biostudies-arrayexpressAnna AlemanyMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-17398We quantified the effects of retinol on the cell type composition of gastruloids at 120 hours after aggregation by single cell RNA sequencing (scRNA-seq). Gastruloids were generated from the Rarb2.LacZ reporter cell line and cultured according to the standard protocol and few modifications: - culture was performed with different concentrations of retinol (0, 300, 600 nM) throughout the protocol. - culture was performed with 300 nM throughout the protocol and addition of a Mek inhibitor (1uM Mirdametinib) for the last 2 days of culture. - culture was performed with 300 nM retinol during the first 3 days and 0 nM retinol for the last 2 days. - culture was performed with 600 nM throughout the protocol and addition of a 20ng/ml FGFb for the last 2 days of culture. Additionally, gastruloids were generated with the Rarb2.LacZ cell line with a Aldh1a2−/− knockout, using the standard protocol and supplementation of 300 nM or 600 nM retinol throughout. For each retinol condition, , 40-45 gastruloids from each experimental group were collected and dissociated into single cells. Live single cells were sorted using FACS (CytoFlex SRT Beckman Coulter). DAPI-negative and singlet populations were gated and collected (Methods). Single-cell RNA sequencing of the 8 samples was done using GEM-X Universal 3’v4 chemistry with on-chip multiplexingbiostudies-arrayexpressSample Collection - Gastruloids were collected and washed with ice-cold PBS three times. They were incubated with TrypLE Express (Gibco, 12605028) for 2X 4 min in a heating block at 37oC, shaking at 300 rpm. After incubations, the gastruloids were dissociated into single cells by pipetting up and down 10 times. The cells were washed with PBS twice, centrifuged at 1300 rpm for 5 min at 4°C, and the supernatant was carefully discarded without disturbing the pellet. The cells were resuspended in PBS containing 0,04 % BSA (Sigma-Aldrich, A3059) and 1:5000 DAPI (Invitrogen, D1306) as a viability dye. The cell suspension was filtered through a FACS (Falcon, 352235), and FACS analysis was performed (CytoFlex SRT Beckman Coulter). DAPI-negative and singlet populations were gated and collectedNucleic Acid Extraction - 10X Chromium Genomics workflow was performed by polyA enrichmentLibrary Construction - 10X Chromium Genomics workflow was performed by polyA enrichment with single cell resolutionSequencing - Sequencing of the prepared libraries on a partial lane of the illumina NovaSeq6000 S4 PE150bpOrganizationMINSEQE ScoreAssays and DataProcessed DataMAGE-TAB FilesData Transformation - Standard 10X Genomics Cell Ranger Pipelines were run (v7.1.0) for alignment, barcode assignment and gene quantificaiton. A custom reference genome was created by adding the LazC and Neomycing genes to the standard mouse reference from 10X Genomics (refdata-gex-nn10-2020-A) according to 10X instructions.MetabolomicsUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000Axial elongation and cell fate decisions during early embryonic development are regulated by gradients of signaling molecules, such as retinoic acid (RA) and fibroblast growth factor (FGF). To assess the role of RA in vitro, studies have relied on its direct addition to the medium, which can produce teratogenic effects. Here, we examined the role of a RA precursor, retinol, on axial elongation, anteroposterior development, and FGF-dependent signaling in murine gastruloids, an in vitro model system for early embryogenesis. Rather than applying RA ectopically, we supplemented a retinoid-free medium with its precursor, retinol, prompting the cells to produce RA endogenously. Our results showed that the spatiotemporal RA and FGF signaling can be manipulated with different doses of retinol, influencing gastruloid elongation and lineage specification. Gastruloids cultured in high doses of retinol showed an enrichment of ectoderm, whereas low doses resulted in early mesoderm specification and increased FGF signaling. We further used our approach to confirm that RA signaling inhibits FGF in gastruloids. Thus, retinol can modulate cellular composition in gastruloids, allowing us to investigate fate commitment of ectoderm and mesoderm progenitors in vitro.RNA-seq of coding RNA from single cellsMus musculusRetinoic acid and FGF signaling interact to control elongation and lineage specification in a mouse gastruloid modelAnna AlemanyMehmet Yildiz, Noëlle Dommann, Tugce Kardelen Hasdemir, Esther de Vries, Anna Alemany, Pascale F. Dijkers, Niels Geijsenfalsesingle cell RNA sequencing of 120h mouse gastruloids cultured with different doses of retinolWe quantified the effects of retinol on the cell type composition of gastruloids at 120 hours after aggregation by single cell RNA sequencing (scRNA-seq). Gastruloids were generated from the Rarb2.LacZ reporter cell line and cultured according to the standard protocol and few modifications: - culture was performed with different concentrations of retinol (0, 300, 600 nM) throughout the protocol. - culture was performed with 300 nM throughout the protocol and addition of a Mek inhibitor (1uM Mirdametinib) for the last 2 days of culture. - culture was performed with 300 nM retinol during the first 3 days and 0 nM retinol for the last 2 days. - culture was performed with 600 nM throughout the protocol and addition of a 20ng/ml FGFb for the last 2 days of culture. Additionally, gastruloids were generated with the Rarb2.LacZ cell line with a Aldh1a2−/− knockout, using the standard protocol and supplementation of 300 nM or 600 nM retinol throughout. For each retinol condition, , 40-45 gastruloids from each experimental group were collected and dissociated into single cells. Live single cells were sorted using FACS (CytoFlex SRT Beckman Coulter). DAPI-negative and singlet populations were gated and collected (Methods). Single-cell RNA sequencing of the 8 samples was done using GEM-X Universal 3’v4 chemistry with on-chip multiplexing2026-07-24T00:00:00Z2026-07-24T01:00:58.651Z2026-07-23T16:26:25.853ZE-MTAB-1739842411133ERP202614E-MTAB-15527EFO_0002944EFO_0004170EFO_0005684EFO_0005518EFO_0003816EFO_000418410.1242/dev.205153