The Cerebellum: Purification and Coculture of Identified Cell Populations

Hatten, Mary E.; Gao, Wei‐Qiang; Morrison, Mary E.; Mason, Carol A.

doi:10.7551/mitpress/4913.003.0023

Cited by 27 publications

(1 citation statement)

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“…We collected and enriched primary CGNs from postnatal day 6-8 (P6-P8) C57BL6 mice cerebella as previously described [109]. Cerebella were dissected from the rest of the brain and the meningeal layer was carefully removed.…”

Section: Co-immunoprecipitationmentioning

confidence: 99%

Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Hallada,

Shirinifard,

Solecki

2024

Preprint

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Sorting maturing neurons into distinct layers is critical for brain development, with disruptions leading to neurological disorders and pediatric cancers. Lamination coordinates where, when, and how cells interact, facilitating events that direct migrating neurons to their destined positions within emerging neural networks and control the wiring of connections in functional circuits. While the role of adhesion molecule expression and presentation in driving adhesive recognition during neuronal migration along glial fibers is recognized, the mechanisms by which the spatial arrangement of these molecules on the cell surface dictates adhesive specificity and translates contact-based external cues into intracellular responses like polarization and cytoskeletal organization remain largely unexplored. We used the cerebellar granule neuron (CGN) system to demonstrate that JAM-C receptor cis-binding on the same cell and trans-binding to neighboring cells controls the recruitment of the Pard3 polarity protein and drebrin microtubule-actin crosslinker at CGN to glial adhesion sites, complementing previous studies that showed Pard3 controls JAM-C exocytic surface presentation. Leveraging advanced imaging techniques, specific probes for cell recognition, and analytical methods to dissect adhesion dynamics, our findings reveal: 1) JAM-C cis or trans mutants result in reduced adhesion formation between CGNs and cerebellar glia, 2) these mutants exhibit delayed recruitment of Pard3 at the adhesion sites, and 3) CGNs with JAM-C mutations experience postponed sorting and entry into the cerebellar molecular layer (ML). By developing a conditional system to image adhesion components from two different cells simultaneously, we made it possible to investigate the dynamics of cell recognition on both sides of neuron-glial contacts and the subsequent recruitment of proteins required for CGN migration. This system and an approach that calculates local correlation based on convolution kernels at the cell adhesions site revealed that CGN to CGN JAM recognition preferentially recruits higher levels of Pard3 and drebrin than CGN to glia JAM recognition. The long latency time of CGNs in the inner external germinal layer (EGL) can be attributed to the combined strength of CGN-CGN contacts and the less efficient Pard3 recruitment by CGN-BG contacts, acting as gatekeepers to ML entry. As CGNs eventually transition to glia binding for radial migration, our research demonstrates that establishing permissive JAM-recognition sites on glia via cis and trans interactions of CGN JAM-C serves as a critical temporal checkpoint for sorting at the EGL to ML boundary. This mechanism integrates intrinsic and extrinsic cellular signals, facilitating heterotypic cell sorting into the ML and dictating the precise spatial organization within the cerebellar architecture.

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Section: Co-immunoprecipitationmentioning

confidence: 99%

Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Hallada,

Shirinifard,

Solecki

2024

Preprint

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Reciprocal actions of ATF5 and Shh in proliferation of cerebellar granule neuron progenitor cells

Lee

Angelastro

Kenney

et al. 2012

Developmental Neurobiology

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Precise regulation of neuroprogenitor cell proliferation and differentiation is required for successful brain development, but the factors that contribute to this are only incompletely understood. The transcription factor ATF5 promotes proliferation of cerebral cortical neuroprogenitor cells and its down-regulation permits their differentiation. Here, we examine the expression and regulation of ATF5 in cerebellar granule neuron progenitor cells (CGNPs) as well as the role of ATF5 in the transition of CGNPs to post-mitotic cerebellar granule neurons (GCNs). We find that ATF5 is expressed by proliferating CGNPs in both the embryonic and post-natal cerebellar external granule layer (EGL) and in the rhombic lip, the embryonic structure from which the EGL arises. In contrast, ATF5 is undetectable in post-mitotic GCNs. In highly enriched dissociated cultures of CGNPs and CGNs, ATF5 is expressed only in CGNPs. Constitutive ATF5 expression in CGNPs does not affect their proliferation or exit from the cell cycle. In contrast, in presence of sonic hedgehog (Shh), a mitogen for CGNPs, constitutively expressed ATF5 promotes CGNP proliferation and delays their cell cycle exit and differentiation. Conversely, ATF5 loss-of-function conferred by a dominant-negative form of ATF5, significantly diminishes Shh-stimulated CGNP proliferation and promotes differentiation. In parallel with its stimulation of CGNP proliferation, Shh enhances ATF5 expression by what appeared to be a post-transcriptional mechanism involving protein stabilization. These findings indicate a reciprocal interaction between ATF5 and Shh in which Shh stimulates ATF5 expression and in which ATF5 contributes to Shh-stimulated CGNP expansion.

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Rapid method for culturing embryonic neuron–glial cell cocultures

Svenningsen

Shan

Colman

et al. 2003

J of Neuroscience Research

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A streamlined, simple technique for primary cell culture from E17 rat tissue is presented. In an attempt to standardize culturing methods for all neuronal cell types in the embryo, we evaluated a commercial medium without serum and used similar times for trypsinization and tested different surfaces for plating. In 1 day, using one method and a single medium, it is possible to produce robust E17 cultures of dorsal root ganglia (DRG), cerebellum, and enteric plexi. Allowing the endogenous glial cells to repopulate the cultures saves time compared with existing techniques, in which glial cells are added to cultures first treated with antimitotic agents. It also ensures that all the cells present in vivo will be present in the culture. Myelination commences after approximately 2 weeks in culture for dissociated DRG and 3-4 weeks in cerebellar cultures. In enteric cultures, glial wrapping of the enteric neurons is seen after 3 weeks (2 weeks in ascorbic acid), suggesting that basal lamina production is important even for glial ensheathment in the enteric nervous system. No overgrowth of fibroblasts or other nonneuronal cells was noted in any cultures, and myelination of the peripheral nervous system and central nervous system cultures was very robust.

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The Cerebellum: Purification and Coculture of Identified Cell Populations

Cited by 27 publications

References 0 publications

Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Junctional Adhesion Molecule (JAM)-C recruitment of Pard3 and drebrin to cell contacts initiates neuron-glia recognition and layer-specific cell sorting in developing cerebella

Reciprocal actions of ATF5 and Shh in proliferation of cerebellar granule neuron progenitor cells

Rapid method for culturing embryonic neuron–glial cell cocultures

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