scholarly journals Dissociation of intact adult mouse cortical projection neurons for single-cell RNA-seq

2021 ◽  
Vol 2 (4) ◽  
pp. 100941
Author(s):  
Noa Golan ◽  
William B. Cafferty
Keyword(s):  
Single Cell ◽  
Adult Mouse ◽  
Projection Neurons ◽  
Rna Seq ◽  
Cortical Projection

Differential RA responsiveness among subsets of mouse late progenitor spermatogonia

Reproduction ◽  
2021 ◽  
Author(s):  
Shinnosuke Suzuki ◽  
John R. McCarrey ◽  
Brian P Hermann
Keyword(s):  
Stem Cells ◽  
Retinoic Acid ◽  
Single Cell ◽  
Adult Mouse ◽  
Retinoic Acid Receptor ◽  
Mouse Testis ◽  
Rna Seq ◽  
Reporter Expression ◽  
Acid Receptor ◽  
Whole Mount

Initiation of spermatogonial differentiation in the mouse testis begins with the response to retinoic acid (RA) characterized by activation of KIT and STRA8 expression. In the adult, spermatogonial differentiation is spatiotemporally coordinated by a pulse of RA every 8.6 days that is localized to stages VII-VIII of the seminiferous epithelial cycle. Dogmatically, progenitor spermatogonia that express retinoic acid receptor gamma (RARG) at these stages will differentiate in response to RA, but this has yet to be tested functionally. Previous single-cell RNA-seq data identified phenotypically and functionally distinct subsets of spermatogonial stem cells (SSCs) and progenitor spermatogonia, where late progenitor spermatogonia were defined by expression of RARG and Dppa3. Here, we found late progenitor spermatogonia (RARGhigh KIT-) were further divisible into two subpopulations based on Dppa3 reporter expression (Dppa3-ECFP or Dppa3-EGFP) and were observed across all stages of the seminiferous epithelial cycle. However, nearly all Dppa3+ spermatogonia were differentiating (KIT+) late in the seminiferous epithelial cycle (stages X-XII), while Dppa3- late progenitors remained abundant, suggesting that Dppa3+ and Dppa3- late progenitors differentially responded to RA. Following acute RA treatment (2-4hr), significantly more Dppa3+ late progenitors induced KIT, including at the midpoint of the cycle (stages VI-IX), than Dppa3- late progenitors. Subsequently, single-cell analyses indicated a subset of Dppa3+ late progenitors expressed higher levels of Rxra, which we confirmed by RXRA whole-mount immunostaining. Together, these results indicate RARG alone is insufficient to initiate a spermatogonial response to RA in the adult mouse testis and suggest differential RXRA expression may discriminate responding cells.

scholarly journals Expression of the Na+/K+-ATPase Subunits in Adult Mouse Brain Analyzed by Single-Cell RNA-Seq Profiling

2019 ◽  
Vol 116 (3) ◽  
pp. 129a
Author(s):  
Song Jiao ◽  
Cristina Moreno Vadillo ◽  
Miguel Holmgren
Keyword(s):  
Single Cell ◽  
Mouse Brain ◽  
Adult Mouse ◽  
Rna Seq ◽  
Adult Mouse Brain ◽  
Atpase Subunits

scholarly journals Single-cell dissection of schizophrenia reveals neurodevelopmental-synaptic axis and transcriptional resilience

2020 ◽  
Author(s):  
W. Brad Ruzicka ◽  
Shahin Mohammadi ◽  
Jose Davila-Velderrain ◽  
Sivan Subburaju ◽  
Daniel Reed Tso ◽  
...  
Keyword(s):  
Single Cell ◽  
Cell Types ◽  
Synaptic Function ◽  
Inhibitory Neurons ◽  
Projection Neurons ◽  
Cell Type ◽  
Cortical Projection ◽  
Human Neurons ◽  
Small Effect Size ◽  
Therapeutic Development

AbstractSchizophrenia is a devastating mental disorder with a high societal burden, complex pathophysiology, and diverse genetic and environmental risk factors. Its complexity, polygenicity, and small-effect-size and cell-type-specific contributors have hindered mechanistic elucidation and the search for new therapeutics. Here, we present the first single-cell dissection of schizophrenia, across 500,000+ cells from 48 postmortem human prefrontal cortex samples, including 24 schizophrenia cases and 24 controls. We annotate 20 cell types/states, providing a high-resolution atlas of schizophrenia-altered genes and pathways in each. We find neurons are the most affected cell type, with deep-layer cortico-cortical projection neurons and parvalbumin-expressing inhibitory neurons showing significant transcriptional changes converging on genetically-implicated regions. We discover a novel excitatory-neuron cell-state indicative of transcriptional resilience and enriched in schizophrenia subjects with less-perturbed transcriptional signatures. We identify key trans-acting factors as candidate drivers of observed transcriptional perturbations, including MEF2C, TCF4, SOX5, and SATB2, and map their binding patterns in postmortem human neurons. These factors regulate distinct gene sets underlying fetal neurodevelopment and adult synaptic function, bridging two leading models of schizophrenia pathogenesis. Our results provide the most detailed map to date for mechanistic understanding and therapeutic development in neuropsychiatric disorders.

scholarly journals Transcription factor expression defines subclasses of developing projection neurons highly similar to single-cell RNA-seq subtypes

2020 ◽  
Vol 117 (40) ◽  
pp. 25074-25084 ◽  
Author(s):  
Whitney E. Heavner ◽  
Shaoyi Ji ◽  
James H. Notwell ◽  
Ethan S. Dyer ◽  
Alex M. Tseng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Transcription Factor ◽  
Single Cell ◽  
Progenitor Cells ◽  
Cell Fate ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Projection Neurons ◽  
Cortical Projection ◽  
Functional Features

We are only just beginning to catalog the vast diversity of cell types in the cerebral cortex. Such categorization is a first step toward understanding how diversification relates to function. All cortical projection neurons arise from a uniform pool of progenitor cells that lines the ventricles of the forebrain. It is still unclear how these progenitor cells generate the more than 50 unique types of mature cortical projection neurons defined by their distinct gene-expression profiles. Moreover, exactly how and when neurons diversify their function during development is unknown. Here we relate gene expression and chromatin accessibility of two subclasses of projection neurons with divergent morphological and functional features as they develop in the mouse brain between embryonic day 13 and postnatal day 5 in order to identify transcriptional networks that diversify neuron cell fate. We compare these gene-expression profiles with published profiles of single cells isolated from similar populations and establish that layer-defined cell classes encompass cell subtypes and developmental trajectories identified using single-cell sequencing. Given the depth of our sequencing, we identify groups of transcription factors with particularly dense subclass-specific regulation and subclass-enriched transcription factor binding motifs. We also describe transcription factor-adjacent long noncoding RNAs that define each subclass and validate the function of Myt1l in balancing the ratio of the two subclasses in vitro. Our multidimensional approach supports an evolving model of progressive restriction of cell fate competence through inherited transcriptional identities.

scholarly journals Epigenomic diversity of cortical projection neurons in the mouse brain

Nature ◽  
2021 ◽  
Vol 598 (7879) ◽  
pp. 167-173 ◽  
Author(s):  
Zhuzhu Zhang ◽  
Jingtian Zhou ◽  
Pengcheng Tan ◽  
Yan Pang ◽  
Angeline C. Rivkin ◽  
...  
Keyword(s):  
Single Cell ◽  
Cortical Neurons ◽  
Neuronal Cell ◽  
Cell Types ◽  
Molecular Properties ◽  
Projection Neurons ◽  
Long Distance ◽  
Cortical Projection ◽  
Retrograde Labelling ◽  
Projection Properties

AbstractNeuronal cell types are classically defined by their molecular properties, anatomy and functions. Although recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain1, neuronal cell types are often studied out of the context of their anatomical properties. To improve our understanding of the relationship between molecular and anatomical features that define cortical neurons, here we combined retrograde labelling with single-nucleus DNA methylation sequencing to link neural epigenomic properties to projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical and cortico-subcortical long-distance projections. Our results showed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. On the basis of their epigenomes, intra-telencephalic cells that project to different cortical targets could be further distinguished, and some layer 5 neurons that project to extra-telencephalic targets (L5 ET) formed separate clusters that aligned with their axonal projections. Such separation varied between cortical areas, which suggests that there are area-specific differences in L5 ET subtypes, which were further validated by anatomical studies. Notably, a population of cortico-cortical projection neurons clustered with L5 ET rather than intra-telencephalic neurons, which suggests that a population of L5 ET cortical neurons projects to both targets. We verified the existence of these neurons by dual retrograde labelling and anterograde tracing of cortico-cortical projection neurons, which revealed axon terminals in extra-telencephalic targets including the thalamus, superior colliculus and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

scholarly journals Epigenomic Diversity of Cortical Projection Neurons in the Mouse Brain

2020 ◽  
Author(s):  
Zhuzhu Zhang ◽  
Jingtian Zhou ◽  
Pengcheng Tan ◽  
Yan Pang ◽  
Angeline Rivkin ◽  
...  
Keyword(s):  
Single Cell ◽  
Cortical Neurons ◽  
Neuronal Cell ◽  
Cell Types ◽  
Molecular Properties ◽  
Projection Neurons ◽  
Long Distance ◽  
Cortical Projection ◽  
Axon Terminals ◽  
Projection Properties

SummaryNeuronal cell types are classically defined by their molecular properties, anatomy, and functions. While recent advances in single-cell genomics have led to high-resolution molecular characterization of cell type diversity in the brain, neuronal cell types are often studied out of the context of their anatomical properties. To better understand the relationship between molecular and anatomical features defining cortical neurons, we combined retrograde labeling with single-nucleus DNA methylation sequencing to link epigenomic properties of cell types to neuronal projections. We examined 11,827 single neocortical neurons from 63 cortico-cortical (CC) and cortico-subcortical long-distance projections. Our results revealed unique epigenetic signatures of projection neurons that correspond to their laminar and regional location and projection patterns. Based on their epigenomes, intra-telencephalic (IT) cells projecting to different cortical targets could be further distinguished, and some layer 5 neurons projecting to extra-telencephalic targets (L5-ET) formed separate subclusters that aligned with their axonal projections. Such separation varied between cortical areas, suggesting area-specific differences in L5-ET subtypes, which were further validated by anatomical studies. Interestingly, a population of CC projection neurons clustered with L5-ET rather than IT neurons, suggesting a population of L5-ET cortical neurons projecting to both targets (L5-ET+CC). We verified the existence of these neurons by labeling the axon terminals of CC projection neurons and observed clear labeling in ET targets including thalamus, superior colliculus, and pons. These findings highlight the power of single-cell epigenomic approaches to connect the molecular properties of neurons with their anatomical and projection properties.

scholarly journals Characterizing Adult cochlear supporting cell transcriptional diversity using single-cell RNA-Seq: Validation in the adult mouse and translational implications for the adult human cochlea

2019 ◽  
Author(s):  
Michael Hoa ◽  
Rafal Olszewski ◽  
Xiaoyi Li ◽  
Ian Taukulis ◽  
Alvin DeTorres ◽  
...  
Keyword(s):  
Single Cell ◽  
Hair Cells ◽  
Adult Mouse ◽  
Significant Proportion ◽  
Supporting Cell ◽  
Rna Seq ◽  
Supporting Cells ◽  
Human Cochlea ◽  
Cell Transcriptome ◽  
Cochlear Supporting Cells

AbstractHearing loss is a problem that impacts a significant proportion of the adult population. Cochlear hair cell loss due to loud noise, chemotherapy and aging is the major underlying cause. A significant proportion of these individuals are dissatisfied with available treatment options which include hearing aids and cochlear implants. An alternative approach to restore hearing would be to regenerate hair cells. Such therapy would require recapitulation of the complex architecture of the organ of Corti, necessitating regeneration of both mature hair cells and supporting cells. Transcriptional profiles of the mature cell types in the cochlea are necessary to can provide a metric for eventual regeneration therapies. To assist in this effort, we sought to provide the first single-cell characterization of the adult cochlear supporting cell transcriptome. We performed single-cell RNA-Seq on FACS-purified adult cochlear supporting cells from the LfngEGFP adult mouse, in which supporting cells express GFP. We demonstrate that adult cochlear supporting cells are transcriptionally distinct from their perinatal counterparts. We establish cell type-specific adult cochlear supporting cell transcriptome profiles, and we validate these expression profiles through a combination of both fluorescent immunohistochemistry and in situ hybridization co-localization and qPCR of adult cochlear supporting cells. Furthermore, we demonstrate the relevance of these profiles to the adult human cochlea through immunofluorescent human temporal bone histopathology. Finally, we demonstrate cell cycle regulator expression in adult supporting cells and perform pathway analyses to identify potential mechanisms for facilitating mitotic regeneration (cell proliferation, differentiation, and eventually regeneration) in the adult mammalian cochlea. Our findings demonstrate the importance of characterizing mature as opposed to perinatal supporting cells.

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