Studying the function of common genetic variants in primary human tissues and during development is challenging. To address this, we use an efficient multiplexing strategy to differentiate 215 human induced pluripotent stem cell (iPSC) lines toward a midbrain neural fate, including dopaminergic neurons, and use single-cell RNA sequencing (scRNA-seq) to profile over 1 million cells across three differentiation time points. The proportion of neurons produced by each cell line is highly reproducible and is predictable by robust molecular markers expressed in pluripotent cells. Expression quantitative trait loci (eQTL) were characterized at different stages of neuronal development and in response to rotenone-induced oxidative stress. Of these, 1,284 eQTL colocalize with known neurological trait risk loci, and 46% are not found in the Genotype-Tissue Expression (GTEx) catalog. Our study illustrates how coupling scRNA-seq with long-term iPSC differentiation enables mechanistic studies of human trait-associated genetic variants in otherwise inaccessible cell states.

Population-scale single-cell RNA-seq profiling across dopaminergic neuron differentiation

Dorsal Raphe Dual Serotonin-Glutamate Neurons Drive Reward by Establishing Excitatory Synapses on VTA Mesoaccumbens Dopamine Neurons.

Non-small cell lung cancer (NSCLC) remains lethal despite the development of numerous drug therapy technologies. About 85% to 90% of lung cancers are NSCLC and the 5-year survival rate is at best still below 50%. Thus, it is important to find drugable target genes for NSCLC to develop an effective therapy for NSCLC. Integrated analysis of publically available gene expression and promoter methylation patterns of two highly aggressive NSCLC cell lines generated by in vivo selection was performed. We selected eleven critical genes that may mediate metastasis using recently proposed principal component analysis based unsupervised feature extraction. The eleven selected genes were significantly related to cancer diagnosis. The tertiary protein structure of the selected genes was inferred by Full Automatic Modeling System, a profile-based protein structure inference software, to determine protein functions and to specify genes that could be potential drug targets. We identified eleven potentially critical genes that may mediate NSCLC metastasis using bioinformatic analysis of publically available data sets. These genes are potential target genes for the therapy of NSCLC. Among the eleven genes, TINAGL1 and B3GALNT1 are possible candidates for drug compounds that inhibit their gene expression.

TINAGL1 and B3GALNT1 are potential therapy target genes to suppress metastasis in non-small cell lung cancer

Mutations in minor spliceosome components such as U12 snRNA (cerebellar ataxia) and U4atac snRNA (microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1)) result in tissue-specific symptoms. Given that the minor spliceosome is ubiquitously expressed, we hypothesized that these restricted phenotypes might be caused by the tissue-specific regulation of the minor spliceosome targets, i.e. minor intron-containing genes (MIGs). The current model of inefficient splicing is thought to apply to the regulation of the ~ 500 MIGs identified in the U12DB. However this database was created more than 10 years ago. Therefore, we first wanted to revisit the classification of minor introns in light of the most recent reference genome. We then sought to address specificity of MIG expression, minor intron retention, and alternative splicing (AS) across mouse and human tissues. We employed position-weight matrices to obtain a comprehensive updated list of minor introns, consisting of 722 mouse and 770 human minor introns. These can be found in the Minor Intron DataBase (MIDB). Besides identification of 99% of the minor introns found in the U12DB, we also discovered ~ 150 new MIGs. We then analyzed the RNAseq data from eleven different mouse tissues, which revealed tissue-specific MIG expression and minor intron retention. Additionally, many minor introns were efficiently spliced compared to their flanking major introns. Finally, we identified several novel AS events across minor introns in both mouse and human, which were also tissue-dependent. Bioinformatics analysis revealed that several of the AS events could result in the production of novel tissue-specific proteins. Moreover, like the major introns, we found that these AS events were more prevalent in long minor introns, while retention was favoured in shorter introns. Here we show that minor intron splicing and AS across minor introns is a highly organised process that might be regulated in coordination with the major spliceosome in a tissue-specific manner. We have provided a framework to further study the impact of the minor spliceosome and the regulation of MIG expression. These findings may shed light on the mechanism underlying tissue-specific phenotypes in diseases associated with minor spliceosome inactivation. MIDB can be accessed at https://midb.pnb.uconn.edu
 .

/pdf/minor-intron-splicing-revisited-identification-of-new-minor-2lfsf6gqlm.pdf

Minor intron splicing revisited: identification of new minor intron-containing genes and tissue-dependent retention and alternative splicing of minor introns

Tracking Calcium Dynamics and Immune Surveillance at the Choroid Plexus Blood-Cerebrospinal Fluid Interface

Among the brainstem raphe nuclei, the dorsal raphe nucleus (DR) contains the greatest number of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout DR subdomains These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR We further show that P2ry1-Pet1 DR neurons - the most molecularly distinct subtype - possess unique efferent projections and electrophysiological properties These data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Mutation in minor spliceosome components is linked to the developmental disorder microcephalic osteodysplastic primordial dwarfism type 1 (MOPD1). Here, we inactivated the minor spliceosome in the developing mouse cortex (pallium) by ablating Rnu11, which encodes the crucial minor spliceosome small nuclear RNA (snRNA) U11. Rnu11 conditional knockout mice were born with microcephaly, which was caused by the death of self-amplifying radial glial cells (RGCs), while intermediate progenitor cells and neurons were produced. RNA sequencing suggested that this cell death was mediated by upregulation of p53 (Trp53 - Mouse Genome Informatics) and DNA damage, which were both observed specifically in U11-null RGCs. Moreover, U11 loss caused elevated minor intron retention in genes regulating the cell cycle, which was consistent with fewer RGCs in S-phase and cytokinesis, alongside prolonged metaphase in RGCs. In all, we found that self-amplifying RGCs are the cell type most sensitive to loss of minor splicing. Together, these findings provide a potential explanation of how disruption of minor splicing might cause microcephaly in MOPD1.

/pdf/minor-spliceosome-inactivation-causes-microcephaly-owing-to-zgoj74l1ro.pdf

Minor spliceosome inactivation causes microcephaly, owing to cell cycle defects and death of self-amplifying radial glial cells.

Author response: A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons

Brainstem median raphe (MR) neurons expressing the serotonergic regulator gene Pet1 send collateralized projections to forebrain regions to modulate affective, memory-related, and circadian behaviors Some Pet1 neurons express a surprisingly incomplete battery of serotonin pathway genes, with somata lacking transcripts for tryptophan hydroxylase 2 (Tph2) encoding the rate-limiting enzyme for serotonin [5-hydroxytryptamine (5-HT)] synthesis, but abundant for vesicular glutamate transporter type 3 (Vglut3) encoding a synaptic vesicle-associated glutamate transporter Genetic fate maps show these nonclassical, putatively glutamatergic Pet1 neurons in the MR arise embryonically from the same progenitor cell compartment-hindbrain rhombomere 2 (r2)-as serotonergic TPH2+ MR Pet1 neurons Well established is the distribution of efferents en masse from r2-derived, Pet1-neurons; unknown is the relationship between these efferent targets and the specific constituent source-neuron subgroups identified as r2-Pet1Tph2 -high versus r2-Pet1Vglut3 -high Using male and female mice, we found r2-Pet1 axonal boutons segregated anatomically largely by serotonin+ versus VGLUT3+ identity The former present in the suprachiasmatic nucleus, paraventricular nucleus of the thalamus, and olfactory bulb; the latter are found in the hippocampus, cortex, and septum Thus r2-Pet1Tph2- high and r2-Pet1Vglut3- high neurons likely regulate distinct brain regions and behaviors Some r2-Pet1 boutons encased interneuron somata, forming specialized presynaptic "baskets" of VGLUT3+ or VGLUT3+/5-HT+ identity; this suggests that some r2-Pet1Vglut3- high neurons may regulate local networks, perhaps with differential kinetics via glutamate versus serotonin signaling Fibers from other Pet1 neurons (non-r2-derived) were observed in many of these same baskets, suggesting multifaceted regulation Collectively, these findings inform brain organization and new circuit nodes for therapeutic considerationsSIGNIFICANCE STATEMENT Our findings match axonal bouton neurochemical identity with distant cell bodies in the brainstem raphe The results are significant because they suggest that disparate neuronal subsystems derive from Pet1 + precursor cells of the embryonic progenitor compartment rhombomere 2 (r2) Of these r2-Pet1 neuronal subsystems, one appears largely serotonergic, as expected given expression of the serotonergic regulator PET1, and projects to the olfactory bulb, thalamus, and suprachiasmatic nucleus Another expresses VGLUT3, suggesting principally glutamate transmission, and projects to the hippocampus, septum, and cortex Some r2-Pet1 boutons-those that are VGLUT3+ or VGLUT3+/5-HT+ co-positive-comprise "baskets" encasing interneurons, suggesting that they control local networks perhaps with differential kinetics via glutamate versus serotonin signaling Results inform brain organization and circuit nodes for therapeutic consideration

https://www.jneurosci.org/content/jneuro/41/12/2581.full.pdf

Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

The dorsal raphe nucleus (DR) contains the largest brain population of Pet1-lineage neurons, a predominantly serotonergic group distributed throughout multiple DR subdomains. These neurons collectively regulate diverse physiology and behavior and are often therapeutically targeted to treat affective disorders. Characterizing Pet1 neuron molecular heterogeneity and relating it to anatomy is vital for understanding DR functional organization, with potential to inform therapeutic separability. Here we use high-throughput and DR subdomain-targeted single-cell transcriptomics and intersectional genetic tools to map molecular and anatomical diversity of DR-Pet1 neurons. We describe up to fourteen neuron subtypes, many showing biased cell body distributions across the DR. We further show that P2ry1-Pet1 DR neurons – the most molecularly distinct of the subtypes – possess unique efferent projections and electrophysiological properties. The present data complement and extend previous DR characterizations, combining intersectional genetics with multiple transcriptomic modalities to achieve fine-scale molecular and anatomic identification of Pet1 neuron subtypes.

https://www.biorxiv.org/content/biorxiv/early/2020/01/29/2020.01.28.923375.full.pdf

Nikita Sturrock

Papers

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons.

Minor spliceosome inactivation causes microcephaly, owing to cell cycle defects and death of self-amplifying radial glial cells.

Author response: A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons

Neurochemically and Hodologically Distinct Ascending VGLUT3 versus Serotonin Subsystems Comprise the r2-Pet1 Median Raphe.

A single-cell transcriptomic and anatomic atlas of mouse dorsal raphe Pet1 neurons