Unknown,Transcriptomics,Genomics,Proteomics

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Slamseq of whole neurons with perturbed mRNA degradation machinery

ABSTRACT: We perturbed mRNA degradation machinery in Ascl1-induced neurons (iNeurons) and investigated the change in mRNA half-lives. We performed SLAMseq Metabolic RNA Labeling on Mutant iNeuron line harbouring a ponasteroneA-inducible heterozygous dominant-negative Caf1 (dnCaf1) and on Wild Type iNeurons (WT). The Slamseq technique provided snapshots of mRNA kinetics allowing to estimate mRNA half-lives and assess the effect of mRNA degradation machinery on the level of mRNA stability.

INSTRUMENT(S): NextSeq 500

ORGANISM(S): Mus musculus

SUBMITTER: Marina Chekulaeva

PROVIDER: E-MTAB-9545 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:Specific neuronal types derived from embryonic stem cells (ESCs) can facilitate mechanistic studies and potentially aid in regenerative medicine. Existing induction methods, however, mostly rely on the effects of growth factors, which generally tend to result in mixed populations of neurons. Here we report that over-expression of specific transcription factors (TFs) in ESCs can rather guide the differentiation of ESCs towards specific neuron types. Analysis of published data on gene expression changes early (two days) after induction of each of 185 induced TFs implicated candidate TFs for further ESC differentiation studies. After induction for 6 days four of them (Ascl1, Smad7, Nr2f1, and Ascl2) generated a high proportion (>35%) of cells with neural progenitor marker PSA-NCAM and clear neural morphology on day 14. The capacity of these TFs to induce neural differentiation is inferred to be most likely linked to early activation of the Notch signaling pathway. Among the neuron-like cells, GABA-positive cells were most abundant (32-97% for 4 top TFs), whereas Isl1-positive cells and TH-positive cells were less abundant (<12% and <5%, respectively). Enrichment of cells obtained with the induction of Ascl1, Smad7, and Nr2f1 using beads with anti-PSA-NCAM antibody resulted in essentially pure population of neuron-like cells with expression profiles similar to neural tissues and highly expressed markers of GABAergic neurons. A time-course experiment with induction of Ascl1 showed early upregulation of most neural-specific and GABAergic-specific mRNA and miRNAs. We identified mRNA and miRNAs, whose expression depended on the induction of Ascl1, and showed that they were enriched in Ascl1 target genes. In summary, this study indicates that induction of transcription factors is a promising approach to generate candidate specific neural cell types from ESCs. Transcription factor Ascl1 was induced in mouse ESCs to facilitate neural differentiation. Expression of transgenic Ascl1 was repressed by doxycycline (Dox); thus, it were induced in Dox- conditions, whereas Dox+ conditions represent control cells with no expression of Ascl1 transgene. For neural differentiation, cells were cultured 3 days in alpha-MEM medium and then - in NeuroCult neural differentiation medium for 2-11 days (total up to 14 days). RNA was extracted with mirVana kit (Thermo Fisher Scientific).

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Slamseq of whole neurons with perturbed mRNA degradation machinery

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