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ATACseq and RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

ABSTRACT: ATACseq and RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

PROVIDER: PRJNA546805 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR9207160_1.fastq.gz	Fastqsanger.gz
	SRR9207160_2.fastq.gz	Fastqsanger.gz
	SRR9207170_1.fastq.gz	Fastqsanger.gz
	SRR9207170_2.fastq.gz	Fastqsanger.gz
	SRR9207180_1.fastq.gz	Fastqsanger.gz

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ATACseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

Project description:ATACseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

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RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

Project description:RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

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ATACseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

Project description:The goal of this study is to analyse the open chromatin regions via ATACseq in different sub-types of the zebrafish embryonic dorsal aorta (DA) at 28-30 hpf using TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos. 2000-3000 cells were FAC-sorted directly into 100 µl of 1x HBSS/10 mM HEPES/0.25% BSA buffer for Tagmentation (as described by Buenrostro et al. (2013), Nat. Methods 10, 1213-1218), but with 1.5 µl Tn5 transposase (Illumina) in a 50 µl reaction volume. DNA was purified using the QIAquick PCR purification kit (Qiagen). Fragments were amplified for 16 cycles. Genomic control DNA was isolated from 30.000 cells using the DNeasy Blood & Tissue kit (Qiagen) and eluted with EB buffer. Tagmentation was performed as described. Fragments were amplified for 9 cycles. Sequencing was performed on a NextSeq machine. Sequenced reads were checked for base qualities, trimmed where 20% of the bases were below quality score 20, and filtered to exclude adapters using Trimmomatic (Version 0.32).

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ATACseq and RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

Project description:This SuperSeries is composed of the SubSeries listed below.

2019-06-06 | GSE132260 | GEO

Unravelling the heterogeneity of haemogenic endothelium in human pluripotent stem cell differentiation cultures

Project description:In embryo, few Endothelial cells from Aorta would be producing Haemogenic endothelium (HE) cells. These HE cells would then be progressing to take kind of a round shape and Undergo EHT. They then take a full round shape to make Intra-aortic haematopoietic clusters (IAHC) cells. These IAHC cells would then diassociate and turn into Hematopoitic Stem Cells (HSC) that ultimately would go to produce different lineage of hematopoitic (blood) cells. We wanted to study the heterogeneity of HE in human pluripotent stem cells.

2020-05-02 | E-MTAB-8271 | biostudies-arrayexpress

RNAseq of haemogenic and aortic endothelium in zebrafish embryos 28-20 hours post fertilization

Project description:The goal of this study is to perform RNAseq in different sub-types of the zebrafish embryonic dorsal aorta (DA) at 28-30 hpf using TgBAC(runx1P2:Citrine);Tg(kdrl:mCherry) double-transgenic zebrafish embryos. A min. of 3000 cells per population were collected via FACS. RNA was isolated with the RNeasy Plus Micro Kit (QIAGEN, Cat No. 74034). High quality RNA (RIN > 8.0) was sent for RNAseq to the Wellcome Trust Centre for Human Genetics (WTCHG). 2.2 ng of total RNA was used to generate SMARTer libraries for low-input RNA. Sequencing was performed on an Illumina HiSeq4000 machine with a 75 bp paired end protocol. Sequenced reads were checked for base qualities, trimmed where 20% of the bases were below quality score 20, and filtered to exclude adapters using Trimmomatic (Version 0.32). Sequences were aligned to the Zebrafish Genome Zv10 with STAR with default parameters. Aligned read features were counted using Subread tool: featureCounts method (version 1.4.5-p1). To determine number of mapped reads we used the trimmed data. The alignment has been performed using STAR with default parameters. The number of mapped reads (QC-passed reads count) has been obtain using Samtools mapping statistics (flagstat tool).

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Unravelling the heterogeneity of haemogenic endothelium in human pluripotent stem cell differentiation cultures

Project description:Unravelling the heterogeneity of haemogenic endothelium in human pluripotent stem cell differentiation cultures

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Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

Project description:The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

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HoxA3 is an apical regulator of haemogenic endothelium.

Project description:During development, haemogenesis occurs invariably at sites of vasculogenesis. Between embryonic day (E) 9.5 and E10.5 in mice, endothelial cells in the caudal part of the dorsal aorta generate haematopoietic stem cells and are referred to as haemogenic endothelium. The mechanisms by which haematopoiesis is restricted to this domain, and how the morphological transformation from endothelial to haematopoietic is controlled are unknown. We show here that HoxA3, a gene uniquely expressed in the embryonic but not yolk sac vasculature, restrains haematopoietic differentiation of the earliest endothelial progenitors, and induces reversion of the earliest haematopoietic progenitors into CD41-negative endothelial cells. This reversible modulation of endothelial-haematopoietic state is accomplished by targeting key haematopoietic transcription factors for downregulation, including Runx1, Gata1, Gfi1B, Ikaros, and PU.1. Through loss-of-function, and gain-of-function epistasis experiments, and the identification of antipodally regulated targets, we show that among these factors, Runx1 is uniquely able to erase the endothelial program set up by HoxA3. These results suggest both why a frank endothelium does not precede haematopoiesis in the yolk sac, and why haematopoietic stem cell generation requires Runx1 expression only in endothelial cells.

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Blood stem cell-forming haemogenic endothelium in zebrafish derives from arterial endothelium.

Project description:Haematopoietic stem cells are generated from the haemogenic endothelium (HE) located in the floor of the dorsal aorta (DA). Despite being integral to arteries, it is controversial whether HE and arterial endothelium share a common lineage. Here, we present a transgenic zebrafish runx1 reporter line to isolate HE and aortic roof endothelium (ARE)s, excluding non-aortic endothelium. Transcriptomic analysis of these populations identifies Runx1-regulated genes and shows that HE initially expresses arterial markers at similar levels to ARE. Furthermore, runx1 expression depends on prior arterial programming by the Notch ligand dll4. Runx1-/- mutants fail to downregulate arterial genes in the HE, which remains integrated within the DA, suggesting that Runx1 represses the pre-existing arterial programme in HE to allow progression towards the haematopoietic fate. These findings strongly suggest that, in zebrafish, aortic endothelium is a precursor to HE, with potential implications for pluripotent stem cell differentiation protocols for the generation of transplantable HSCs.

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