Project description:The aim of the experiment was to compare newly defined CD44Neg, CD44LoKitNeg, CD44LoKitPos and CD44High populations from mouse Aorta-Gonad-Mesonephros (AGM) region
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.
Project description:The aim of this experiment was to compare by single-cell RNA sequencing the aorta-gonad-mesonephros (AGM) region and yolk sac (YS), the two major sites of embryonic hematopoietic, between 9.5 and 11.5 days of mouse embryonic development.
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 and refers 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. AGM regions were obtained from E11.5 embryos by dissection and digested with 0.1% collagenase. Cells were stained with anti-CD31, anti-ckit, anti-CD45 and anti-Ter119 antibodies. Sorting of the CD31+CD45-Ter119- population was performed, and cells were separated into c-kit+ and c-kit-. 3 replicates each of c-kit+ and c-kit- cells.
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 and refers 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.
Project description:We profile the transcriptional landscape of mouse fetal placenta and identify four distinct types of haematopoietic stem/progenitor cells (HSPCs) during mid-gestation using single-cell RNA sequencing. We experimentally validate and uncover that a subpopulation of placental endothelial cells exhibits hemogenic endothelial (HE) potential and shares transcriptional features with HE cells in the aorta-gonad-mesonephros (AGM) region.
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:In this study we have examined the role of Tet enzymes in regulation of embryonic Endothelial cells (ECs) and Hematopoietic stem and progenitor cells (HSPCs). We have performed transcriptomic and methylation analyses of Tet deficient and wild type ECs and HSPCs isolated from E11.5 embryonic aorta-gonad-mesonephros (AGM) to identify differentially expressed genes and differentially methylated regions.
Project description:Hematopoietic stem cells (HSC) are generated from specialized endothelial cells of the embryonic aorta. Previously, inflammatory factors have been implicated in regulating mouse HSC development, but it is unclear what cells in the embryonic aorta-gonad- mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our CyTOF results indicate two abundant myeloid cell types - mannose-receptor positive AGM- associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1. AGM-aM express a pro-inflammatory signature, localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult- repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM- associated macrophages in regulating the embryonic development of HSCs.