Single-cell transcriptome analysis of EpCAM+ cells from P3.25 mouse fetal livers
Ontology highlight
ABSTRACT: We employed marker-free single-cell RNA-Seq to characterize comprehensive transcriptional profiles of 52 EpCAM+ cells from postnatal day 3.25 mouse fetal livers. These cells show characters of cholangiocytes. Combined with our previous single cell data of E11.5~P2.5 mouse fetal livers, our data depict the dynamic trajectories with transcriptional profiles at single-cell resolution during mouse liver development, and provide insights into the fate decision and transcriptional control of self-renewal, differentiation and maturation of liver stem/progenitor cells.
Project description:The Nrf2 transcription factor is a key player in the cellular stress response, which regulates the expression of important antioxidant enzymes and other cytoprotective proteins. We recently generated a novel transgenic mouse model to determine the function of Nrf2 in the skin. These mice show reduced number of apoptotic keratinocytes after UVB irradiation due to enhanced ROS detoxification. They also show enhanced tumorigenesis in the HPV8 tumor model. RNA from whole skin of 3 single P2.5 K5cre-caNrf2 (tg/tg) and 3 single P2.5 K5cre-wt (tg/wt=control) mice were used
Project description:The Nrf2 transcription factor is a key player in the cellular stress response, which regulates the expression of important antioxidant enzymes and other cytoprotective proteins. We recently generated a novel transgenic mouse model to determine the function of Nrf2 in the skin. These mice revealed interesting phenotypic abnormalities, including hyperkeratosis and acanthosis. To gain insight into the underlying molecular mechanisms, we wanted to identify genes, which are differentially expressed in the skin of wild-type and mutant mice before the onset of phenotypic abnormalities. RNA from whole skin of 3 single P2.5 K5cre-caNrf2 (tg/tg) and 3 single P2.5 K5cre-wt (tg/wt=control) mice were used
Project description:Purpose: The goals of this study are to identify dysregulated mRNAs in the heart after deletion of miR-1-1 and miR-1-2. Methods: Total RNAs were extracted from hearts at embryonic E15.5 (E15.5) or postnatal 2.5 days (P2.5) of miR-1s dHET and miR-1s dKO mice. mRNAs were purified using a poly-A selection approach and sequenced using Illumina HiSeq 2000. The sequence reads were aligned to the mouse reference genome (NCBI Build 37/mm9) using TopHat program (Bowtie algorithm). Transcript assembles and identification of differentially expressed genes were achieved using Cufflinks package. To account for expression bias due to transcript length, each sample transcript expression was normalized by using cufflinks algorithm with a FDR of 0.05. Results: Using 1.5-fold change as a cutoff, 997 and 653 transcripts were found to be upregulated and downregulated, respectively, in miR-1s dKO heart at P2.5. 423 transcripts were found to be upregulated and 653 were down-regulated in miR-1s dKO heart at E15.5. Many upregulated genes are directly involved in a fetal gene program. Conclusions: miR-1 directly represses a fetal gene program. We performed RNA deep-seq using postnatal 2.5 days (P2.5) heart from miR-1-1 and miR-1-2 double knockout mice and compared it to that of littermate control.
Project description:Whole livers were collected from mouse fetuses at embryonic day 13.5 (E13.5), and single-cell suspensions were prepared by successive passage through 25-gauge needles. Fetal liver cells were maintained in RPMI1640 (Wako, Osaka, Japan) supplemented with 20% charcoal-stripped fetal bovine serum (FBS), 100 U/ml penicillin, 100µg/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO) (generously provided by Kyowa Hakko Kirin Co. Ltd.). Megakaryocytes were harvested for RNA purification from a day-3 culture. CD41+ cells were enriched using biotinylated anti-CD41 antibody (Serotec; clone MWReg30) and streptavidin-coupled Dynabeads (Dynal Biotech ASA) from a culture of E13.5 fetal liver cells of WT and p45–/– mice. Total RNA was purified from the sorted CD41+ cells. Gene expression was measured in primary megakaryocytes cultured from p45-/- fetal livers and wild type fetal livers at E13.5. Three independent experiments were performed.
Project description:Purpose: The goals of this study are to identify dysregulated mRNAs in the heart after deletion of miR-1-1 and miR-1-2. Methods: Total RNAs were extracted from hearts at embryonic E15.5 (E15.5) or postnatal 2.5 days (P2.5) of miR-1s dHET and miR-1s dKO mice. mRNAs were purified using a poly-A selection approach and sequenced using Illumina HiSeq 2000. The sequence reads were aligned to the mouse reference genome (NCBI Build 37/mm9) using TopHat program (Bowtie algorithm). Transcript assembles and identification of differentially expressed genes were achieved using Cufflinks package. To account for expression bias due to transcript length, each sample transcript expression was normalized by using cufflinks algorithm with a FDR of 0.05. Results: Using 1.5-fold change as a cutoff, 997 and 653 transcripts were found to be upregulated and downregulated, respectively, in miR-1s dKO heart at P2.5. 423 transcripts were found to be upregulated and 653 were down-regulated in miR-1s dKO heart at E15.5. Many upregulated genes are directly involved in a fetal gene program. Conclusions: miR-1 directly represses a fetal gene program.
Project description:Whole fetal livers were collected from mouse fetuses at embryonic day 14.5 (E14.5), and single-cell suspensions were prepared by successive passage through 18-, 21 and 23-gauge needles. Fetal liver cells were maintained in Dulbecco modified Eagle medium (DMEM; Invitrogen) supplemented with 10% fetal bovine serum (FBS; Invitrogen), 100 U/ml penicillin, 100M-BM-5g/ml streptomycin, and 50ng/ml recombinant human thrombopoietin (TPO; Peprotech). After 5 days of culture, megakaryocytes were purified using a discontinuous bovine serum albumin gradient (BSA, SigmaM-bM-^@M-^SAldrich; 3%, 1.5%, and 0%). Total RNA was isolated with TriM-bM-^@M-^SReagent (MRC) following manufacturerM-bM-^@M-^Ys instructions, and its quality was assessed with NDM-bM-^@M-^S1000 Nanodrop (Peqlab) and on a 1.5% agarose gel. Gene expression was measured in primary megakaryocytes cultured from miR-142-/- fetal livers and wild type fetal livers at E14.5. Two independent experiments were performed.
Project description:Although circadian clocks oscillate in most cells, it has been difficult to detect canonical expression of clock genes in fetal rodent tissues, including the fetal liver. The oscillation status of fetal clocks and the maternal influence on these clocks have not yet been conclusively defined. Here we report that, when the mother mice are under restricted feeding, the expression rhythms of several clock genes can be detected in fetal mouse livers. Those rhythms, although of low amplitude, reversed their profiles under opposite feeding cycles, suggesting maternal entrainment of the weak fetal liver clocks. However, Bmal1 could show biphasic expression in the fetal livers. The expression of some metabolic genes (e.g. Insig1) also showed biphasic daily changes in fetal livers, possibly as a result of interactions between the unique in utero milieu and the fetal liver clocks. Regular rhythms of clock gene expression were detected in dissociated fetal hepatocytes in culture. Differential expression of metabolic genes were found between fetal and adult livers, suggesting that metabolic features affected clock amplitude. Genome-wide differences in DNA methylation were also found between adult and fetal livers. Some of those epigenetic changes were likely critical for the developmental changes in clock amplitudes.