Project description:Exposure to ionizing radiation around the time of neural tube closure results in a number of developmental defects, including neural tube defects (e.g. exencephaly, cleft palate) and eye defects (e.g. microphtalmos, anaphtalmos). These defects result from perturbed neural tube closure and development of the presumptive eye. In order to better understand the underlying molecular mechanisms, gene expression profiling was performed in heads of mouse embryos at embryonic day 9, after they had been irradiated at embryonic day 7.5 with 1 Gy of X-rays. Non-irradiated mice served as controls for differential gene expression.
Project description:We found that conventional targeted disruption of the entire Cables2 locus caused post-gastrulation embryonic lethality in mouse. To examine global gene changes during gastrulation, we performed the RNA-seq experiment using WT- and Cables2-null embryos at embryonic day 6.5 and 7.5.
Project description:To characterize the effect of cell cyle elongation on nascent transcription during zygotic genome activation (ZGA) of Xenopus laevis embryos, we microinjected 5-ethynyl uridine (EU) into 1-cell stage embryos and treated the embryos with 0.2 mg/ml of cycloheximide (CHX) to arrest them in interphase from 5 hpf to 7.5 hpf. Control and CHX-arrested embryos were collected for isolating total RNAs, followed by biotinylation using disulfide biotin azide via click reaction and purification of nascent transcripts using streptavidin beads. Libraries were constructed and sequenced on illumina NextSeq 500.
Project description:Alternative splicing (AS) and alternative promoter (AP) usage expand the repertories of mammalian transcriptome profiles and thus diversify gene functions. However, our knowledge about the extent and functions of AS and AP usage in mouse early embryogenesis remains elusive. Here, by performing whole-transcriptome splicing profiling with high-throughput next generation sequencing, we report that AS extensively occurs in embryonic day (E) 7.5 mouse primary germ layers, and may be involved in multiple developmental processes. In addition, numerous RNA splicing factors are differentially expressed and alternatively spliced across the three germ layers, implying the potential importance of AS machinery in shaping early embryogenesis. Notably, AP usage is remarkably frequent at this stage, accounting for more than one quarter (430/1648) of the total significantly different AS events. Genes generating the 430 AP events participate in numerous biological processes, and include important regulators essential for mouse early embryogenesis, suggesting that AP usage is widely used and might be relevant to mouse germ layer specification. Our data underline the potential significance of AP usage in mouse gastrulation, providing a rich data source and opening another dimension for understanding the regulatory mechanisms of mammalian early development.
Project description:Embryonic day (E)12.5 whole murine embryos, E11.5 - E14.5 whole murine embryos, E11.5 - E14.5, post-natal day (P)3 and P35 murine forelimbs, E14.5 brains, and COL1A2-mutant and COL1A2-WT forelimbs were fractionated and specific fractions were analyzed via LC-MS/MS. Aha-enrichment experiments consisted of in vivo protein labeling with azidohomoalanine (Aha) followed by tissue fractionation of the forelimbs and enrichment of labeled ECM proteins from the final IN pellet ('enriched'). 'Unenriched samples', or the background from which newly synthesized proteins were enriched from, were also analyzed via LC-MS/MS.