Single-cell transcriptomic interrogation of hematopoietic progenitors within the E11.5 AGM of the mouse
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ABSTRACT: To clarify the relationship between eMPPs and other progenitors, including pre-HSCs, within the AGM landscape, we performed single cell RNA-sequencing of hematopoietic progenitors within the E11.5 AGM.
Project description:We sorted endothelial (EC), hematopoietic (HC) and hematopoietic stem/progenitor (HSPC) cell populations from AGM of E11.5 C57Bl6 embryos, established and compared their transcriptome to highlight specific regulators of hematopoietic emergence.
Project description:The aim of this study was to analyse the heterogeneity within the first haematopoietic stem cells generated during mouse development using single-cell RNA-Seq. Freshly dissected aorta-gonads-mesohenphros (AGM) regions from E11.5 mouse embryos were dissociated by collagenase and the cells stained for the cell surface markers EPCR and CD45. EPCR+CD45+ cells were index-sorted as single cells into 96-well plates and processed based on the SmartSeq2 protocol. PCA and tSNE analyses identified 2 main subpopulations which are segregated based on differences in cell cycle. Known HSC markers such as Gata2 and Runx1 show little correlation.
Project description:Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Hematopoiesis depends on the Notch pathway and the identification of Notch-targets is important for the understanding of blood origin. Hematopoietic Stem Cells (HSCs) specification occurs in the embryonic aorta and requires Notch activation, however which are the elements regulated by Notch that control this process are mainly unknown. Here, we took a genome-wide approach to identify putative direct Notch targets by precipitating the chromatin that binds to the Notch partner RBPj in the Aorta-Gonad-Mesonephros (AGM) tissue from E11.5 mouse embryos. This assay revealed 701 gene promoter regions as candidates to be regulated by Notch in the AGM. Chromatin was obtained from a pool of 40 dissected AGMs at E11.5. Chromatin immunoprecipitation (ChIP) was performed as previously described (Aguilera et al, PNAS 2004) with minor modifications. In brief, cross-linked chromatin was sonicated for 10 minutes, medium-power, 0.5-interval; with a Bioruptor (Diagenode) and precipitated with anti-RBPJ (Chu and Bresnick, 2004). After crosslinkage reversal, DNA was used as a template for PCR or for array hybridization. Mouse promoter chip on chip microarray SET (Agilent) was used to identify RBPj targets. It covers 70,000 best identified gene regions with a-5.5 kb to + 2.5 kb range, and has on average 25 probes per gene with an average probe to probe distance of 200 bp. The ChIP-on-chip was performed with dye swaps and one IgG control was brought along. Enrichment analysis was done by comparing the precipitation normalized dye swap signal with input control signal.
Project description:By chemical modulation of the PKA/CREB and BMP pathways in isolated AGM VE-cadherin+ cells from mid-gestation embryos, we demonstrate that PKA/CREB regulates hematopoietic engraftment and clonogenicity of hematopoietic progenitors and is dependent on secreted BMP ligands through the type I BMP receptor. We used microarray to document upregulation of PKA/CREB-BMP pathway as well as global BMP target upregulation upon PKA/CREB activation. Isolated VE+ cells from E11.5 AGM were treated with BMP4 (4ng/ml), forskolin (25uM) or both for 8 hours before RNA isolation.
Project description:Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Cells that emerge from the endothelium and show hematopoietic traits can be distinguished by the expression of the c-kit receptor and finally acquire the CD45 marker. The effect of Notch activation by each one of the ligands has been tested on c-kit+ and ckit- cells from the endothelium of the E11.5 AGM . Incubations with stromal cell lines exposed the cells to high levels of ligand and induced changes in the transcriptome of these cells.
Project description:Purpose: The goals of this study are to identify the different gene expresion level in dorsal skin cells or AGM region at different embryonic stages Methods: Dorsal skin cells were collected from different-staged embryo: E9.5, E10.5 E12.5 and E14.5. AGM were collected from different-staged embryo: E9.5, E10.5 and E11.5. In duplicate. RNA were extrated and analyzed using RNA sequencing. For dorsal skine tissues, we screened the genes were highly expressed at early embryonic stage E9.5, but not later stages (E10.5, E12.5, and E14.5). For embryonic AGM tissues, we selected the genes were highly expressed at early embryonic stage E9.5, but not later stages (E10.5 and E11.5). The gene list were filtered based on secreted potein candidates. These gene candidates might function as novel embryonic anti-tumor factors Results: Using an optimized data analysis workflow, we mapped the gene list for potential secretory anti-tumor factors. Conclusions: One of the top gene from the gene lists is NEPN which we hypotherze it has unique anti-tumor function in the early embryonic microenvirnemt and might be used as a novel cancer therapeutic drug