Project description:The separation of the first two lineages - trophectoderm (TE) and inner cell mass (ICM) - is a crucial event in the development of the early embryo. The ICM, which constitutes the pluripotent founder cell population, develops into the embryo proper, whereas the TE, which comprises the surrounding outer layer, supports the development of the ICM before and after implantation. Cdx2, the first transcription factor expressed specifically in the developing TE, is crucial for the differentiation of cells into the TE, as lack of zygotic Cdx2 expression leads to a failure of embryos to hatch and implant into the uterus. However, speculation exists as to whether maternal Cdx2 is required for initiation of TE lineage separation. Here, we show that effective elimination of both maternal and zygotic Cdx2 transcripts by an RNA interference approach resulted in failure of embryo hatching and implantation, but the developing blastocysts exhibited normal gross morphology, indicating that TE differentiation had been initiated. Expression of keratin 8, a marker for differentiated TE, further confirmed the identity of the TE lineage in Cdx2-deficient embryos. However, these embryos exhibited low mitochondrial activity and abnormal ultrastructure, indicating that Cdx2 plays a key role in the regulation of TE function. Furthermore, we found that embryonic compaction does not act as a 'switch' regulator to turn on Cdx2 expression. Our results clearly demonstrate that neither maternal nor zygotic Cdx2 transcripts direct the initiation of ICM/TE lineage separation.
Project description:CDX2 plays a crucial role in the formation and maintenance of the trophectoderm epithelium in preimplantation embryos. Follistatin supplementation during the first 72?hr of in vitro culture triggers a significant increase in blastocyst rates, CDX2 expression, and trophectoderm cell numbers. However, the underlying epigenetic mechanisms by which follistatin upregulates CDX2 expression are not known. Here, we investigated whether stimulatory effects of follistatin are linked to alterations in DNA methylation within key regulatory regions of the CDX2 gene. In vitro-fertilized (IVF) zygotes were cultured with or without 10?ng/ml of recombinant human follistatin for 72?hr, then cultured without follistatin until Day 7. The bisulfite-sequencing analysis revealed differential methylation (DM) at specific CpG sites within the CDX2 promoter and intron 1 following follistatin treatment. These DM CpG sites include five hypomethylated sites at positions -1384, -1283, -297, -163, and -23, and four hypermethylated sites at positions -1501, -250, -243, and +20 in the promoter region. There were five hypomethylated sites at positions +3060, +3105, +3219, +3270, and +3545 in intron 1. Analysis of transcription factor binding sites using MatInspector combined with a literature search revealed a potential association between differentially methylated CpG sites and putative binding sites for key transcription factors involved in regulating CDX2 expression. The hypomethylated sites are putative binding sites for FXR, STAF, OCT1, KLF, AP2 family, and P53 protein, whereas the hypermethylated sites are putative binding sites for NRSF. Collectively, our results suggest that follistatin may increase CDX2 expression in early bovine embryos, at least in part, by modulating DNA methylation at key regulatory regions.
Project description:The AMP-activated protein kinase (AMPK) mediates rapid, stress-induced loss of the inhibitor of differentiation (Id)2 in blastocysts and trophoblast stem cells (TSC), and a lasting differentiation in TSC. However, it is not known if AMPK regulates other potency factors or regulates them before the blastocyst stage. The caudal-related homeodomain protein (Cdx)2 is a regulatory gene for determining TSC, the earliest placental lineage in the preimplantation mouse embryo, but is expressed in the oocyte and in early cleavage stage embryos before TSC arise. We assayed the expression of putative potency-maintaining phosphorylated Cdx2 ser60 in the oocyte, two-cell stage embryo, blastocyst, and in TSC. We studied the loss of Cdx2 phospho ser60 expression induced by hyperosmolar stress and its underlying mechanisms. Hyperosmolar stress caused rapid loss of nuclear Cdx2 phospho ser60 and Id2 in the two-cell stage embryo by 0.5?h. Stress-induced Cdx2 phospho ser60 and Id2 loss is reversed by the AMPK inhibitor compound C and is induced by the AMPK agonist 5-amino-1-?-d-ribofuranosyl-imidazole-4-carboxamide in the absence of stress. In the two-cell stage embryo and TSC hyperosmolar, stress caused AMPK-mediated loss of Cdx2 phospho ser60 as detected by immunofluorescence and immunoblot. We propose that AMPK may be the master regulatory enzyme for mediating stress-induced loss of potency as AMPK is also required for stress-induced loss of Id2 in blastocysts and TSC. Since AMPK mediates potency loss in embryos and stem cells it will be important to measure, test mechanisms for, and manage the AMPK function to optimize the stem cell and embryo quality in vitro and in vivo.
Project description:Neuroretina and retinal pigment epithelium (RPE) are differentiated from the progenitors in optic vesicles, but it is unclear when and how the two lineages are segregated. Manipulation of chick embryos reveals that the early anteroventral optic vesicle is crucial for neuroretinal development, but the molecular mechanism is unclear. Homeodomain transcription factor Six3 is required for neuroretinal specification and is dispensable for RPE formation, but the cell fates of Six3-deficient progenitors and the origins of remnant RPE are unknown. Here, we performed lineage tracing of Six3-Cre positive cells in wild-type and Six3-deficient mouse embryos. Six3-Cre positive progenies were found in a population of progenitors in the anteroventral optic pits/vesicles starting at E8.5, and were found in neuroretina, optic stalk, ventral forebrain, but not RPE, at E10.5. Six3-deletion in the small population of progenitors at E8.5 was sufficient to cause rostral expansion of Wnt8b and drastic reduction of Fgf8/MAPK signaling, ablating neuroretinal specification without affecting RPE. Lineage tracing revealed Six3-deficient progenitors at E8.5 were eventually lost and the remnant RPE was derived from Six3-Cre negative cells. Thus, Six3 in a small population of progenitors expressing Six3-Cre at E8.5 is required for neuroretinal specification via regulating cell signaling and survival in mice.
Project description:Understanding processes how the early stage kidney precursor gives rise to metanephric mesenchyme, which is a committed progenitor cells of adult kidney is important for the regeneration of kidney in vitro. The combination of fluorescent activated cell sorting (FACS) plus microarray analysis offers a powerful, efficient and effective method for the creation of global gene expression profiles of the developing kidney precursors. Those gene expression data provides insights into not only the stage specific marker genes but also the signals working in each population, which should be informative for the directed differentiation of pluripotent stem cells in vitro. Osr1-GFP knock-in mice were used to isolate kidney precursor cells from embryos at E8.5, E9.5 and E11.5. At E9.5 and E11.5 embryos, to identify the differences between nephron progenitors and surrounding mesenchyme, nephron progenitor populations were further enriched by gating Osr1-GFP positive Integrin alpha8 positive Pdgfr alpha negative population and compared with Osr1-GFP positive cells other than that gate. RNA was isolated from cells and the gene expression profiles were determined by microarrays.
Project description:Non-canonical Wnt/planar cell polarity (PCP) signaling plays a primary role in the convergent extension that drives neural tube closure and body axis elongation. PCP signaling gene mutations cause severe neural tube defects (NTDs). However, the role of canonical Wnt/?-catenin signaling in neural tube closure and NTDs remains poorly understood. This study shows that conditional gene targeting of ?-catenin in the dorsal neural folds of mouse embryos represses the expression of the homeobox-containing genes Pax3 and Cdx2 at the dorsal posterior neuropore (PNP), and subsequently diminishes the expression of the Wnt/?-catenin signaling target genes T, Tbx6 and Fgf8 at the tail bud, leading to spina bifida aperta, caudal axis bending and tail truncation. We demonstrate that Pax3 and Cdx2 are novel downstream targets of Wnt/?-catenin signaling. Transgenic activation of Pax3 cDNA can rescue the closure defect in the ?-catenin mutants, suggesting that Pax3 is a key downstream effector of ?-catenin signaling in the PNP closure process. Cdx2 is known to be crucial in posterior axis elongation and in neural tube closure. We found that Cdx2 expression is also repressed in the dorsal PNPs of Pax3-null embryos. However, the ectopically activated Pax3 in the ?-catenin mutants cannot restore Cdx2 mRNA in the dorsal PNP, suggesting that the presence of both ?-catenin and Pax3 is required for regional Cdx2 expression. Thus, ?-catenin signaling is required for caudal neural tube closure and elongation, acting through the transcriptional regulation of key target genes in the PNP.
Project description:A) Chromatins were prepared from Cdx2-inducible ES cells cultured for 48 - 60 hours in the Dox+ and Dox- conditions. Chromatin immunoprecipitation (ChIP) was carried out by using anti-FLAG M2 affinity gel. ChIP product was tested by Western blotting using anti-FLAG antibody. Nuclear extract from ES cells cultured for 48 - 60 hours in Dox+ and Dox- condition was used for the Western blot. B) CDX2 ChIP-Seq peaks in the Hoxa7 gene region. UCSC Mouse Mm9 browser view of Hoxa7 gene locus after mapping CDX2 ChIP-Seq tags locations in the wiggle format. CDX2 ChIP-Seq peaks are shown in red color. C) Cdx2 ChIP-Seq result was verified by qPCR. Target genes were indicated in (G). Primers flanking a promoter region of Hbb-b1 and Pou5f1 as well as a gene desert region in chromosome 3 were used as negative controls. Primers flanking of Actb gene promoter were used for normalization. The relative enrichment of CDX2 binding was indicated as fold change. (D) CDX2-binding motifs identified with CisFinder using 200 bp sequences centered at ChIP sites. (F) Potential CDX2-direct target genes based on ChIP-Seq and the alteration of expression by Cdx2-overexpression. (G) Identification of CDX2 target genes by combining information on binding sites with gene expression response to Cdx2 over-expression Chromatin IP against CDX2-Flag fusion protein. MC1 ES cells were genetically modified for ROSA26 locus to have Tet-Off expression cassette for C-terminal FLAG tagged Cdx2. The peaks are obtained from the Eland Multi Alignment file. The number of tags in peaks was compared with the number of tags in the control sample for the same region corrected by the total coverage of tags. See supplemental file of the paper for details.
Project description:Separation of cell lineages during early mammalian development is required to establish the pluripotent founder cell population that will give rise to the embryo proper and a functional trophoblast to support its development. We systemically assessed the role of the homeobox gene Cdx2 in vivo and in vitro development with an RNAi approach. Effective elimination of both maternal and zygotic Cdx2 resulted in typical phenotypes of Cdx2-mutant embryos, such as failure of hatching and implantation. However, the blastulation and expression of TE specific markers in these Cdx2-deficient embryos excluded the possibility of Cdx2 to act as a TE determinant, although compromised structure and functioning of TE was observed and the resulted embryos were not viable. Strikingly, the efficiency of stem cell derivation was significantly higher than control when embryos were put on MEF at the 8-cell stage and the derived stem cells were fully pluripotent as shown by chimera and tetraploid complementation experiments. Comparative genomic hybridization of wild type and Cdx2 mutant at 8-cell and blastocyst mouse embryos were performed. Overall design: 8-cell biological duplicates and blastocyst stage biological triplicates embryos were used.The hybridization experiments were duplicated in a reciprocal labeling manner to reduce dye integration bias (dye-swaps).