biostudies-arrayexpressRoel QuintensMus musculushttps://www.ebi.ac.uk/biostudies/studies/E-MTAB-14528Embryonic DNA damage, e.g. resulting from exposure to moderate and high doses of ionizing radiation, has important consequences on the normal neurodevelopment, often resulting in microcephaly that can be associated with seizures. The effects of prenatal radiation exposure have previously been investigated with a focus on the neocortex, which is the birthplace of excitatory neurons. The possible effects on interneurons and specifically their progenitors are currently unknown. Here, we generated primary neural progenitor cell (NPC) cultures from the neocortex (NCX) and the medial ganglionic eminences (MGE), the origin of the majority of interneurons, of embryonic day 13 mice. Cells were irradiated with a dose of 0 Gy (sham) or 1 Gy and RNA was extracted at 6 h and 24 h following exposure, and subsequently used for RNA-sequencing (PE150).biostudies-arrayexpressSample Collection - Mouse primary neural progenitor cells (NPCs) were irradiated and lysed at 6 h or 24 h following IR exposure. Cell lysis was performed in RLT Plus lysis buffer (Qiagen, #74136) and samples were stored at -80°C until further use.Nucleic Acid Extraction - Total RNA extraction was performed by using the RNeasy Mini Kit (Qiagen, #74136).Sample Treatment - Acute irradiation (1 Gy) was performed using an X-strahl 320 kV (0.13 Gy/min, inherent filtration: 0.21 mmAl, additional filtration: 3.8 mmAl + 1.4 mm Cu + DAP, tube voltage: 250 kV, tube current: 12 mA, source distance: 100 cm, vertical beam orientation) in accordance to ISO 4037. For sham (0 Gy) irradiation, cells were moved to the irradiation facility but not placed under the X-ray beam.Library Construction - RNA quality was determined using the 5400 Agilent fragment analyzer system (Agilent Technologies, United States). Most of the samples had RNA integrity numbers >9.0. cDNA library construction and sequencing were performed by NovoGene (Cambridge, United Kingdom). Messenger RNA was purified from total RNA using poly-T oligo-attached magnetic beads. After fragmentation, the first strand cDNA was synthesized using random hexamer primers, followed by the second strand cDNA synthesis using either dUTP for directional library or dTTP for non-directional library. For the non-directional library, it was ready after end repair, A-tailing, adapter ligation, size selection, amplification, and purification. The library was checked with Qubit and real-time PCR for quantification and bioanalyzer for size distribution detection.Sequencing - Libraries were sequenced on Illumina NovaSeq 6000 S4 flowcell with PE150 according to results from library quality control and expected data volume.Growth Protocol - The prefrontal cortex (NCX) and medial ganglionic eminence (MGE) of the right hemisphere of C57Bl/6J mice at E13 were dissected and mechanically dissociated by gentle pipetting in Accutase (Stemcell Technologies, 07920). Primary mouse NPCs were cultured as monolayers onto Poly-D-Lysine (PDL) coated plates (Corning) in Dulbecco’s Modified Eagle Medium (DMEM)/F-12 (Gibco, 11330-032) supplemented with B-27 (1:50, Gibco, 17504-044), N-2 (1:100, Gibco, 17502-048), Recombinant Human Fibroblast Growth Factor-basic (FGF-2, 20 ng/ml, Peprotech, 100-18C) and with or without Recombinant Murine Epidermal Growth Factor (EGF, 10 ng/ml, Peprotech, 315-09), respectively. Cells were passaged every 3 to 4 days (~80% confluent) by centrifugation at 300 x g for 3 min, followed by mechanical redissociation and replating as described above.OrganizationMINSEQE ScoreAssays and DataMAGE-TAB FilesUnknownTranscriptomicsGenomicsProteomicsIllumina NovaSeq 6000<h4> Abstract </h4> Prenatal radiation-induced DNA damage poses a significant threat to normal brain development, resulting in microcephaly which primarily affects the cerebral cortex. It is unclear which molecular mechanisms are at the basis of this defect in humans as the few mechanistic studies performed so far were done in animals. Here, we leveraged human embryonic stem cell- derived forebrain organoids as a model for human corticogenesis. Organoids were X-irradiated with a moderate and a high dose at different time points, representing very early and mid corticogenesis. Irradiation caused a dose- and developmental-timing-dependent reduction in organoid size, which was more prominent in developmentally younger organoids. This coincided with a dose-dependent canonical p53-DREAM-dependent DNA damage response (DDR), consisting of cell cycle arrest, DNA repair and apoptosis. The DDR was delayed and less pronounced in the older organoids. Besides the DDR, we observed radiation-induced premature differentiation of neural progenitors and changes in metabolism. Importantly, our transcriptomic analysis furthermore demonstrated a concerted p53-E2F4-dependent repression of primary microcephaly genes. We found that this was a human-specific feature, as it was not observed in mouse embryonic brains or primary mouse neural progenitor cells. Thus, human forebrain organoids are an excellent model to investigate prenatal DNA damage-induced microcephaly and to uncover potentially targetable human-specific pathways.RNA-seq of coding RNAMus musculusA human-specific, concerted repression of microcephaly genes contributes to radiation-induced growth defects in forebrain organoidsInterneuron migration impairment and brain region-specific DNA damage response following irradiation during early neurogenesis in miceLisa BerdenLisa Berden, Nicholas Rajan, André-Claude Mbouombouo Mfossa, Isabeau De Bie, Emre Etlioglu, Mohammed Abderrafi Benotmane, Mieke Verslegers, Najat Aourz, Ilse Smolders, Jean-Michel Rigo, Bert Brône, Roel QuintensRoel QuintensNicholas RajanEmre EtliogluJessica Honorato Ribeiro, Emre Etlioglu, Jasmine Buset, Ann Janssen, Hanne Puype, Lisa Berden, André Claude Mbouombouo Mfossa, Winnok H. De Vos, Vanessa Vermeirssen, Sarah Baatout, Nicholas Rajan, Roel QuintensfalseBulk RNA-seq of primary mouse neural progenitor cells derived from the embryonic neocortex or medial ganglionic eminence at different time points after exposure to ionizing radiationEmbryonic DNA damage, e.g. resulting from exposure to moderate and high doses of ionizing radiation, has important consequences on the normal neurodevelopment, often resulting in microcephaly that can be associated with seizures. The effects of prenatal radiation exposure have previously been investigated with a focus on the neocortex, which is the birthplace of excitatory neurons. The possible effects on interneurons and specifically their progenitors are currently unknown. Here, we generated primary neural progenitor cell (NPC) cultures from the neocortex (NCX) and the medial ganglionic eminences (MGE), the origin of the majority of interneurons, of embryonic day 13 mice. Cells were irradiated with a dose of 0 Gy (sham) or 1 Gy and RNA was extracted at 6 h and 24 h following exposure, and subsequently used for RNA-sequencing (PE150).2025-10-01T00:00:00Z2025-10-01T01:06:00.388Z2024-10-10T14:02:19.002ZE-MTAB-14528ERP165007EFO_0002944EFO_0004170EFO_0003789EFO_0005518EFO_0003738EFO_0004184EFO_000396910.1101/2024.06.27.600564