Dissection of anterior mesendoderm segregation at single cell level in zebrafish
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PROVIDER: PRJNA1048453 | ENA |
REPOSITORIES: ENA
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Project description:Purpose: To investigate Ripply1 binding site in zebrafish embryo Methods: HA-ripply-injected embryos were harvested at 6hpf. CUT&Tag were performed on the collected cells using Hyperactive Universal CUT&Tag Assay Kit for Illumina Pro (Vazyme, TD904) following the protocol.The products were sent for paired-end sequencing at 150 bp read length on a NovaSeq 6000 system. Results: 72,683 peaks were enriched in HA-ripply1-injected samples. And Ripply1 binding peaks were found to be enriched in the immediate upstream region of the gene body of sox17 and sox32. Conclusions: Ripply1 may repress sox17 and sox32 expression through direct binding.
2023-12-06 | GSE249292 | GEO
Project description:Differential Nodal level promotes mesendoderm cell fate segregation mediated by chromatin organization
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Project description:Purpose: To investigate the mechanism of prechordal plate and anterior endoderm separation . Methods: Nodal injected explants (injected with 10pg ndr2 mRNA) constructed from lft1 mutants and ndr1 morphants were harvested at 6hpf. Libraries were prepared using Chromium Controller and Chromium Single Cell 3’Library & Gel Bead Kit v3 (10x Genomics, PN-1000075) according to the manufacturer’s protocol for 10000 cells recovery. For single-cell multiomics, zebrafish embryos at 6 hpf were harvested. Libraries were prepared using Chromium Next GEM Single Cell Multiome ATAC + Gene Expression Reagent Bundle (10x Genomics, 4 rxns PN-1000285) according to the manufacturer’s protocol for 10000 cells recovery. Results: A total of 10,614 single-cell transcriptomes and 4,335 multiomics were collected after stringent quality control measures. Conclusions: A slight bias in Nodal signaling promotes a differential chromatin structure between prechordal plate and endoderm, which drives a differential expression of those key regulators, such as gsc and ripply1 in these two cell lineages, and further regulates mesendoderm cell fate separation.
2023-01-28 | GSE223636 | GEO
Project description:ES cells are differentiated into mesenderm cell linage by Activin A treatment. During this differentaition, gene expression profile of ES cells, day 4 mesendoderm cell, day 5 mesendoderm cells and mesoderm cells are examined.
2009-06-12 | E-MEXP-1672 | biostudies-arrayexpress
Project description:TORC2 is a serine-threonine kinase complex conserved through evolution that recently emerged as a new regulator of actin dynamics and cell migration. However, knockout in mice of its core components Sin1 and Rictor is embryonic lethal, which has limited in vivo analyses. Here, we analysed TORC2 function during early zebrafish development, using a morpholino-mediated loss of function of sin1. Sin1 appears required during gastrulation for migration of the prechordal plate, the anterior most mesoderm. In absence of Sin1, cells migrate both slower and less persistently, which can be correlated to a reduction in actin-rich protrusions and a randomisation of the remaining protrusions. These results demonstrate that, as established in vitro, the TORC2 component Sin1 controls actin dynamics and cell migration in vivo. We furthermore establish that Sin1 is required for protrusion formation downstream of PI3K, and is acting upstream of the GTPase Rac1, since expression of an activated form of Rac1 is sufficient to rescue sin1 loss of function.
| S-EPMC4339552 | biostudies-literature
Project description:We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted 34k expression arrays were generated by printing Oligo nucleotides from three different sets (MWG, Compugen and Operon) onto epoxy slides. Total RNA from Kaede protein-injected and FAM-P treated zebrafish embryos was amplified using an Ambion kit (Cat#1753), labeled with Cy dyes and hybridized overnight to the oligo chip in a MAUI chamber. The experiment comparing mesendoderm was performed four times (twice with WIK/AB hybrid strain embryos, once with EK strain embryos and once with AB strain embryos) each time including a dye swap, for a total of eight (four forward and four dye-swap) hybridizations. Three control cRNA probes were also generated from AB-strain embryos, namely: (1) whole 40% epiboly stage embryos, (2) FACS-sorted green cells from dissociated 40% epiboly embryos that were Kaede-injected but NOT photolabeled and (3) FACS-sorted red cells from dissociated 40% epiboly embryos that were Kaede-injected AND photolabeled. These probes were each generated three times, each time including a dye swap and co-hybridized as follows: (1) whole embryo cRNA vs green cell cRNA (three forward and three dye-swap hybridiizations) and (2) green cell cRNA vs. red cell cRNA (three forward and three dye-swap hybridizations).
2010-05-26 | E-GEOD-8654 | biostudies-arrayexpress
Project description:We have developed a method for rapidly identifying early, regionally-expressed molecules: FACS-Assisted Microdissection of Photolabeled cells (FAM-P). For our first FAM-P project, we injected embryos with purified Kaede protein, a stony coral protein that fluoresces at 514 nm (green) prior to- and 582 nm (red) after photoconversion at 405 nm. We used the scanning laser of a confocal microscope to photolabel late blastula-stage embryos along 4-6 tiers of marginal cells, comprising the mesoderm and endoderm germ layer precursors. This procedure left the neurectoderm precursor cells marked by green fluorescence and the mesendoderm precursor cells marked by red fluorescence. Embryonic cells were dissociated and subjected to FACS, separating red mesendoderm precursors from green neurectoderm precursors. RNA was extracted and linearly amplified once, then dye-labeled and co-hybridized to a microarray with over 30,000 oligos representing more than 20,000 unique zebrafish genes. In validation of our strategy, many genes known to have elevated expression in the late blastula margin (e.g., squint, fgf8, lim1 and gata5) and several genes known to be specific to the early neurectoderm (e.g., sox31 and otx2) were independently identified by this approach. In addition to known genes, our method has identified dozens of previously uncharacterized genes that are enriched in the pre-mesendoderm or pre-neurectoderm. Keywords: 40% epiboly-stage embryos, injected with purified Kaede protein, differentially photolabeled, disaggregated and FACS sorted
2008-06-17 | GSE8654 | GEO
Project description:Postnatal neuroblasts were microdissected by laser micro dissection from posterior and anterior rostral migratory stream (RMS). Neuroblasts in the posterior RMS are just started their migration. Neuroblasts in the anterior RMS are already migrated for several days in the stream
2008-09-01 | E-MEXP-1430 | biostudies-arrayexpress
Project description:Liver Cancer Evolution - Lesion segregation
| PRJEB37808 | ENA
Project description:The TGF-β superfamily member Nodal triggers mesendoderm differentiation in embryonic stem (ES) cells. This transition however requires cooperating Wnt signaling inputs. Here we report that p53, a powerful tumor suppressor in adult tissues, orchestrates this cooperation. We show that p53, which is released from inhibition as mouse ES cells exit from pluripotency, acts as a direct inducer of Wnt3 expression. Wnt-activated Tcf3 then converges with Nodal-activated Smad transcriptional complexes on mesendoderm specification super-enhancers. p53 and its homolog p73 act redundantly in Wnt induction. When p53 and p73 are depleted, the ES cells fail to secrete autocrine Wnt ligands, Tcf and Nodal-activated Smads fail to bind to mesendoderm gene enhancers, and the cells adopt ectoderm rather than mesendoderm identity. Enforced Wnt3 expression or addition of Wnt3a rescues mesendoderm differentiation in p53/p73-depleted ES cells. Thus, independently of their established role as tumor suppressors that guard genome integrity in mature cells, p53 and p73 serve a primordial role in ES cell differentiation by driving a cooperation of Wnt and TGF-β transcriptional inputs on mesendoderm identity gene enhancers.
2017-01-06 | GSE70486 | GEO