{"database":"biostudies-arrayexpress","file_versions":[],"scores":null,"additional":{"submitter":["Jielin Yang"],"organism":["Rattus norvegicus"],"full_dataset_link":["https://www.ebi.ac.uk/biostudies/studies/E-MTAB-16069"],"description":["Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset."],"repository":["biostudies-arrayexpress"],"sample_protocol":["Nucleic Acid Extraction - Spatially barcoded mRNA molecules were captured in situ on 10x Genomics Visium Spatial Gene Expression Slides. Tissue sections (10 µm thick) were fixed in 3.7% formaldehyde, stained with hematoxylin and eosin, and imaged before permeabilization. Permeabilization was optimized for embryonic tissue using 0.5 U/ml collagenase and 0.2 mg/ml BSA in HBSS at 37 °C for 20 min, followed by digestion in 0.1% pepsin in 0.1 M HCl for 7 min. Released mRNA hybridized to the oligo-dT primers within each spatial capture spot. Reverse transcription generated spatially barcoded cDNA molecules, which were enzymatically released using a Proteinase K/PKD buffer (Qiagen) mix at 56 °C for 1.5 h, then collected for library construction.","Sample Collection - Embryos were collected from pregnant Wistar rats at gestational day 14–16 under isoflurane anesthesia, following institutional ethical approval (No. 2020PS357K, Shengjing Hospital, China Medical University). Immediately after cesarean section, embryos were rinsed in ice-cold sterile saline to remove blood and blotted dry with RNase-free gauze. Each intact embryo was embedded in pre-cooled optimal cutting temperature (OCT) compound (SAKURA, Japan) in sagittal orientation and snap-frozen in isopentane chilled with liquid nitrogen for 1 min. Samples were stored at −80 °C until sectioning.","Library Construction - cDNA libraries were prepared following the 10x Genomics Visium Spatial Gene Expression User Guide and previously published protocols (Salmén et al., 2018; Ståhl et al., 2016). Released cDNA underwent second-strand synthesis, in vitro transcription, adaptor ligation, and second reverse transcription to generate indexed sequencing libraries. The libraries were purified, quantified, and quality-checked before sequencing. All spatial library preparation steps were performed on an automated MBS 8000 system (Jemt et al., 2016) by Biomarker Technologies Corporation (Beijing, China).","Sequencing - Final Visium spatial gene expression libraries were diluted to 4 nM and sequenced on an Illumina NovaSeq 6000 platform using 150 bp paired-end (PE150) reads, achieving a minimum depth of 50,000 reads per spot."],"figure_sub":["Organization","MINSEQE Score","Assays and Data","Processed Data","MAGE-TAB Files"],"data_protocol":["Data Transformation - Raw fastq files were processed with 10X Genomics SpaceRanger v3.1.3 with mRatBN7-2-2024-A as the reference genome to generate the filtered feature barcode matrix."],"omics_type":["Metabolomics","Unknown","Transcriptomics","Genomics","Proteomics"],"instrument_platform":["Illumina NovaSeq 6000"],"study_type":["spatial transcriptomics by high-throughput sequencing"],"species":["Rattus norvegicus"],"pubmed_authors":["Chen-Yi Wang","Jielin Yang"],"additional_accession":[]},"is_claimable":false,"name":"Rack1-mediated ferroptosis affects hindgut development in rats with anorectal malformations: Spatial transcriptome insights","description":"Anorectal malformation (ARM), a common congenital anomaly of the digestive tract, is a result of insufficient elongation of the urorectal septum. The cytoplasmic protein Receptor of Activated C-Kinase 1 (Rack1) is involved in embryonic neural development; however, its role in embryonic digestive tract development and ARM formation is unexplored. Our study explored the hindgut development and cell death mechanisms in ARM-affected rats using spatial transcriptome analysis. We induced ARM in rats by administering ethylenethiourea via gavage on gestational day (GD) 10. On GDs 14-16, embryos from both normal and ARM groups underwent spatial transcriptome sequencing, which identified key genes and signalling pathways. Rack1 exhibited significant interactions among differentially expressed genes on GDs 15 and 16. Reduced Rack1 expression in the ARM-affected hindgut, verified by Rack1 silencing in intestinal epithelial cells, led to increased P38 phosphorylation and activation of the MAPK signalling pathway. The suppression of this pathway downregulated Nqo1 and Gpx4 expression, resulting in elevated intracellular levels of ferrous ions, reactive oxygen species (ROS) and lipid peroxides. Downregulation of Gpx4 expression in the ARM hindgut, coupled with Rack1 co-localisation and consistent mitochondrial morphology, indicated ferroptosis. In summary, Rack1, acting as a hub gene, modulates ferrous ions, lipid peroxides, and ROS via the P38-MAPK/Nqo1/Gpx4 axis. This modulation induces ferroptosis in intestinal epithelial cells, potentially influencing hindgut development during ARM onset.","dates":{"release":"2026-01-01T00:00:00Z","modification":"2026-05-27T13:38:31.235Z","creation":"2025-11-13T15:12:10.228Z"},"accession":"E-MTAB-16069","cross_references":{"ENA":["ERP184190"],"EFO":["EFO_0002944","EFO_0004170","EFO_0030005","EFO_0005518","EFO_0003816","EFO_0004184"]}}