scholarly journals Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Hourinaz Behesti ◽  
Arif Kocabas ◽  
David E Buchholz ◽  
Thomas S Carroll ◽  
Mary E Hatten
Keyword(s):  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Affinity Purification ◽  
Brain Regions ◽  
Temporal Shift ◽  
Cerebellar Neurons ◽  
Cerebellar Granule ◽  
Human Cerebellum ◽  
Species Specific ◽  
Cerebellar Histogenesis

Brain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, human cerebellar histogenesis differs in complexity compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. We report a rapid protocol for the derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC). Upon transplantation into juvenile mice, hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification-seq, we identified an unexpected temporal shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, in the human outer external granule layer. This molecular divergence may enable the protracted development of the human cerebellum compared to mice.

scholarly journals Altered temporal sequence of transcriptional regulators in the generation of human cerebellar granule cells

2021 ◽  
Author(s):  
Hourinaz Behesti ◽  
Arif Kocabas ◽  
David E. Buchholz ◽  
Thomas S. Carroll ◽  
Mary E. Hatten
Keyword(s):  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Affinity Purification ◽  
Brain Regions ◽  
Molecular Characteristics ◽  
Cerebellar Neurons ◽  
Cerebellar Granule ◽  
Cerebellar Tissue ◽  
Species Specific

SummaryBrain development is regulated by conserved transcriptional programs across species, but little is known about divergent mechanisms that create species-specific characteristics. Among brain regions, the cerebellum is now recognized to contribute to human cognitive evolution having a broad range of non-motor cognitive functions in addition to motor control. Emerging studies highlight the complexity of human cerebellar histogenesis, compared with non-human primates and rodents, making it important to develop methods to generate human cerebellar neurons that closely resemble those in the developing human cerebellum. Here we report a rapid and simple protocol for the directed derivation of the human ATOH1 lineage, the precursor of excitatory cerebellar neurons, from human pluripotent stem cells (hPSC), and strategies to decrease culture variability; a common limitation in hPSC studies. Upon transplantation into juvenile mice, early postmitotic hPSC-derived cerebellar granule cells migrated along glial fibers and integrated into the cerebellar cortex. By Translational Ribosome Affinity Purification (TRAP)-seq, the ATOH1 lineage most closely resembled human cerebellar tissue in the second trimester. Unexpectedly, TRAP-seq identified a heterochronic shift in the expression of RBFOX3 (NeuN) and NEUROD1, which are classically associated with differentiated neurons, within granule cell progenitors (GCPs) in the human external granule layer. This molecular divergence may provide the mechanism by which the GCP pool persists into year two post birth in humans, but only lasts for two weeks in mice. Our approach provides a scalable in vitro model of the human ATOH1 lineage that yields cerebellar granule cells within 48 days as well as a strategy for identifying uniquely human cellular and molecular characteristics.

The 270 kDa splice variant of erythrocyte beta-spectrin (beta I sigma 2) segregates in vivo and in vitro to specific domains of cerebellar neurons

1993 ◽  
Vol 106 (1) ◽  
pp. 67-78 ◽  
Author(s):  
F. Malchiodi-Albedi ◽  
M. Ceccarini ◽  
J.C. Winkelmann ◽  
J.S. Morrow ◽  
T.C. Petrucci
Keyword(s):  
Purkinje Cells ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Membrane Surface ◽  
Granular Cells ◽  
Cerebellar Neurons ◽  
Cerebellar Granule ◽  
Sigma 1 ◽  
Alpha Spectrin ◽  
Alpha Beta

Spectrin isoforms arise from four distinct genes, three of which generate multiple alternative transcripts. With no biochemical restrictions on the assembly of alpha beta heterodimers, more than 25 distinct heterodimeric spectrin species may exist. Whether (and why) this subtle but substantial diversity is realized in any single cell is unknown. To address this question, sequence-specific antibodies to alternatively spliced regions of alpha- and beta-spectrin have been prepared. Reported here is the localization in rat cerebellar neurons at light and electron microscopic levels of an antibody against a unique sequence (beta I sigma 2-A = PGQHKDGQKSTGDERPT) from the 270 kDa transcript of the red cell beta-spectrin gene (spectrin beta I sigma 2). In this version, the 3′ sequence of erythroid beta-spectrin (beta I sigma 1) is replaced with an alternative sequence that shares substantial homology with the 3′ sequence of non-erythroid beta-spectrin (beta II sigma 1). The antibody to beta I sigma 2-A stains a single protein band at 270 kDa, determined by western blotting, in both rat cerebellum and in cultured cerebellar granule cells, and does not react with beta II sigma 1 spectrin (beta-fodrin). This antibody stains the dendritic spines of Purkinje cells in the molecular layer, and is concentrated at postsynaptic densities (PSDs) adjacent to synapsin I (which is confined to the presynaptic membrane). The soma of Purkinje cells do not stain. In the granular layer, cytoplasmic organelles and the postsynaptic densities of granular cells stain strongly. Astrocytes are also stained. In all cells, plasma membrane staining is confined to postsynaptic densities (PSD). The beta I sigma 2 isoform co-immunoprecipitates with non-erythroid alpha-spectrin (alpha II sigma), even though the distribution of alpha II sigma within neurons only partially overlaps that of beta I sigma 2. No hybrid beta I sigma 2 and beta II sigma 1 (beta-fodrin) spectrin complexes appear to exist. Spectrin beta I sigma 2 is also polarized in cultured rat cerebellar granule cells, where it is abundant in cell bodies but not neurites. The overall distribution of beta I sigma 2 is as a subset of the distribution of spectrins 240/235E previously detected with a generally reactive erythrocyte alpha beta-spectrin antibody. These findings establish the highly precise segregation of a beta-spectrin isoform to distinct cytoplasmic and membrane surface domains, indicate that it is complexed (partially) with non-erythroid alpha-spectrin, and demonstrate that cytoskeletal targeting mechanisms are preserved in cultured granular cells.(ABSTRACT TRUNCATED AT 400 WORDS)

scholarly journals Single-cell spatial transcriptomic analysis reveals common and divergent features of developing postnatal granule cerebellar cells and medulloblastoma

BMC Biology ◽  
2021 ◽  
Vol 19 (1) ◽  
Author(s):  
Wenqin Luo ◽  
Guan Ning Lin ◽  
Weichen Song ◽  
Yu Zhang ◽  
Huadong Lai ◽  
...  
Keyword(s):  
Single Cell ◽  
Rna Sequencing ◽  
Granule Cells ◽  
Cerebellar Granule Cells ◽  
Granule Neurons ◽  
Cerebellar Granule ◽  
Granule Neuron ◽  
Proliferation And Differentiation ◽  
Single Cell Rna Sequencing ◽  
Cerebellar Cells

Abstract Background Cerebellar neurogenesis involves the generation of large numbers of cerebellar granule neurons (GNs) throughout development of the cerebellum, a process that involves tight regulation of proliferation and differentiation of granule neuron progenitors (GNPs). A number of transcriptional regulators, including Math1, and the signaling molecules Wnt and Shh have been shown to have important roles in GNP proliferation and differentiation, and deregulation of granule cell development has been reported to be associated with the pathogenesis of medulloblastoma. While the progenitor/differentiation states of cerebellar granule cells have been broadly investigated, a more detailed association between developmental differentiation programs and spatial gene expression patterns, and how these lead to differential generation of distinct types of medulloblastoma remains poorly understood. Here, we provide a comparative single-cell spatial transcriptomics analysis to better understand the similarities and differences between developing granule and medulloblastoma cells. Results To acquire an enhanced understanding of the precise cellular states of developing cerebellar granule cells, we performed single-cell RNA sequencing of 24,919 murine cerebellar cells from granule neuron-specific reporter mice (Math1-GFP; Dcx-DsRed mice). Our single-cell analysis revealed that there are four major states of developing cerebellar granule cells, including two subsets of granule progenitors and two subsets of differentiating/differentiated granule neurons. Further spatial transcriptomics technology enabled visualization of their spatial locations in cerebellum. In addition, we performed single-cell RNA sequencing of 18,372 cells from Patched+/− mutant mice and found that the transformed granule cells in medulloblastoma closely resembled developing granule neurons of varying differentiation states. However, transformed granule neuron progenitors in medulloblastoma exhibit noticeably less tendency to differentiate compared with cells in normal development. Conclusion In sum, our study revealed the cellular and spatial organization of the detailed states of cerebellar granule cells and provided direct evidence for the similarities and discrepancies between normal cerebellar development and tumorigenesis.

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