A mammalian brain is composed of numerous cell types organized in an intricate manner to form functional neural circuits. Single-cell RNA sequencing allows systematic identification of cell types ...based on their gene expression profiles and has revealed many distinct cell populations in the brain
. Single-cell epigenomic profiling
further provides information on gene-regulatory signatures of different cell types. Understanding how different cell types contribute to brain function, however, requires knowledge of their spatial organization and connectivity, which is not preserved in sequencing-based methods that involve cell dissociation. Here we used a single-cell transcriptome-imaging method, multiplexed error-robust fluorescence in situ hybridization (MERFISH)
, to generate a molecularly defined and spatially resolved cell atlas of the mouse primary motor cortex. We profiled approximately 300,000 cells in the mouse primary motor cortex and its adjacent areas, identified 95 neuronal and non-neuronal cell clusters, and revealed a complex spatial map in which not only excitatory but also most inhibitory neuronal clusters adopted laminar organizations. Intratelencephalic neurons formed a largely continuous gradient along the cortical depth axis, in which the gene expression of individual cells correlated with their cortical depths. Furthermore, we integrated MERFISH with retrograde labelling to probe projection targets of neurons of the mouse primary motor cortex and found that their cortical projections formed a complex network in which individual neuronal clusters project to multiple target regions and individual target regions receive inputs from multiple neuronal clusters.
To facilitate scalable profiling of single cells, we developed split-pool ligation-based transcriptome sequencing (SPLiT-seq), a single-cell RNA-seq (scRNA-seq) method that labels the cellular origin ...of RNA through combinatorial barcoding. SPLiT-seq is compatible with fixed cells or nuclei, allows efficient sample multiplexing, and requires no customized equipment. We used SPLiT-seq to analyze 156,049 single-nucleus transcriptomes from postnatal day 2 and 11 mouse brains and spinal cords. More than 100 cell types were identified, with gene expression patterns corresponding to cellular function, regional specificity, and stage of differentiation. Pseudotime analysis revealed transcriptional programs driving four developmental lineages, providing a snapshot of early postnatal development in the murine central nervous system. SPLiT-seq provides a path toward comprehensive single-cell transcriptomic analysis of other similarly complex multicellular systems.
Most transcription factor families contain highly related paralogs generated by gene duplication, and functional divergence is generally accomplished by activation of distinct sets of genes by each ...member. Here we compare the molecular functions of Myf5 and MyoD, two highly related bHLH transcription factors that regulate skeletal muscle specification and differentiation. We find that MyoD and Myf5 bind the same sites genome-wide but have distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits Pol II, and robustly activates gene transcription. Therefore, the initial specification of the muscle lineage by Myf5 occurs without significant induction of gene transcription. Transcription of the skeletal muscle program is then achieved by the subsequent expression of MyoD, which binds to the same sites as Myf5, indicating that each factor regulates distinct steps in gene initiation and transcription at a shared set of binding sites.
•Myf5 and MyoD bind a nearly identical set of sites genome-wide•Myf5 and MyoD induce comparable levels of H4 acetylation around their binding sites•Myf5 lacks a strong activation domain and does not induce robust transcription•MyoD and Myf5 have diverged in function rather than DNA motif binding specificity
Conerly et al. show that MyoD and Myf5 have overlapping binding sites but distinct functions: Myf5 induces histone acetylation without Pol II recruitment or robust gene activation, whereas MyoD induces histone acetylation, recruits Pol II, and robustly activates transcription. Domain swaps demonstrate that histone acetylation and transcriptional induction are separable.
In the present study we show that the master myogenic regulatory factor, MYOD1, is a positive modulator of molecular clock amplitude and functions with the core clock factors for expression of ...clock-controlled genes in skeletal muscle. We demonstrate that MYOD1 directly regulates the expression and circadian amplitude of the positive core clock factor
. We identify a non-canonical E-box element in
and demonstrate that is required for full MYOD1-responsiveness. Bimolecular fluorescence complementation assays demonstrate that MYOD1 colocalizes with both BMAL1 and CLOCK throughout myonuclei. We demonstrate that MYOD1 and BMAL1:CLOCK work in a synergistic fashion through a tandem E-box to regulate the expression and amplitude of the muscle specific clock-controlled gene, Titin-cap (
). In conclusion, these findings reveal mechanistic roles for the muscle specific transcription factor MYOD1 in the regulation of molecular clock amplitude as well as synergistic regulation of clock-controlled genes in skeletal muscle.
Facioscapulohumeral dystrophy (FSHD) is caused by decreased epigenetic repression of the D4Z4 macrosatellite array and recent studies have shown that this results in the expression of low levels of ...the DUX4 mRNA in skeletal muscle. Several other mechanisms have been suggested for FSHD pathophysiology and it remains unknown whether DUX4 expression can account for most of the molecular changes seen in FSHD. Since DUX4 is a transcription factor, we used RNA-seq to measure gene expression in muscle cells transduced with DUX4, and in muscle cells and biopsies from control and FSHD individuals. We show that DUX4 target gene expression is the major molecular signature in FSHD muscle together with a gene expression signature consistent with an immune cell infiltration. In addition, one unaffected individual without a known FSHD-causing mutation showed the expression of DUX4 target genes. This individual has a sibling with FSHD and also without a known FSHD-causing mutation, suggesting the presence of an unidentified modifier locus for DUX4 expression and FSHD. These findings demonstrate that the expression of DUX4 accounts for the majority of the gene expression changes in FSHD skeletal muscle together with an immune cell infiltration.
The diverse progenitors that give rise to the human neocortex have been difficult to characterize because progenitors, particularly radial glia (RG), are rare and are defined by a combination of ...intracellular markers, position and morphology. To circumvent these problems, we developed Fixed and Recovered Intact Single-cell RNA (FRISCR), a method for profiling the transcriptomes of individual fixed, stained and sorted cells. Using FRISCR, we profiled primary human RG that constitute only 1% of the midgestation cortex and classified them as ventricular zone-enriched RG (vRG) that express ANXA1 and CRYAB, and outer subventricular zone-localized RG (oRG) that express HOPX. Our study identified vRG and oRG markers and molecular profiles, an essential step for understanding human neocortical progenitor development. FRISCR allows targeted single-cell profiling of any tissues that lack live-cell markers.
Recent studies have demonstrated that MyoD initiates a feed-forward regulation of skeletal muscle gene expression, predicting that MyoD binds directly to many genes expressed during differentiation. ...We have used chromatin immunoprecipitation and high-throughput sequencing to identify genome-wide binding of MyoD in several skeletal muscle cell types. As anticipated, MyoD preferentially binds to a VCASCTG sequence that resembles the in vitro-selected site for a MyoD:E-protein heterodimer, and MyoD binding increases during differentiation at many of the regulatory regions of genes expressed in skeletal muscle. Unanticipated findings were that MyoD was constitutively bound to thousands of additional sites in both myoblasts and myotubes, and that the genome-wide binding of MyoD was associated with regional histone acetylation. Therefore, in addition to regulating muscle gene expression, MyoD binds genome wide and has the ability to broadly alter the epigenome in myoblasts and myotubes.
► ChIP-seq identifies tens of thousands of MyoD binding sites in myoblasts and myotubes ► Developmentally regulated binding is associated with genes regulated by MyoD ► Genome-wide constitutive MyoD binding is associated with histone acetylation ► Motif analysis indicates both positive and negative feed forward regulation
Interneurons expressing cholecystokinin (CCK) and parvalbumin (PV) constitute two key GABAergic controllers of hippocampal pyramidal cell output. Although the temporally precise and millisecond-scale ...inhibitory regulation of neuronal ensembles delivered by PV interneurons is well established, the in vivo recruitment patterns of CCK-expressing basket cell (BC) populations has remained unknown. We show in the CA1 of the mouse hippocampus that the activity of CCK BCs inversely scales with both PV and pyramidal cell activity at the behaviorally relevant timescales of seconds. Intervention experiments indicated that the inverse coupling of CCK and PV GABAergic systems arises through a mechanism involving powerful inhibitory control of CCK BCs by PV cells. The tightly coupled complementarity of two key microcircuit regulatory modules demonstrates a novel form of brain-state-specific segregation of inhibition during spontaneous behavior.
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•CA1 CCK basket cells are selectively labeled in the Sncg-Flp mouse line•CCK basket and PV cells display complementary activity during spontaneous behaviors•PV cells inhibit CCK basket cells in vivo•CCK basket cell activity inversely scales with the pyramidal cell ensemble activity
Dudok et al. show a complementary perisomatic inhibitory system consisting of CCK- and PV-expressing interneurons that are active at distinct times in an alternating fashion during spontaneous behaviors. Due to PV to CCK inhibition, CCK basket cells are the most active when PV and pyramidal cells are relatively silent.
Mammalian cortex is a laminar structure, with each layer composed of a characteristic set of cell types with different morphological, electrophysiological, and connectional properties. Here, we ...define chromatin accessibility landscapes of major, layer-specific excitatory classes of neurons, and compare them to each other and to inhibitory cortical neurons using the Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-seq). We identify a large number of layer-specific accessible sites, and significant association with genes that are expressed in specific cortical layers. Integration of these data with layer-specific transcriptomic profiles and transcription factor binding motifs enabled us to construct a regulatory network revealing potential key layer-specific regulators, including
,
,
,
, and
. This dataset is a valuable resource for identifying candidate layer-specific cis-regulatory elements in adult mouse cortex.
Facioscapulohumeral dystrophy (FSHD) is one of the most common inherited muscular dystrophies. The causative gene remains controversial and the mechanism of pathophysiology unknown. Here we identify ...genes associated with germline and early stem cell development as targets of the DUX4 transcription factor, a leading candidate gene for FSHD. The genes regulated by DUX4 are reliably detected in FSHD muscle but not in controls, providing direct support for the model that misexpression of DUX4 is a causal factor for FSHD. Additionally, we show that DUX4 binds and activates LTR elements from a class of MaLR endogenous primate retrotransposons and suppresses the innate immune response to viral infection, at least in part through the activation of DEFB103, a human defensin that can inhibit muscle differentiation. These findings suggest specific mechanisms of FSHD pathology and identify candidate biomarkers for disease diagnosis and progression.
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► DUX4 binds a double homeodomain motif in MaLR retrotransposons and nonrepetitive DNA ► DUX4 activates germline genes, retrotransposons, and immune mediators ► DUX4 target genes are expressed in FSHD skeletal muscle ► DUX4 targets are candidate biomarkers for FSHD and suggest disease mechanisms
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