Project description:Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial- mesenchymal transition (EMT) is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs Snai1 does not respond to TGFα or BMP4 signalling but it is induced by retinoic acid (RA) treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα the dissociation of the Polycomb repressor compex 2 (PRC2) which results in the decrease of H3K27me3 and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c-Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes. microarray analysis of embryonic stem cells (ESC) expressing Snail-ER at various time points of induction with 4-OHT
Project description:Although much is known about the pluripotency self-renewal circuitry, the molecular events that lead embryonic stem cells (ESCs) exit from pluripotency and begin differentiation are largely unknown. We found that the zinc finger transcription factor Snai1, involved in gastrulation and epithelial- mesenchymal transition (EMT) is already expressed in the inner cell mass of the preimplantation blastocysts. In ESCs Snai1 does not respond to TGFα or BMP4 signalling but it is induced by retinoic acid (RA) treatment, which induces the binding, on the Snai1 promoter, of the retinoid receptors RARγ and RXRα the dissociation of the Polycomb repressor compex 2 (PRC2) which results in the decrease of H3K27me3 and the increase of histone H3K4me3. Snai1 mediates the repression of pluripotency genes by binding directly to the promoters of Nanog, Nr5a2, Tcl1, c-Kit, and Tcfcp2l1. The transient activation of Snai1 in embryoid bodies induces the expression of the markers of all three germ layers. These results suggest that Snai1 is a key factor that triggers ESCs exit from the pluripotency state and initiate their differentiation processes.
Project description:Embryonic stem cells (ESC) are derived from blastocyst-stage embryos and are thought to be functionally equivalent to the inner cell mass in their developmental potential. ESCs pluripotency is maintained through a complex interplay of different signaling pathways and a network of transcription factors, which is centered around Oct3/4, Sox2 and Nanog. Although, in general, much is known about this pluripotency self-renewal circuitry, the molecular events that lead ESC to exit from pluripotency and begin differentiation are currently less known. Retinoic acid, an active metabolite of the vitamin A (retinol), plays important and pleiotropic roles in vertebrate embryonic development and ESC differentiation. Here we demonstrate that RA promotes early steps of ESC differentiation, and that ESC increase their capacity to synthesize RA during spontaneous differentiation as embryoid bodies, up-regulating the RA biosynthetic pathway components RDH1, RDH10, ADH3, RALDH2, and CRABP2. Microarray derived from total RNA of mESC not treated or treated with all-trans retinoic acid (ATRA) for 2 hours.
Project description:A unique embryonic stem cells showing naïve state was established from primplantation mouse blastocyst but maintaind their self renew under FGF2 stimulus condition We used microarray to compare gene expression patterns of our pluripotent stem cell line (named FGF-ESC) with ESCs or EpiSCs in addition to inner cell mass from E3.5 preimplantation blastocyst as in vivo control. Total RNA was extracted from FGF-ESC, ESC, EpiSC and inner cell mass from E3.5 preimplantation blastocyst, and served for microarray analysis using Affymetrix Mouse Gene 2.1 Array
Project description:A bioenergetic balance between glycolysis and mitochondrial respiration is particularly important for stem cell fate specification. It however remains to be determined whether undifferentiated spermatogonia switch their preference of bioenergy production during differentiation. In this study, we found that ATP generation in spermatogonia was gradually increased upon retinoic acid-induced differentiation. To accommodate this elevated energy demand, retinoic acid signaling concomitantly switched ATP production in spermatogonia from glycolysis to mitochondrial respiration, accompanied by increased levels of reactive oxygen species. In addition, inhibition of glucose conversion to glucose-6-phosphate or pentose phosphate pathway blocked the formation of c-Kit+ differentiating germ cells, suggesting that metabolites produced from glycolysis are required for spermatogonial differentiation. We further demonstrated that the expression levels of several metabolic regulators and enzymes were significantly altered upon retinoic acid-induced differentiation by both RNA-seq analyses and quantitative proteomics. Taken together, our data unveil a critically regulated bioenergetic balance between glycolysis and mitochondrial respiration which is required for spermatogonial proliferation and differentiation.
Project description:Preimplantation embryo development is a precisely regulated process organized by maternally inherited and newly synthesized proteins. Recently, some studies have reported that blastocyst-like structures, named blastoids, can be generated from mouse ESCs (embryonic stem cells) or EPSCs (extended pluripotent stem cells). In this study, to explore the dynamic expression characteristics of proteins and their PTMs in mouse EPS blastoids, we revealed the protein expression profile of EPS-blastoids and metabolite characteristics by TMT-based quantitative mass spectrometry (MS) strategy. Furthermore, the protein phosphorylation sites were identified to show the phosphoproteomic analysis in blastoids compared with mouse early embryos. Above all, our study revealed the protein expression profile of EPS blastoids compared with mouse embryos during preimplantation development and indicated that glucose metabolism is key to blastoid formation.
Project description:Among its many roles in development, retinoic acid determines the anterior-posterior identity of differentiating motor neurons by activating Retinoic Acid Receptor (RAR)-mediated transcription. RAR is thought to bind the genome constitutively, and only induce transcription in the presence of the retinoid ligand. However, little is known about where RAR binds to the genome or how it selects target sites. We tested the constitutive RAR binding model using the retinoic acid-driven differentiation of mouse embryonic stem cells into differentiated motor neurons. We find that retinoic acid treatment results in widespread changes in RAR genomic binding, including novel binding to genes directly responsible for anterior-posterior specification, as well as the subsequent recruitment of the basal polymerase machinery. Finally, we discovered that the binding of transcription factors at the embryonic stem cell stage can accurately predict where in the genome RAR binds after initial differentiation. We have characterized a ligand-dependent shift in RAR genomic occupancy at the initiation of neurogenesis. Our data also suggests that enhancers active in pluripotent embryonic stem cells may be preselecting regions that will be activated by RAR during neuronal differentiation. The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid. Here, ChIP-seq is used to profile the genome-wide occupancy of RAR isofroms both immediately prior to and during exposure of the cells to retinoic acid. ChIP-seq is also used to profile the genomic occupancy of Pol2 with phosphorylated serine 5 (Pol2-S5P) and phosphorylated serine 2 (Pol2-S2P) after exposure to retinoic acid.