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A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells.

ABSTRACT: Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca(2+) and activates Ca(2+)/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca(2+) signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cells in vivo. We investigated the genome-wide expression profiles of depolarization- and CaN-regulated genes in cultured mouse granule cells and addressed their relevance to gene regulation in developing granule cells in vivo. Granule cells were cultured under a nondepolarization condition (5 mM KCl) and a depolarization condition (25 mM KCl) with and without the CaN inhibitor FK506. Gene expression profiles between depolarization and nondepolarization and between FK506 treatment and untreatment were analyzed by microarray techniques. Both depolarization and FK506 treatment influence expression levels of a large number of genes, most of which are overlapping, however, are conversely regulated by these two treatments. Importantly, many of the FK506-responsive genes are up- or down-regulated in parallel with gene expression in postnatal granule cells in vivo. The FK506-down-regulated genes are highly expressed in proliferating/premigratory granule cells and many of these genes encode cellular components involved in cell proliferation, migration, and differentiation. In contrast, the FK506-up-regulated genes are predominantly expressed in postmigratory granule cells, including many functional molecules implicated in synaptic transmission and modulation. This investigation demonstrates that the CaN signaling plays a pivotal role in development and synaptic organization of granule cells during the postnatal period.

SUBMITTER: Sato M

PROVIDER: S-EPMC556295 | biostudies-literature | 2005 Apr

REPOSITORIES: biostudies-literature

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A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells.

Sato Masaaki M Suzuki Kazunori K Yamazaki Hiroshi H Nakanishi Shigetada S

Proceedings of the National Academy of Sciences of the United States of America 20050404 16

Primary culture of postnatal cerebellar granule cells provides a model system that recapitulates many molecular events of developing granule cells in vivo. Depolarization of cultured granule cells increases intracellular Ca(2+) and activates Ca(2+)/calmodulin-dependent calcineurin (CaN) phosphatase. This Ca(2+) signaling mimics some of the signaling events for proliferation, migration, and differentiation of granule cells in vivo. We investigated the genome-wide expression profiles of depolariza ...[more]

PMID: 15809415

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Project description:BackgroundCerebellar 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.ResultsTo 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.ConclusionIn 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.

Project description:Cerebellar granule cell precursors (GCPs) and granule cells (GCs) represent good models to study neuronal development. Here, we report that the transcription factor myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse GC development. We found that Meis1 is expressed in GC lineage cells and astrocytes in the cerebellum during development. Targeted disruption of the Meis1 gene specifically in the GC lineage resulted in smaller cerebella with disorganized lobules. Knock-down/knock-out (KO) experiments for Meis1 and in vitro assays showed that Meis1 binds to an upstream sequence of Pax6 to enhance its transcription in GCPs/GCs and also suggested that the Meis1-Pax6 cascade regulates morphology of GCPs/GCs during development. In the conditional KO (cKO) cerebella, many Atoh1-positive GCPs were observed ectopically in the inner external granule layer (EGL) and a similar phenomenon was observed in cultured cerebellar slices treated with a bone morphogenic protein (BMP) inhibitor. Furthermore, expression of Smad proteins and Smad phosphorylation were severely reduced in the cKO cerebella and Meis1-knock-down GCPs cerebella. Reduction of phosphorylated Smad was also observed in cerebellar slices electroporated with a Pax6 knock-down vector. Because it is known that BMP signaling induces Atoh1 degradation in GCPs, these findings suggest that the Meis1-Pax6 pathway increases the expression of Smad proteins to upregulate BMP signaling, leading to degradation of Atoh1 in the inner EGL, which contributes to differentiation from GCPs to GCs. Therefore, this work reveals crucial functions of Meis1 in GC development and gives insights into the general understanding of the molecular machinery underlying neural differentiation from neural progenitors.SIGNIFICANCE STATEMENT We report that myeloid ectopic viral integration site 1 homolog (Meis1) plays pivotal roles in the regulation of mouse granule cell (GC) development. Here, we show Meis1 is expressed in GC precursors (GCPs) and GCs during development. Our knock-down and conditional knock-out (cKO) experiments and in vitro assays revealed that Meis1 is required for proper cerebellar structure formation and for Pax6 transcription in GCPs and GCs. The Meis1-Pax6 cascade regulates the morphology of GCs. In the cKO cerebella, Smad proteins and bone morphogenic protein (BMP) signaling are severely reduced and Atoh1-expressing GCPs are ectopically detected in the inner external granule layer. These findings suggest that Meis1 regulates degradation of Atoh1 via BMP signaling, contributing to GC differentiation in the inner EGL, and should provide understanding into GC development.

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A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells.

Publications

A pivotal role of calcineurin signaling in development and maturation of postnatal cerebellar granule cells.

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