Notch Signaling between Cerebellar Granule Cell Progenitors

Abstract

Cerebellar granule cells (GCs) are cells which comprise over 50 percent 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 granule cell progenitors (GCPs) divide to 5 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, 2, 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 and in silico analyses indicate that ON-GCPs have more proliferative and immature properties, while OFF-GCPs have opposite characteristics. Overexpression as well as knockdown 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. Significance StatementThis study is the first to succeed in visualization of Notch signaling in vivo during cerebellar development. Granule cell progenitors (GCPs) in the outermost layer of the developing cerebellum are a seemingly homogenous cell population, but this study revealed two types of GCPs; more proliferative Notch-ON-GCPs and more differentiative Notch-OFF-GCPs, the latter of which gradually give rise to postmitotic granule cells. Our experiments suggest 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, suppressing their differentiation.This study gives new insights into the mechanisms controlling the differences within homogenous cell populations that direct proper and coordinated cell differentiation.

A gene dosage‐dependent effect unveils NBS1 as both a haploinsufficient tumour suppressor and an essential gene for SHH‐medulloblastoma

“…A role for the Notch pathway in SHH‐MB had been previously established by Hallahan et al, reporting increased expression of Notch targets in primary MBs and impaired growth/survival of SHH‐MB cells upon inhibition of Notch signalling [48]. Interestingly, Notch signalling appears to be mostly active in GCPs with more proliferative and immature properties, suggesting its expression is involved in regulating the proportion between GCPs and postmitotic GCs [49]. Our data, indicating the role of Nbn HZ on the enhanced and Notch‐dependent clonogenicity of GCPs, are also in line with the upregulation of the Notch pathway in lymphoblastoid cell lines from NBS heterozygous carriers as reported by Cheung et al .…”

Developmental Basis of SHH Medulloblastoma Heterogeneity

“…Loss of 9q or 10q can activate the SHH pathway, but these CNVs may also promote tumorigenesis by inhibiting differentiation. PTEN and NEURL1 are both located on chromosome 10q and negatively regulate GCP cycling [65][66][67] . Additionally, chromosome 9q contains NTRK2, which plays a vital role in granule cell migration 68,69 and maturation 38 .…”

Transit Amplifying Progenitors in the Cerebellum: Similarities to and Differences from Transit Amplifying Cells in Other Brain Regions and between Species

“…The diversity of GCPs, which is marked by the activity of Notch signaling, has also been reported recently [ 62 ]. Adachi et al [ 62 ] proposed a model in which GCPs with high Notch-signaling activity continue to proliferate (ON-GCPs) by the HES1-mediated suppression of NEUROD1 protein expression; this suppresses the differentiation of GCPs into GCs [ 63 ]. In contrast, GCPs with low Notch-signaling activity (OFF-GCPs) start to differentiate.…”

“… Novel developmental machinery of mammalian granule cells in the EGL. Schematic illustrations of the previous model ( A ) and our model ( B , referencing our recent studies [ 9 , 62 ]) of GC development in the EGL. Notch-signaling activity and the regulation of the NEUROD1 protein in the AT + GCPs ( C ).…”

“…SOX2 expression is a common feature of stem cells in every region (the cerebral cortex [ 85 ], the SGZ [ 86 ], the V-SVZ [ 35 , 86 ], and the cerebellum [ 61 , 87 ]), and DCX is a common feature of neurons (the cerebral cortex [ 88 ], the SGZ, the V-SVZ, and the cerebellum [ 88 ]). In addition, Hes family members are expressed in more undifferentiated progenitors (the cerebral cortex [ 89 , 90 ], the SGZ [ 91 , 92 ], the V-SVZ [ 93 ], and the cerebellum [ 62 , 94 ]). The cerebellum has interesting gene expression features compared to the other regions: (1) PAX6 expression is observed in the most immature progenitor and sustained after differentiation (the cerebral cortex [ 95 ], the SGZ [ 96 , 97 ], the V-SVZ [ 98 , 99 ], and the cerebellum [ 56 , 100 ]), whereas (2) expression of NGN2 (the cerebral cortex [ 101 ], the SGZ [ 99 ], and the V-SVZ [ 99 ]) and EOMES/TBR2 (the cerebral cortex [ 95 ], the SGZ [ 102 ], and the V-SVZ [ 99 , 103 ]) are not observed.…”