Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development

Tiklová, Katarína; Björklund, Åsa K.; Lahti, Laura; Fiorenzano, Alessandro; Nolbrant, Sara; Gillberg, Linda; Volakakis, Nikolaos; Yokota, Chika; Hilscher, Markus M.; Hauling, Thomas; Holmstrom, F Ross; Joodmardi, Eliza; Nilsson, Mats; Parmar, Malin; Perlmann, Thomas

doi:10.1038/s41467-019-08453-1

Cited by 238 publications

(274 citation statements)

References 31 publications

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“…Understanding 3D chromatin structure of rare cell populations is of exceptional importance in the brain, where connecting disease-associated genetic variants in non-coding genomic regions with their target genes remains particularly challenging [11][12][13] , and where cell state and cell localization within the tissue can influence both transcriptional and physiological outcomes [14][15][16][17] .…”

Section: Introductionmentioning

confidence: 99%

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Winick-Ng

Kukalev

Harabula

et al. 2020

Preprint

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Neurons and oligodendrocytes are terminally differentiated cells that perform highly specialized functions, which depend on cascades of gene activation and repression to retain homeostatic control over a lifespan. Gene expression is regulated by threedimensional (3D) genome organisation, from local levels of chromatin compaction to the organisation of topological domains and chromosome compartments. Whereas our understanding of 3D genome architecture has vastly increased in the past decade, it remains difficult to study specialized cells in their native environment without disturbing their activity. To develop the application of Genome Architecture Mapping (GAM) in small numbers of specialized cells in complex tissues, we combined GAM with immunoselection. We applied immunoGAM to map the genome architecture of specific cell populations in the juvenile/adult mouse brain: dopaminergic neurons (DNs) from the midbrain, pyramidal glutamatergic neurons (PGNs) from the hippocampus, and oligodendrocyte lineage cells (OLGs) from the cortex. We integrate 3D genome organisation with single-cell transcriptomics data, and find specific chromatin structures that relate with cell-type specific patterns of gene expression. We discover abundant changes in compartment organisation, especially a strengthening of heterochromatin compartments which establish strong contacts spanning tens of megabases, especially in brain cells. These compartments contain olfactory and taste receptor genes, which are de-repressed in a subpopulation of PGNs with molecular signatures of long-term potentiation (LTP). We also show extensive reorganisation of topological domains where activation of neuronal or oligodendrocyte genes coincides with formation of new TAD borders.Finally, we discover loss of TAD organisation, or 'TAD melting', at long (>1Mb) neuronal genes when they are most highly expressed. Our work shows that the 3D 3 organisation of the genome is highly cell-type specific in terminally differentiated cells of the brain, and essential to better understand brain-specific mechanisms of gene regulation.

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Section: Introductionmentioning

confidence: 99%

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Winick-Ng

Kukalev

Harabula

et al. 2020

Preprint

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“…Further delineation of the continuous cell cycle trajectory and the uncoupling of cytokinesis from karyokinesis require a systematic molecular classification at the transcriptome level. The scRNA-seq approach has revolutionized the possibilities for defining distinct cellular states and diversity, [77][78][79] thus further exploration of temporal changes in gene expression during mitosis could elucidate the transition from proliferation to a terminally differentiated binucleated phenotype.…”

Section: Discussionmentioning

confidence: 99%

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

Milliron

Weiland

Kort

et al. 2019

Preprint

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Rationale-Adult human cardiomyocytes (CMs) do not complete cytokinesis despite passing through the S-phase of the cell cycle. As a result polyploidization and multinucleation occur. In order to get a deeper understanding of the mechanisms surrounding division of CMs there is a crucial need for a technique to isolate CMs that complete cell division/cytokinesis.Objective-Markers of cell cycle progression based on DNA content cannot distinguish between mitotic CMs that fail to complete cytokinesis from those cells that undergo true cell division. With the use of molecular beacons (MB) targeting specific mRNAs we aimed to identify truly proliferative CMs derived from hiPSCs.Methods and Results-Fluorescence activated cell-sorting combined with molecular beacons was performed to sort CM populations enriched for mitotic cells. Expressions of cell-cycle specific genes were confirmed by means of RT-qPCR, single-cell RNA sequencing (scRNA-seq). We further characterized the sorted groups by proliferation assays and time-lapse microscopy which confirmed the proliferative advantage of MB-positive cell populations relative to MB-negative and G2/M populations. Gene expression analysis revealed that the MB-positive CM subpopulation exhibited patterns consistent with the biological processes of nuclear division, chromosome segregation, and transition from M to G1 phase. The use of dual-MBs targeting CDC20 and SPG20 mRNAs (CDC20 + SPG20 + ) enabled the enrichment of cytokinetic events. Interestingly, cells that did not complete cytokinesis and remained binucleated were found to be CDC20 -SPG20 + while polyploid CMs that replicated DNA but failed to complete karyokinesis were found to be CDC20 -SPG20 -.Conclusions-This study demonstrates a novel alternative to existing DNA content-based approaches for sorting CMs with true mitotic potential that can be used to study in detail the unique dynamics of CM nuclei during mitosis. Together with high-throughput scRNA-seq, our technique for sorting live CMs undergoing cytokinesis would provide a basis for future studies to uncover mechanisms underlying the development and regeneration of heart tissue.

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“…The main techniques available to obtain single cell suspensions from brain tissue are enzymatic digestion (ED) and mechanical dissociation (MD). The latter is typically carried out at 4°C (Mattei et al, 2017), whereas ED requires incubation at temperatures between 30-37°C (Saunders et al, 2018; Tiklova et al, 2019; Zhang et al, 2014). With both techniques, cells such as neurons and microglia will lose their processes and suffer harsh conditions whereby a proportion of cells die during the dissociation process.…”

Section: Introductionmentioning

confidence: 99%

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

Mattei

Ivanov

Oostrum

et al. 2020

Preprint

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2Different cell isolation techniques exist for transcriptomic and proteotype profiling of brain cells. 3Here, we provide a systematic investigation of the influence of different cell isolation protocols on 4 transcriptional and proteotype profiles in mouse brain tissue by taking into account single-cell 5 transcriptomics of brain cells, proteotypes of microglia and astrocytes, and flow cytometric analysis 6 of microglia. We show that standard enzymatic digestion of brain tissue at 37°C induces profound 7 and consistent alterations in the transcriptome and proteotype of neuronal and glial cells, as compared 8 to an optimized mechanical dissociation protocol at 4°C. These findings emphasize the risk of 9 introducing technical biases and biological artefacts when implementing enzymatic digestion-based 10 isolation methods for brain cell analyses. 11

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Single-cell RNA sequencing reveals midbrain dopamine neuron diversity emerging during mouse brain development

Cited by 238 publications

References 31 publications

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Cell-type specialization in the brain is encoded by specific long-range chromatin topologies

Isolation of cardiomyocytes undergoing mitosis with complete cytokinesis

Enzymatic dissociation induces transcriptional and proteotype bias in brain cell populations

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