Tracing the transitions from pluripotency to germ cell fate with CRISPR screening
Ontology highlight
ABSTRACT:
ORGANISM(S): unidentified plasmid Mus musculus
PROVIDER: GSE117473 | GEO | 2018/10/01
REPOSITORIES: GEO
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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).
2021-12-10 | PXD023802 | Pride
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155069 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155062 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155058 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155015 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155079 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155088 | GEO
Project description:Germline specification in mammals occurs through an inductive process whereby competent cells in the post-implantation epiblast differentiate into primordial germ cells (PGC). The intrinsic factors that endow epiblast cells with the competence to respond to germline inductive signals remain unknown. Single-cell RNA sequencing across multiple stages of an in vitro PGC-like cells (PGCLC) differentiation system shows that PGCLC genes initially expressed in the naïve pluripotent stage become homogeneously dismantled in germline competent epiblast like-cells (EpiLC). In contrast, the decommissioning of enhancers associated with these germline genes is incomplete. Namely, a subset of these enhancers partly retain H3K4me1, accumulate less heterochromatic marks and remain accessible and responsive to transcriptional activators. Subsequently, as in vitro germline competence is lost, these enhancers get further decommissioned and lose their responsiveness to transcriptional activators. Importantly, using H3K4me1 deficient cells, we show that the loss of this histone modification reduces the germline competence of EpiLC and decreases PGCLC differentiation efficiency. Our work suggests that, although H3K4me1 might not be essential for enhancer function, it can facilitate the (re)activation of enhancers and the establishment of gene expression programs during specific developmental transitions.
2020-07-27 | GSE155083 | GEO
Project description:Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. Refer to individual Series
2016-01-11 | E-GEOD-71933 | biostudies-arrayexpress
Project description:Nanog, a core pluripotency factor in the inner cell mass of blastocysts, is also expressed in unipotent primordial germ cells (PGC) in mice1, where its precise role is yet unclear2-4. We investigated this in an in vitro model, where naïve pluripotent embryonic stem cells (ESCs) cultured in bFGF/ActivinA develop as epiblast-like cells (EpiLCs), and gain competence for PGC-like fate5. Consequently, bone morphogenetic protein (BMP4), or ectopic expression of key germline transcription factors Prdm1/ Prdm14/ Tfap2c, directly induce PGC-like cells (PGCLCs) in EpiLCs, but not in ESCs6-8. Here we report an unexpected discovery that Nanog alone can induce PGCLCs in EpiLCs, independently of BMP4. We propose that following the dissolution of the naïve ESC pluripotency network during establishment of EpiLCs9,10, the epigenome is reset for cell fate determination. Indeed, we found genome-wide changes in NANOG binding pattern between ESCs and EpiLCs, indicating epigenetic resetting of regulatory elements. Accordingly, we show that NANOG can bind and activate enhancers of Prdm1 and Prdm14 in EpiLCs in vitro; BLIMP1 (encoded by Prdm1) then directly induces Tfap2c. Furthermore, while SOX2 and NANOG promote the pluripotent state in ESCs, they show contrasting roles in EpiLCs since Sox2 specifically represses PGCLC induction by Nanog. This study demonstrates a broadly applicable mechanistic principle for how cells acquire competence for cell fate determination, resulting in the context-dependent roles of key transcription factors during development. Nanog ChIP-seq
2016-01-11 | E-GEOD-71932 | biostudies-arrayexpress