High-throughput single-cell gene expression experiments can be used to uncover branching dynamics in cell populations undergoing differentiation through pseudotime methods. We develop the branching ...Gaussian process (BGP), a non-parametric model that is able to identify branching dynamics for individual genes and provide an estimate of branching times for each gene with an associated credible region. We demonstrate the effectiveness of our method on simulated data, a single-cell RNA-seq haematopoiesis study and mouse embryonic stem cells generated using droplet barcoding. The method is robust to high levels of technical variation and dropout, which are common in single-cell data.
There is a paucity of information about longitudinal patterns of IgE responses to allergenic proteins (components) from multiple sources.
This study sought to investigate temporal patterns of ...component-specific IgE responses from infancy to adolescence, and their relationship with allergic diseases.
In a population-based birth cohort, we measured IgE to 112 components at 6 follow-ups during childhood. We used a Bayesian method to discover cross-sectional sensitization patterns and their longitudinal trajectories, and we related these patterns to asthma and rhinitis in adolescence.
We identified 1 sensitization cluster at age 1, 3 at age 3, 4 at ages 5 and 8, 5 at age 11, and 6 at age 16 years. “Broad” cluster was the only cluster present at every follow-up, comprising components from multiple sources. “Dust mite” cluster formed at age 3 years and remained unchanged to adolescence. At age 3 years, a single-component “Grass” cluster emerged, which at age 5 years absorbed additional grass components and Fel d 1 to form the “Grass/cat” cluster. Two new clusters formed at age 11 years: “Cat” cluster and “PR-10/profilin” (which divided at age 16 years into “PR-10” and “Profilin”). The strongest contemporaneous associate of asthma at age 16 years was sensitization to dust mite cluster (odds ratio: 2.6; 95% CI: 1.2-6.1; P < .05), but the strongest early life predictor of subsequent asthma was sensitization to grass/cat cluster (odds ratio: 3.5; 95% CI: 1.6-7.4; P < .01).
We describe the architecture of the evolution of IgE responses to multiple allergen components throughout childhood, which may facilitate development of better diagnostic and prognostic biomarkers for allergic diseases.
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional ...inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
During embryogenesis cells make fate decisions within complex tissue environments. The levels and dynamics of transcription factor expression regulate these decisions. Here, we use single cell live ...imaging of an endogenous HES5 reporter and absolute protein quantification to gain a dynamic view of neurogenesis in the embryonic mammalian spinal cord. We report that dividing neural progenitors show both aperiodic and periodic HES5 protein fluctuations. Mathematical modelling suggests that in progenitor cells the HES5 oscillator operates close to its bifurcation boundary where stochastic conversions between dynamics are possible. HES5 expression becomes more frequently periodic as cells transition to differentiation which, coupled with an overall decline in HES5 expression, creates a transient period of oscillations with higher fold expression change. This increases the decoding capacity of HES5 oscillations and correlates with interneuron versus motor neuron cell fate. Thus, HES5 undergoes complex changes in gene expression dynamics as cells differentiate.
In vitro generation and expansion of hematopoietic stem cells (HSCs) holds great promise for the treatment of any ailment that relies on bone marrow or blood transplantation. To achieve this, it is ...essential to resolve the molecular and cellular pathways that govern HSC formation in the embryo. HSCs first emerge in the aorta-gonad-mesonephros (AGM) region, where a rare subset of endothelial cells, hemogenic endothelium (HE), undergoes an endothelial-to-hematopoietic transition (EHT). Here, we present full-length single-cell RNA sequencing (scRNA-seq) of the EHT process with a focus on HE and dorsal aorta niche cells. By using Runx1b and Gfi1/1b transgenic reporter mouse models to isolate HE, we uncovered that the pre-HE to HE continuum is specifically marked by angiotensin-I converting enzyme (ACE) expression. We established that HE cells begin to enter the cell cycle near the time of EHT initiation when their morphology still resembles endothelial cells. We further demonstrated that RUNX1 AGM niche cells consist of vascular smooth muscle cells and PDGFRa+ mesenchymal cells and can functionally support hematopoiesis. Overall, our study provides new insights into HE differentiation toward HSC and the role of AGM RUNX1+ niche cells in this process. Our expansive scRNA-seq datasets represents a powerful resource to investigate these processes further.
Oscillatory genes, with periodic expression at the mRNA and/or protein level, have been shown to play a pivotal role in many biological contexts. However, with the exception of the circadian clock ...and cell cycle, only a few such genes are known. Detecting oscillatory genes from snapshot single-cell experiments is a challenging task due to the lack of time information. Oscope is a recently proposed method to identify co-oscillatory gene pairs using single-cell RNA-seq data. Although promising, the current implementation of Oscope does not provide a principled statistical criterion for selecting oscillatory genes.
We improve the optimisation scheme underlying Oscope and provide a well-calibrated non-parametric hypothesis test to select oscillatory genes at a given FDR threshold. We evaluate performance on synthetic data and three real datasets and show that our approach is more sensitive than the original Oscope formulation, discovering larger sets of known oscillators while avoiding the need for less interpretable thresholds. We also describe how our proposed pseudo-time estimation method is more accurate in recovering the true cell order for each gene cluster while requiring substantially less computation time than the extended nearest insertion approach.
OscoNet is a robust and versatile approach to detect oscillatory gene networks from snapshot single-cell data addressing many of the limitations of the original Oscope method.
Previous studies have failed to identify mutations in the Wilson's disease gene ATP7B in a significant number of clinically diagnosed cases. This has led to concerns about genetic heterogeneity for ...this condition but also suggested the presence of unusual mutational mechanisms. We now present our findings in 181 patients from the United Kingdom with clinically and biochemically confirmed Wilson's disease. A total of 116 different ATP7B mutations were detected, 32 of which are novel. The overall mutation detection frequency was 98%. The likelihood of mutations in genes other than ATP7B causing a Wilson's disease phenotype is therefore very low. We report the first cases with Wilson's disease due to segmental uniparental isodisomy as well as three patients with three ATP7B mutations and three families with Wilson's disease in two consecutive generations. We determined the genetic prevalence of Wilson's disease in the United Kingdom by sequencing the entire coding region and adjacent splice sites of ATP7B in 1000 control subjects. The frequency of all single nucleotide variants with in silico evidence of pathogenicity (Class 1 variant) was 0.056 or 0.040 if only those single nucleotide variants that had previously been reported as mutations in patients with Wilson's disease were included in the analysis (Class 2 variant). The frequency of heterozygote, putative or definite disease-associated ATP7B mutations was therefore considerably higher than the previously reported occurrence of 1:90 (or 0.011) for heterozygote ATP7B mutation carriers in the general population (P < 2.2 × 10(-16) for Class 1 variants or P < 5 × 10(-11) for Class 2 variants only). Subsequent exclusion of four Class 2 variants without additional in silico evidence of pathogenicity led to a further reduction of the mutation frequency to 0.024. Using this most conservative approach, the calculated frequency of individuals predicted to carry two mutant pathogenic ATP7B alleles is 1:7026 and thus still considerably higher than the typically reported prevalence of Wilson's disease of 1:30 000 (P = 0.00093). Our study provides strong evidence for monogenic inheritance of Wilson's disease. It also has major implications for ATP7B analysis in clinical practice, namely the need to consider unusual genetic mechanisms such as uniparental disomy or the possible presence of three ATP7B mutations. The marked discrepancy between the genetic prevalence and the number of clinically diagnosed cases of Wilson's disease may be due to both reduced penetrance of ATP7B mutations and failure to diagnose patients with this eminently treatable disorder.
Genes with similar transcriptional activation kinetics can display very different temporal mRNA profiles because of differences in transcription time, degradation rate, and RNA-processing kinetics. ...Recent studies have shown that a splicing-associated RNA production delay can be significant. To investigate this issue more generally, it is useful to develop methods applicable to genome-wide datasets. We introduce a joint model of transcriptional activation and mRNA accumulation that can be used for inference of transcription rate, RNA production delay, and degradation rate given data from high-throughput sequencing time course experiments. We combine a mechanistic differential equation model with a nonparametric statistical modeling approach allowing us to capture a broad range of activation kinetics, and we use Bayesian parameter estimation to quantify the uncertainty in estimates of the kinetic parameters. We apply the model to data from estrogen receptor α activation in the MCF-7 breast cancer cell line. We use RNA polymerase II ChIP-Seq time course data to characterize transcriptional activation and mRNA-Seq time course data to quantify mature transcripts. We find that 11% of genes with a good signal in the data display a delay of more than 20 min between completing transcription and mature mRNA production. The genes displaying these long delays are significantly more likely to be short. We also find a statistical association between high delay and late intron retention in pre-mRNA data, indicating significant splicing-associated production delays in many genes.
Gene expression programs determine cell fate in embryonic development and their dysregulation results in disease. Transcription factors (TFs) control gene expression by binding to enhancers, but how ...TFs select and activate their target enhancers is still unclear. HOX TFs share conserved homeodomains with highly similar sequence recognition properties, yet they impart the identity of different animal body parts. To understand how HOX TFs control their specific transcriptional programs in vivo, we compared HOXA2 and HOXA3 binding profiles in the mouse embryo. HOXA2 and HOXA3 directly cooperate with TALE TFs and selectively target different subsets of a broad TALE chromatin platform. Binding of HOX and tissue-specific TFs convert low affinity TALE binding into high confidence, tissue-specific binding events, which bear the mark of active enhancers. We propose that HOX paralogs, alone and in combination with tissue-specific TFs, generate tissue-specific transcriptional outputs by modulating the activity of TALE TFs at selected enhancers.
An intronic GGGGCC-repeat expansion of C9ORF72 is the most common genetic variant of amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. The mechanism of neurodegeneration is unknown, ...but a direct effect on RNA processing mediated by RNA foci transcribed from the repeat sequence has been proposed.
Gene expression profiling utilised total RNA extracted from motor neurons and lymphoblastoid cell lines derived from human ALS patients, including those with an expansion of C9ORF72, and controls. In lymphoblastoid cell lines, expansion length and the frequency of sense and antisense RNA foci was also examined.
Gene level analysis revealed a number of differentially expressed networks and both cell types exhibited dysregulation of a network functionally enriched for genes encoding 'RNA splicing' proteins. There was a significant overlap of these genes with an independently generated list of GGGGCC-repeat protein binding partners. At the exon level, in lymphoblastoid cells derived from C9ORF72-ALS patients splicing consistency was lower than in lines derived from non-C9ORF72 ALS patients or controls; furthermore splicing consistency was lower in samples derived from patients with faster disease progression. Frequency of sense RNA foci showed a trend towards being higher in lymphoblastoid cells derived from patients with shorter survival, but there was no detectable correlation between disease severity and DNA expansion length.
Up-regulation of genes encoding predicted binding partners of the C9ORF72 expansion is consistent with an attempted compensation for sequestration of these proteins. A number of studies have analysed changes in the transcriptome caused by C9ORF72 expansion, but to date findings have been inconsistent. As a potential explanation we suggest that dynamic sequestration of RNA processing proteins by RNA foci might lead to a loss of splicing consistency; indeed in our samples measurement of splicing consistency correlates with disease severity.
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