Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs

Hubbard, Kyle; Gut, Ian; Lyman, Megan; McNutt, Patrick

doi:10.12688/f1000research.2-35.v1

Cited by 62 publications

(83 citation statements)

References 28 publications

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“…To investigate the potential physiological roles of IR during cell differentiation, we analyzed alternative retained introns using RNA-seq data generated across a time series of differentiation of cortical glutamatergic neurons from murine embryonic stem (ES) cells (Hubbard et al 2013). Strikingly, the vast majority (88.7%) of detected differentially retained introns between ES and mature neurons display a progressive increase in retention during differentiation (Fig.…”

Section: Ir Down-regulates Nonphysiologically Relevant Transcriptsmentioning

confidence: 99%

“…For in vitro differentiation of ES cells into neurons, CGR8 mouse ES cells were maintained at subconfluent conditions on gelatincoated plates and were differentiated into neurons as previously described (Hubbard et al 2013). RNA was extracted at different time points during the differentiation protocol using the RNeasy Mini Kit (Qiagen) as recommended by the manufacturer.…”

Section: Cell Culturementioning

confidence: 99%

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Widespread intron retention in mammals functionally tunes transcriptomes

et al. 2014

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Alternative splicing (AS) of precursor RNAs is responsible for greatly expanding the regulatory and functional capacity of eukaryotic genomes. Of the different classes of AS, intron retention (IR) is the least well understood. In plants and unicellular eukaryotes, IR is the most common form of AS, whereas in animals, it is thought to represent the least prevalent form. Using high-coverage poly(A)+ RNA-seq data, we observe that IR is surprisingly frequent in mammals, affecting transcripts from as many as three-quarters of multiexonic genes. A highly correlated set of cis features comprising an ''IR code'' reliably discriminates retained from constitutively spliced introns. We show that IR acts widely to reduce the levels of transcripts that are less or not required for the physiology of the cell or tissue type in which they are detected. This ''transcriptome tuning'' function of IR acts through both nonsense-mediated mRNA decay and nuclear sequestration and turnover of IR transcripts. We further show that IR is linked to a cross-talk mechanism involving localized stalling of RNA polymerase II (Pol II) and reduced availability of spliceosomal components. Collectively, the results implicate a global checkpoint-type mechanism whereby reduced recruitment of splicing components coupled to Pol II pausing underlies widespread IR-mediated suppression of inappropriately expressed transcripts.

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Section: Ir Down-regulates Nonphysiologically Relevant Transcriptsmentioning

confidence: 99%

Section: Cell Culturementioning

confidence: 99%

Widespread intron retention in mammals functionally tunes transcriptomes

et al. 2014

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“…RNA-seq datasets have provided reliable quantitative measurements of the dynamics of AS patterns during neuronal differentiation Hubbard et al, 2013;Irimia et al, 2014;Raj et al, 2014) and have been further utilized to investigate the contribution of AS to transcriptome complexity in brain tissues (Barbosa-Morais et al, 2012;Merkin et al, 2012). Furthermore, multiple variations of in vivo UV-induced crosslinking immunoprecipitation coupled to high-throughput sequencing methods (herein referred to as CLIP-Seq) have revolutionized transcriptome-wide studies of RNA-protein interactions (Darnell, 2010;Kö nig et al, 2011).…”

Section: Introductionmentioning

confidence: 99%

“…Microexon Regulation and Function(A) Median percent spliced in (PSI) levels of alternative neural microexons (top panel), and changes in gene expression (bottom panel) of Rbfox (blue), Ptbp1 (orange), and nSR100 (green) proteins during in vitro differentiation of embryonic stem cells (ESCs) to cortical glutamatergic neurons(Hubbard et al, 2013). Opposing activities and expression levels of Rbfox, nSR100, and Ptbp1 regulate microexon inclusion.…”

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confidence: 99%

Alternative Splicing in the Mammalian Nervous System: Recent Insights into Mechanisms and Functional Roles

Raj

Blencowe

2015

Neuron

431

392

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High-throughput transcriptomic profiling approaches have revealed that alternative splicing (AS) of precursor mRNAs, a fundamental process by which cells expand their transcriptomic diversity, is particularly widespread in the nervous system. AS events detected in the brain are more highly conserved than those detected in other tissues, suggesting that they more often provide conserved functions. Our understanding of the mechanisms and functions of neural AS events has significantly advanced with the coupling of various computational and experimental approaches. These studies indicate that dynamic regulation of AS in the nervous system is critical for modulating protein-protein interactions, transcription networks, and multiple aspects of neuronal development. Furthermore, several underappreciated classes of AS with the aforementioned roles in neuronal cells have emerged from unbiased, global approaches. Collectively, these findings emphasize the importance of characterizing neural AS in order to gain new insight into pathways that may be altered in neurological diseases and disorders.

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“…Longitudinal expression profiling using RNA-sequencing corroborated morphological and proteomic evidence of neuronal specification and maturation 11,15 . Representative markers of developmental progression exhibited stage-specific expression profiles, including Oct3/4, Nestin, DCX, NeuN and KCC2 ( Figure 2B).…”

Section: Representative Resultsmentioning

confidence: 76%

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

Hubbard

Beske

Lyman

et al. 2015

JoVE

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Therapeutic and mechanistic studies of the presynaptically targeted clostridial neurotoxins (CNTs) have been limited by the need for a scalable, cell-based model that produces functioning synapses and undergoes physiological responses to intoxication. Here we describe a simple and robust method to efficiently differentiate murine embryonic stem cells (ESCs) into defined lineages of synaptically active, networked neurons. Following an 8 day differentiation protocol, mouse embryonic stem cell-derived neurons (ESNs) rapidly express and compartmentalize neurotypic proteins, form neuronal morphologies and develop intrinsic electrical responses. By 18 days after differentiation (DIV 18), ESNs exhibit active glutamatergic and γ-aminobutyric acid (GABA)ergic synapses and emergent network behaviors characterized by an excitatory:inhibitory balance. To determine whether intoxication with CNTs functionally antagonizes synaptic neurotransmission, thereby replicating the in vivo pathophysiology that is responsible for clinical manifestations of botulism or tetanus, whole-cell patch clamp electrophysiology was used to quantify spontaneous miniature excitatory post-synaptic currents (mEPSCs) in ESNs exposed to tetanus neurotoxin (TeNT) or botulinum neurotoxin (BoNT) serotypes / A-/G. In all cases, ESNs exhibited near-complete loss of synaptic activity within 20 hr. Intoxicated neurons remained viable, as demonstrated by unchanged resting membrane potentials and intrinsic electrical responses. To further characterize the sensitivity of this approach, dosedependent effects of intoxication on synaptic activity were measured 20 hr after addition of BoNT/A. Intoxication with 0.005 pM BoNT/A resulted in a significant decrement in mEPSCs, with a median inhibitory concentration (IC 50 ) of 0.013 pM. Comparisons of median doses indicate that functional measurements of synaptic inhibition are faster, more specific and more sensitive than SNARE cleavage assays or the mouse lethality assay. These data validate the use of synaptically coupled, stem cell-derived neurons for the highly specific and sensitive detection of CNTs.

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Longitudinal RNA sequencing of the deep transcriptome during neurogenesis of cortical glutamatergic neurons from murine ESCs

Cited by 62 publications

References 28 publications

Widespread intron retention in mammals functionally tunes transcriptomes

Widespread intron retention in mammals functionally tunes transcriptomes

Alternative Splicing in the Mammalian Nervous System: Recent Insights into Mechanisms and Functional Roles

Functional Evaluation of Biological Neurotoxins in Networked Cultures of Stem Cell-derived Central Nervous System Neurons

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