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Notch Signaling between Cerebellar Granule Cell Progenitors.

ABSTRACT: Cerebellar granule cells (GCs) are cells which comprise over 50% of the neurons in the entire nervous system. GCs enable the cerebellum to properly regulate motor coordination, learning, and consolidation, in addition to cognition, emotion and language. During GC development, maternal GC progenitors (GCPs) divide to produce not only postmitotic GCs but also sister GCPs. However, the molecular machinery for regulating the proportional production of distinct sister cell types from seemingly uniform GCPs is not yet fully understood. Here we report that Notch signaling creates a distinction between GCPs and leads to their proportional differentiation in mice. Among Notch-related molecules, Notch1, Notch2, Jag1, and Hes1 are prominently expressed in GCPs. In vivo monitoring of Hes1-promoter activities showed the presence of two types of GCPs, Notch-signaling ON and OFF, in the external granule layer (EGL). Single-cell RNA sequencing (scRNA-seq) and in silico analyses indicate that ON-GCPs have more proliferative and immature properties, while OFF-GCPs have opposite characteristics. Overexpression as well as knock-down (KD) experiments using in vivo electroporation showed that NOTCH2 and HES1 are involved cell-autonomously to suppress GCP differentiation by inhibiting NEUROD1 expression. In contrast, JAG1-expressing cells non-autonomously upregulated Notch signaling activities via NOTCH2-HES1 in surrounding GCPs, eventually suppressing their differentiation. These findings suggest that Notch signaling results in the proportional generation of two types of cells, immature and differentiating GCPs, which contributes to the well-organized differentiation of GCs.

SUBMITTER: Adachi T

PROVIDER: S-EPMC8121261 | biostudies-literature |

REPOSITORIES: biostudies-literature

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Project description:During cerebellar development, granule cell progenitors (GCPs) proliferate exponentially for a fixed period, promoted by paracrine mitogenic factor Sonic Hedgehog (Shh) secreted from Purkinje cells (PCs). Dysregulation of Shh signaling leads to uncontrolled GCP proliferation and medulloblastoma. Serendipitously our previous work discovered insulin-like growth factor 1 (IGF1) as another key driver for medulloblastoma, which led to the current investigation into the role of IGF1 in GCPs during normal development. While the IGF1R conditional knockout model revealed GCP defects in anterior cerebellum, the posterior cerebellum was mostly intact, likely owing to incomplete excision of floxed alleles. To circumvent this hurdle, we enlisted a mouse genetic system called Mosaic Analysis of Double Markers (MADM), which sporadically generates homozygous null cells unequivocally labeled with GFP and their wildtype sibling cells labeled with RFP, enabling phenotypic analysis at single-cell resolution. Using MADM, we found that loss of IGF1R resulted in a 10-fold reduction of GCs in both anterior and posterior cerebellum; and that hindered S phase entry and increased cell cycle exit collectively led to this phenotype. Genetic interaction studies showed that IGF1 signaling prevents GCP cell cycle exit at least partially through suppressing the level of p27kip1, a negative regulator of cell cycle. Finally, we found that IGF1 is produced by PCs in a temporally regulated fashion: it is highly expressed early in development when GCPs proliferate exponentially, then gradually decline as GCPs commit to cell cycle exit. Taken together, our studies reveal IGF1 as a paracrine factor that positively regulates GCP cell cycle in cooperation with Shh, through dampening the level of p27 to prevent precocious cell cycle exit. Our work not only showcases the power of phenotypic analysis by the MADM system but also provides an excellent example of multi-factorial regulation of robust developmental programs.

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Notch Signaling between Cerebellar Granule Cell Progenitors.

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