Retinoic acid mediated neural and mesoderm specification during vertebrate trunk development
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500, Linux computer, Linux Beowulf cluster; AMD Opteron Linux Beowulf cluster; AMD Opteron 6168
ORGANISM(S): Mus musculus
SUBMITTER: James Briscoe
PROVIDER: E-MTAB-5208 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:To define the sequence of events that lead to the generation of somatic motor neurons (MNs), we took advantage of ESCs, which can be directed to differentiate into spinal neural progenitors (NPs) in vitro (Gouti et al. 2014). This method relies on the exposure of ESCs, cultured as a monolayer, to a brief pulse of Wnt signalling prior to neural induction. Subsequently, removal of Wnt signalling and exposure to retinoic acid (RA) results in the generation of NPs. Exposure of these NPs to Shh signalling agonist SAG induces the expression of genes expressed in the ventral spinal cord and the induction of MNs.
2018-02-01 | E-MTAB-5466 | biostudies-arrayexpress
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX proteins play a fundamental role in promoting the expression of PM genes, including the master regulator Mesogenin1 (Msgn1). To address the role of PBX proteins in PM differentiation, RNA-seq was performed on wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (12 hours and 24 hours). To establish the direct transcriptional regulation of Msgn1 by PBX/HOX, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed RNA-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at 24 hours. All differentiation experiments were performed in biological triplicates with WT and Pbx1/2-DKO lines, and in biological duplicates with pMsgn1-mut lines.
2021-07-21 | E-MTAB-9773 | biostudies-arrayexpress
Project description:Exposure of mouse ESCs to a sequence of extrinsic signals, which recapitulates in vivo development, generates a bipotential neuromesodermal precursor (NMP) that can be directed to differentiate into either spinal cord or paraxial mesodermal tissue. To induce differentiation,mouse ES cells were plated on CellBINDSurface dishes (Corning) precoated with 0.1% gelatin (Sigma) at a density of 5x10^3 cells cm-2 in N2B27 medium. Cells were grown in N2B27 supplemented with 10ng/ml bFGF (R&D) for 3 days (D1-D3) and then were transferred into serum free media without bFGF (D3-D5). To induce ventral hindbrain identity NPCs (NH) 100nM RA (Sigma) and 500nM SAG (Calbiochem) was added from D3-D5. Spinal cord identity (NP) was induced by the addition of 5nM CHIR 99021 (Axon) from D2 to D3 followed by 100nM RA, 500nM SAG from D3-D5. To induce mesodermal differentiation the cells were treated with CHIR99021 from D2-D5.
2014-07-18 | E-MTAB-2268 | biostudies-arrayexpress
Project description:ATAC-seq was performed, mapped, and analyzed as previously described (PMID: 34446717: \\"ATAC-seq was performed on 50,000 cells per replicate as described in Buenrostro et al. (with modifications based on Corces et al.), on EpiSCs and PSM-differentiated cell populations at desired time-points. Libraries were generated using the Ad1_noMX and Ad2.1–2.16 barcoded primers64 and amplified for 10 total PCR cycles. Libraries were purified with AMPure XP beads to remove contaminating primer dimers and fragments >1,000 bp. Library quality was assessed using the Fragment analyzer and quantitated by Qubit assay. The libraries were sequenced with 50 bp paired-end reads on a Next-Seq 500 Sequencer (Illumina) at the DanStem Genomics Platform (University of Copenhagen, Copenhagen, Denmark).\\"). For all conditions, two biological replicate samples were collected from independent experiments. Library quality was assessed using the Fragment Analyzer and quantitated by Qubit assay. The libraries were sequenced with 50 bp paired-end reads on a NextSeq 500 Sequencer (Illumina) at the DanStem/reNEW Genomics Platform (University of Copenhagen, Copenhagen, Denmark). Prediction of cis-regulatory elements (CREs) and gene annotation was done using rGREAT (v4.0.4) [PMID: 20436461],[PMID: 36040971] or HOMER (v.4.7)[PMID: 20513432]
2023-12-01 | E-MTAB-13407 | biostudies-arrayexpress
Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). WNT signalling sustains both NMP expansion and PM differentiation, but the mechanism by which it distinguishes between these alternative fates is unknown. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX/HOX complexes establish a permissive chromatin landscape for de novo recruitment of the WNT-effector LEF1 on PM genes, including the master regulator Mesogenin1 (Msgn1). To assess the PBX-dependent changes in chromatin accessibility during PM differentiation, we performed ATAC-seq of wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours). To assess the direct consequence of PBX/HOX binding on chromatin accessibility, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed ATAC-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at different time-points (12 hours, 24 hours, 36 hours, 48 hours).
2021-07-21 | E-MTAB-9776 | biostudies-arrayexpress
Project description:Aging improves pancreatic β-cell function in mice. This is a surprising finding since aging is typically associated with functional decline. We performed single-cell RNA sequencing of β-cells from 3 and 26 month old mice to explore how changes in gene expression contribute to improved function with age. The old mice were healthy, had reduced blood glucose levels and increased β-cell mass, which correlated to their body weight. β-cells from young and old mice had similar transcriptome profiles. In fact, only 193 genes (0.89% of all detected genes) were significantly regulated (⥠2-fold; false discovery rate < 0.01; normalized counts > 5). Of these, 183 were downregulated and mainly associated with pathways regulating gene expression, cell cycle, cell death and survival as well as cellular movement, function and maintenance. Collectively, our data show that β-cells from very old mice have transcriptome profiles similar to those of young mice. These data support previous findings that aging is not associated with reduced β-cell mass or functional β-cell decline in mice. Single-cell RNA sequencing of mouse pancreatic islet beta cells
2016-06-30 | E-GEOD-83146 | biostudies-arrayexpress
Project description:This study provides an assessment of the Fluidigm C1 platform for RNA sequencing of single mouse pancreatic islet cells. The system combines microfluidic technology and nanoliter-scale reactions. We sequenced 622 cells allowing identification of 341 islet cells with high-quality gene expression profiles. The cells clustered into populations of alpha-cells (5%), beta-cells (92%), delta-cells (1%) and PP-cells (2%). We identified cell-type specific transcription factors and pathways primarily involved in nutrient sensing and oxidation and cell signaling. Unexpectedly, 281 cells had to be removed from the analysis due to low viability (23%), low sequencing quality (13%) or contamination resulting in the detection of more than one islet hormone (64%). Collectively, we provide a resource for identification of high-quality gene expression datasets to help expand insights into genes and pathways characterizing islet cell types. We reveal limitations in the C1 Fluidigm cell capture process resulting in contaminated cells with altered gene expression patterns. This calls for caution when interpreting single-cell transcriptomics data using the C1 Fluidigm system. Single-cell RNA sequencing of mouse C57BL/6 pancreatic islet cells
2016-02-19 | E-GEOD-77980 | biostudies-arrayexpress
Project description:We encapsulated common marmoset pluripotent stem cells (cmPSCs) in agarose microgels and optimised culture conditions to generate epiblast and amnion spheroids. Using this system, we investigate the effects of signalling perturbations on epiblast and amnion spheroids as a model for early lineage specification
2022-07-06 | E-MTAB-10639 | biostudies-arrayexpress
Project description:PolyA RNAseq of mouse ESCs and EpiLCs carrying a homozygous point mutation in the catalytic domain of mll2[Y2602A]. Total RNA was isolated from cell pellets using NEB Monarch Total RNA Miniprep Kit and libraries prepared using NEBNext RNA Ultra II kit (NEB E7770S, input: 350 ng of total RNA). Libraries were multiplexed and sequenced on a NextSeq500 (Paired-End; read length 40). Each sample is present in 3 biological replicates.
2023-11-15 | E-MTAB-13467 | biostudies-arrayexpress
Project description:In this study, we performed bulk RNA-seq of pluripotent stem cells (PSCs) and induced pre-somitic mesoderm (PSM) cells of six different mammalian species. The species studied are: mouse (Mus musculus), marmoset (Callithrix jacchus), rabbit (Oryctolagus cuniculus), human (Homo sapiens), cattle (Bos taurus) and southern white rhinoceros (Ceratotherium simum). We used mouse ESCs, marmoset iPSCs, rabbit ESCs, human iPSCs, bovine ESCs and rhinoceros ESCs to induce PSM-like cells from these species following protocols already described. PSC samples were extracted under maintenance conditions. PSM samples were extracted on the day when the differentiation efficiency was higher based on the percentage of cells expressing the PSM marker TBX6. We used identical culture conditions when extracting the induced PSM cells to minimize the effect of external factors on our results. Two replicates per each cell type and species were collected for a total of 24 samples. We compared the expression levels of more than 10,000 orthologous protein-coding genes across the six species. With this, we determined that the species-specific segmentation clock periods might be derived from species-specific gene expression profiles controlling basic biological processes.
2023-05-04 | E-MTAB-12263 | biostudies-arrayexpress