Next Generation Sequencing Facilitates Quantitative Analysis of Transcriptomes of Yolk Sacs of E16.5 Wild Type and Rsu1-/- mouse embryos
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ABSTRACT: Purpose: Next-generation sequencing (NGS) has revolutionized systems-based analysis of cellular pathways. The goals of this study are to compare NGS-derived yolk sac transcriptome profiling (RNA-seq) of E16.5 Rsu1-/- mouse embryos to that of the wild-type controls Methods: Yolk sac mRNA profiles of yolk sac isolated from E16.5 wild-type (WT) and ras suppressor 1 (Rsu1−/−) emdbryos were generated by deep sequencing, in triplicate, using Illumina Hiseq 2500 platform. The sequence reads that passed quality filters were were mapped to human reference genome GRCh38 by HISAT2 v2.2.1 with default parameters. Results: Using an optimized data analysis workflow, we mapped about 40 million sequence reads per sample to the mouse genome and identified ___ transcripts in the yolk sacs of E16.5 WT and Rsu1−/− embryos with HISAT2 v2.2.1 workflow . Approximately __ % of the transcripts showed differential expression between the WT and Rsu1−/− yolk sac, with a fold change ≥2.0 and p value <0.05. Altered expression of 12 genes was confirmed with qRT–PCR, demonstrating the high degree of sensitivity of the RNA-seq method. Conclusions: Our study represents the first detailed analysis of E16.5 Rsu1-/- yolk sac transcriptomes, which would expedite genetic network analyses and permit the dissection of complex biologic functions of Rsu1 during late embryogenesis.
Project description:This study aimed at exploring the physiological function of mammalian HYPB by means of knockout mouse model. Homogenous disruption of mouse Hypb gene leads to embryonic lethality at E10.5-E11.5. Severe vascular defects were observed in the Hypb-/- embryos, yolk sac and placenta.In the mutant embryo and yolk sac, disorganized and abnormally dilated capillaries cannot be remodeled into large blood vessels or intricate networks. Thus, our results suggest that the mammalian HYPB HMT plays an important role in embryonic vascularization. Keywords: knockout, mouse embryo development, angiogenesis, yolk sac, E9.0, E10.5
Project description:Yolk sac is an important site for early embryonic hematopoiesis. However, our understanding of early hematopoietic development is still very limited. Single cell transcriptome sequencing provides us with a good research method. Here, we performed single cell RNA-seq analysis for Carnegie stage 11 (CS11) and Carnegie stage 15 (CS15) human yolk sacs.
Project description:Primitive erythropoiesis in the mouse yolk sac is followed by definitive erythropoiesis resulting in adult erythrocytes. In comparison to definitive erythropoiesis little is known about the genes that control the embryonic erythroid program. The purpose of this study was to generate a profile of mouse embryonic yolk sac erythroid cells and identify novel regulatory genes differentially expressed in erythroid compared to non-erythroid (epithelial cells). The identification of these genes will contribute to a greater understanding of how the primitive erythroid program is controlled. This work will have clinical implications for treating sickle cell anemia and β-thalassemia. Activating genes in adult erythroid cells that increase embryonic or fetal globin gene expression may be a therapeutic approach to treat individuals with these disorders. Experiment Overall Design: Embryonic day 9.5 (E9.5) yolk sacs were dissected from the embryos of timed-pregnant FVB/N mice. These tissues were frozen in OCT media and 8-micron frozen sections were obtained. Laser capture microdissection (LCM) was used to isolate primitive erythroid precursors and epithelial cells from these E9.5 yolk sac frozen sections using 2 to 4 yolk sacs from 2 different litters per biological replicate. Paired erythroid and epithelial samples were collected from the same microscope slides. Total RNA was isolated from 4 different pairs of erythroid and epithelial samples and hybridized to Affymetrix 430 A 2.0 microarrays.
Project description:Purpose: The goal of this study is to compare the expression profile of wild type and Tbx1-nmf219/nmf219 mice with NGS-derived inner ear transcriptome profiling (RNA-seq) Methods: inner ear mRNA profiles of E16.5 wild-type (WT) and Tbx1-nmf219/nmf219mice were generated by RNA-seq Results: Using an optimized data analysis workflow, we mapped about 30 million sequence reads per sample to the mouse genome and identified transcripts in the inner ear of WT and Tbx1nmf219/nmf219 mice. Approximately 10% of the transcripts showed differential expression between the WT and Tbx1nmf219/nmf219 inner ear, with a fold change ≥1.5 and p value <0.05. Conclusions: Our study represents the detailed analysis of inner ear transcriptomes, with biologic replicates, generated by RNA-seq technology.
Project description:Purpose: The goal of this study is to integrate NGS-derived parotid salivary gland transcriptome profiling (RNA-seq) to metabolomics (UPLC-MS/MS) methods and identify mechanisms driving radiation-induced xerostomia, with potential clinical application to head and neck cancer patients. Methods: Parotid salvary gland mRNA profiles of 28-day-old wild-type (WT) and 5 Gy radiation treated mice were generated by deep sequencing, in triplicate, using Illumina TruSeq. The sequenced reads that passed quality filters were trimmed with Trimmomatic, mapped to mm10 genome with HISAT2, and summarized at the gene level with HTSeq-count and analyzed with DESeq2. Results: Using an optimized data analysis workflow, we mapped sequenced reads per sample to the mouse genome (build mm10) and identified 25422 genes with at least 1 read in the parotid glands of WT and radiation treated mice with HISTA2 workflow. RNA-seq data QA/QC by Principal Component Analysis showed a clear separation between conditions. 155 genes showed significant differential expression between the WT and radiation treated salivary gland, with an adjsuted p-value < 0.05. Pre ranked Gene Set Enrichment Analysis against the CPDB database led to 859 altered pathways with a p-value < 0.05. Integrative analysis pointed to 103 RNA-Seq/Metabolite joint enriched pathways with a p-value < 0.05.
Project description:Investigating the blood, immune and stromal cells present in a human fetal embryo in a world first single cell transcriptomic atlas. The embryo was dissected into 12 coronal sections, yolk sac, and yolk sac stalk. Live single cells sorted, with cell suspension then undergoing 10x chromium 5 prime scRNA-seq. This accession contains the yolk sac and yolk sac stalk data from this embryo. A matched accession contains the coronal section data. Lane "WS_wEMB12142156" (from yolk sac) was excluded from downstream analysis due to low fraction reads in cells post-CellRanger QC. Termination procedure for this embryo was medical. The F158_[features...barcodes...matrix].[tsv...mtx].gz files attached to this accession represent raw count data from all the 10x lanes in this accession combined, and as output from CellRanger filtered matrices (CellRanger version 6.0.1 using human reference genome GRCh38-2020-A). One set of count matrices relates to the yolk sac data, and one set of count matrices relates to the yolk sac stalk data.
Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix).
Project description:The human definitive yolk sac is an important organ supporting the early developing embryo through nutrient supply and by facilitating the establishment of the embryonic circulatory system. However, the molecular and cellular biology of the human yolk sac remains largely obscure due to the lack of suitable in vitro models. Here, we show that human induced pluripotent stem cells (hiPSCs) co-cultured with various types of stromal cells as spheroids self-organize into yolk sac-like organoids without the addition of exogenous factors. Yolk sac-like organoids recapitulated a yolk sac specific cellular complement and structures as well as the functional ability to generate definitive hematopoietic progenitor cells (HPCs). Furthermore, sequential hemato-vascular ontogenesis could be observed during organoid formation. Notably, our organoid system can be performed in a scalable, autologous, and xeno-free condition, thereby providing an important model of human definitive yolk sac development and allows for efficient bulk generation of hiPSC-derived HPCs.
Project description:GW182 (Tnrc6a) is a key component of RISC (miRNA-Induced Silencing Complex) that plays a critical role in miRNA-mediated gene silencing. Here, we show that GW182 is expressed in the yolk sac endoderm, and that gene-trap disruption of GW182 leads to growth arrest of yolk sac endoderm, impaired hematopoiesis and embryonic lethality. To investigate roles of GW182 in the yolk sac endoderm, we assessed changes in mRNA expression in the yolk sac of E9.5 GW182gt/gt embryos using microarrays (Affymetrix). Yolk sac of wild type littermates and GW182gt/gt embryos at E9.5 was collected for total RNA isolation using Trizol (Invitrogen). RNAs were purified according to the manufacturer’s protocol before subjected to Mouse Gene 1.0 ST Whole Genome Array (Affymetrix) for mRNA expression profiling. Experiments were performed in triplicate. Differentially expressed mRNAs were identified using a two-sample t-test (P<0.05 considered significant).
Project description:The embryonic site of definitive hematopoietic stem cell (dHSC) origination has been debated for decades. Although an intra-embryonic origin is supported, recent data suggest that a large fraction of adult blood derives from the yolk sac (YS). Investigating the origins of hematopoiesis before heartbeat onset (i.e. 5-7 somite pairs (sp)) is precluded by a lack of assays that can distinguish dHSC precursors in early embryos. Here, we report robust, multi-lineage and serially transplantable dHSC activity from cultured 2-7sp murine embryonic explants (Em-Ex). dHSC were undetectable in 2-7sp YS explants (YS-Ex). Our work supports a model in which the embryo, not the YS, is the major source of lifelong hematopoiesis.