embryoid bodies, exposed to signalling cocktails, that do not recapitulate the native embryonic environment during PGC formation. Here we show that mouse gastruloids, three-dimensional in vitro models of gastrulation, generate a population of Gastruloid-derived PGC-like cells (Gld-PGCLCs) that resemble early PGCs in vivo. Importantly, the conserved organisation of mouse gastruloids leads to coordinated spatial and temporal localisation of Gld-PGCLCs relative to surrounding somatic cells, even in the absence of exogenous PGC-specific signalling or extraembryonic tissues. In gastruloids, self-organised interactions between cells and tissues, including the endodermal epithelium, enables the specification, migration, and subsequent maturation of a pool of Gld-PGCLCs. As such, mouse gastruloids represent a new source of PGCLCs in vitro and, due to their inherent co-development, serve as a novel model of dynamic PGC development within integrated tissue environments.
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Project description:Reconstitution of female germ-cell development in vitro is a key challenge in reproductive biology and medicine. We show here that female (XX) embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) in mice are induced into primordial germ cell-like cells (PGCLCs), which, when aggregated with female gonadal somatic cells as reconstituted ovaries, develop pre-meiotic germ cell characteristics, including X-reactivation, imprint erasure, and cyst formation. Upon transplantation under ovarian bursa, PGCLCs in the reconstituted ovaries mature into germinal vesicle-stage oocytes, which, through in vitro maturation and fertilization, contribute to fertile offspring. Our culture system serves as a robust foundation for the investigation of key properties of female germ cells, including the acquisition of totipotency, and for the reconstitution of whole female germ-cell development in vitro. We performed expression analysis of female PGC-like cells [PGCLCs; day 3 (d3), day 6 (d6), and day 3 followed by day 6 in aggregation with female embryonic day (E) 12.5 gonadal somatic cells (d3ag6)] in comparison to in vivo embryonic PGCs (E12.5 female PGCs and E9.5 PGCs) and male d6 PGCLCs. PGCLCs were marked with fluorescence of Blimp1-mVenus, stella-ECFP transgenic reporters (BVSC). PGCs were marked with fluorescence of the stella-EGFP transgenic reporter.
2012-11-05 | E-GEOD-40716 | biostudies-arrayexpress
Project description:The generation of properly functioning gametes in vitro, a key goal in developmental/reproductive biology, requires multi-step reconstitutions of complex germ cell development. Based on the logic of primordial germ cell (PGC)-specification, we demonstrate here the generation of PGC-like cells (PGCLCs) in mice with robust capacity for spermatogenesis from embryonic stem cells (ESCs) and induced pluripotent stem cells (iPSCs) through epiblast-like cells (EpiLCs), a cellular state highly similar to pre-gastrulating epiblasts, but distinct from epiblast stem cells (EpiSCs). The global transcription profiles, epigenetic reprogramming, and cellular dynamics during PGCLC induction from EpiLCs are a meticulous capture of those associated with PGC specification from the epiblasts. Furthermore, we identify Integrin-beta 3 and SSEA1 as markers that purify PGCLCs with spermatogenic capacity free from tumorigenic undifferentiated cells. With the reconstitution of PGC specification pathway from the naive inner cell mass state, our study defines a paradigm for the essential step of in vitro gametogenesis. We performed this analysis to reveal the characters of the cells that we created in this study, epiblast-like cells (EpiLCs) and primordial germ cells-like cells (PGCLCs). Because EpiLCs were induced from embryonic stem cells (ESCs), and equivalent to pre-gastrulating epiblast (embryonic day [E] 5.5-6.0) in vivo (embryo), ESCs and epiblast were included in this analysis. Epiblast stem cells (EpiSCs) are a culture cell type derived from epiblast, and were also included. PGCLCs were supposed to be equivalent to E9.5 PGCs based on reporter fluorescent transgene expressions and epigenetic properties, and therefore E9.5 PGCs were also inckuded in this analysis. Because epiblast and E9.5 PGCs are of a small number of cells in embryos (a few hundred to thousand cells), cDNAs were amplified with a quantitative global PCR method (Kurimoto et al., 2006, Nucleic Acids Research) for microarray analyses. We took two biological replicate for each cell type.
2011-08-04 | E-GEOD-30056 | biostudies-arrayexpress
Project description:Primordial germ cells (PGCs) are the embryonic precursors to egg and sperm. When removed from the embryonic gonad, PGCs can give rise to embryonic germ cell lines (EGs), pluripotent stem cells that display most of the characteristics of embryonic stem cells (ESCs) including the ability to form teratomas and to contribute to chimera formation. In mice, EG cells can be derived between E8.5 up to E12.5 of embryonic development, at which point the PGCs undergo sexual differentiation and in the male transition into unipotent gonocytes. Dazl, a germ cell-specific RNA-binding protein, is specifically expressed in developing PGCs and is required for proper germ cell development. Dazl knockout mice are infertile, but the molecular mechanisms underlying this phenotype are still unknown. Here we demonstrate that Dazl localizes in granular structures in mammalian PGCs but not in ESCs. We demonstrate Dazl plays a central role in a large mRNA/protein interactive network that includes members of Fragile-X family RNA-binding proteins. We demonstrate that Dazl and Fxr1 play a central role in these granules and directly regulate the translation of specific core pluripotency factors, including Sox2 and Suz12. RNA species interacting specifically with Dazl in primordial germ cells were identified by RNA-IP microarray analysis. This dataset contains data from UV-crosslinked RNA-IPs from in vitro PGC lysates. UV-crosslinked RNA-IP (anti-GFP and anti-PABP1) experiments from in vitro derived primordial germ cells expressing Dazl-GFP-V5. Input Total RNA and polyA-binding protein 1 (PABP1) IP were used as controls. Control and IP-enriched RNA samples were analyzed by Agilent Mouse Whole Genome 4X44K one-color microarrays. Two replicates for each condition were done.
2013-12-31 | E-GEOD-39541 | biostudies-arrayexpress