Biology subjects

Stephen, D.

Publications and source records attributed to Stephen, D..

7 recordsLinked to original sources

bioRxiv2026

A Rare Nestin-Expressing Granule Cell Precursor Subpopulation Underlies SHH Medulloblastoma Formation

Granule cell precursors (GCPs) drive the major postnatal expansion of the cerebellum and are the cells of origin of sonic hedgehog medulloblastoma (SHH MB). Although GCPs are often treated as a uniform population, increasing evidence suggests they are heterogeneous, and whether specific subpopulations show distinct tumorigenic competence remains unclear. Here, we identified a rare Nestin-expressing GCP subpopulation in the normal early postnatal cerebellum and showed that it is spatially restricted, molecularly distinct and highly competent to form tumors. These cells are enriched in the posterior external granule layer and co-express Atoh1. Using different SHH MB mouse models, we showed that when this rare subpopulation of GCPs is targeted they can give rise to SHH MB with an efficiency comparable to targeting a larger number of Atoh1-expressing GCPs and that tumors derived from Nestin-expressing GCPs arise in the posterior-lateral cerebellum. Single cell RNA sequencing revealed that Nestin-expressing GCP have a transcriptome indicating reduced neuronal differentiation and enrichment for stem cell genes compared to bulk GCPs and more closely align with SHH MB cells. Together, our findings reveal functionally important heterogeneity within the GCP lineage and suggest that SHH MB arises preferentially from a small subpopulation of GCPs that express Nestin.

cancer biology↗

bioRxiv2025

The regenerative potential of adult Nestin+ cerebellar astroglia is limited compared to in neonates

There is a crucial need for strategies that stimulate repair in the adult brain. The neonatal mouse cerebellum can regenerate via the adaptive reprogramming of nestin (Nes)-expressing progenitors (NEPs). However, analysis of Nes+ cells of the adult cerebellum is limited. Using reporter lines and genetic inducible fate mapping, we show that adult Nes+ cells are mainly Bergmann glia (Nes+ Bg) that have in vitro sphere-forming ability. Following injury, Nes+ Bg increase in number due to upregulation of Nes in Hopx-expressing Bg, but the cells exhibit limited regeneration. ATAC-seq of Nes+ Bg reveals that silencing of developmental genes compared to neonatal NEPs contributes to the impaired regeneration. Activating sonic hedgehog signalling augments the number of Nes+ Bg after injury but not neurogenesis, showing additional cues are required. Our results demonstrate an age-dependent decline in the regenerative potential of NEPs and highlight Nes+ Bg as potential injury-responsive cells that could facilitate regeneration.

developmental biology↗

bioRxiv2025

PTEN restrains SHH medulloblastma growth through cell autonomous and nonautonomous mechanisms

A third of patients with the pediatric cerebellar tumor Medulloblastoma (MB) have mutations that activate Sonic hedgehog (SHH) signaling (SHH-MB subgroup). The contribution of secondary mutations to tumor severity, however is not clear. PTEN mutations are enriched in the SHH-1 subtype that has the lowest survival rate. Widespread heterozygous loss of Pten in two SHH-MB mouse models increases penetrance and excellerates onset of differentiated tumors. We delineated cellular and transcriptional changes that accelerate tumor growth and cause differentiation using a sporadic SHH-MB mouse model expressing oncogenic SmoM2 in rare cerebellar granule cell precursors (GCPs) and scRNA-seq analysis. Homozygous but not heterozygous sporadic loss of Pten resulted in rapid acceleration of tumor growth and end stage disease by 40 days, compared to [~]25% survival in control SmoM2 mice at 100 days. Heterozygous PTEN mutations therefore should negatively impact disease outcome primarily with germline mutations. Loss of Pten in normal or SmoM2-expressing GCPs increased proliferation and enhanced progenitor state initially but by 12 days Pten mutant SmoM2 tumors were highly differentiated due to increased survival of non-proliferating GCPs. Furthermore, macrophage infiltration and cytotoxicity appeared reduced in differentiated regions of tumors lacking Pten, indicating cell nonautonomous changes could also contribute to accelerated tumor growth.

cancer biology↗

bioRxiv2024

An increase in reactive oxygen species underlies neonatal cerebellum repair

The neonatal mouse cerebellum shows remarkable regenerative potential upon injury at birth, wherein a subset of Nestin-expressing progenitors (NEPs) undergoes adaptive reprogramming to replenish granule cell progenitors that die. Here, we investigate how the microenvironment of the injured cerebellum changes upon injury and contributes to the regenerative potential of normally gliogenic-NEPs and their adaptive reprogramming. Single cell transcriptomic and bulk chromatin accessibility analyses of the NEPs from injured neonatal cerebella compared to controls show a temporary increase in cellular processes involved in responding to reactive oxygen species (ROS), a known damage-associated molecular pattern. Analysis of ROS levels in cerebellar tissue confirm a transient increased one day after injury at postanal day 1, overlapping with the peak cell death in the cerebellum. In a transgenic mouse line that ubiquitously overexpresses human mitochondrial catalase (mCAT), ROS is reduced 1 day after injury to the granule cell progenitors, and we demonstrate that several steps in the regenerative process of NEPs are curtailed leading to reduced cerebellar growth. We also provide preliminary evidence that microglia are involved in one step of adaptive reprogramming by regulating NEP replenishment of the granule cell precursors. Collectively, our results highlight that changes in the tissue microenvironment regulate multiple steps in adaptative reprogramming of NEPs upon death of cerebellar granule cell progenitors at birth, highlighting the instructive roles of microenvironmental signals during regeneration of the neonatal brain.

developmental biology↗

bioRxiv2024

Cerebellar output neurons impair non-motor behaviors by altering development of extracerebellar connectivity

The capacity of the brain to compensate for insults during development depends on the type of cell loss, whereas the consequences of genetic mutations in the same neurons are difficult to predict. We reveal powerful compensation from outside the cerebellum when the excitatory cerebellar output neurons are ablated embryonically and demonstrate that the minimum requirement for these neurons is for motor coordination and not learning and social behaviors. In contrast, loss of the homeobox transcription factors Engrailed1/2 (EN1/2) in the cerebellar excitatory lineage leads to additional deficits in adult learning and spatial working memory, despite half of the excitatory output neurons being intact. Diffusion MRI indicates increased thalamo-cortico-striatal connectivity in En1/2 mutants, showing that the remaining excitatory neurons lacking En1/2 exert adverse effects on extracerebellar circuits regulating motor learning and select non-motor behaviors. Thus, an absence of cerebellar output neurons is less disruptive than having cerebellar genetic mutations.

neuroscience↗

bioRxiv2019

A bestiary of genetic mouse lines and related tools for inducible and reversible intersectional misexpression

Thanks to many advances in genetic manipulation, mouse models have become very powerful in their ability to interrogate biological processes. In order to precisely target expression of a gene of interest to particular cell types, intersectional genetic approaches utilizing two promoter/enhancers unique to a cell type are ideal. Within these methodologies, variants that add temporal control of gene expression are the most powerful. We describe the development, validation and application of an intersectional approach that involves three transgenes, requiring the intersection of two promoter/enhancers to target gene expression to precise cell types. Furthermore, the approach utilizes available lines expressing tTA/rTA to control timing of gene expression based on whether doxycycline is absent or present, respectively. We also show that the approach can be extended to other animal models, using chicken embryos. We generated three mouse lines targeted at the Tigre (Igs7) locus with TRE-loxP-tdTomato-loxP upstream of three genes (p21, DTA and Ctgf) and combined them with Cre and tTA/rtTA lines that target expression to the cerebellum and limbs. Our tools will facilitate unraveling biological questions in multiple fields and organisms. Summary statementAhmadzadeh et al. present a collection of four mouse lines and genetic tools for misexpression-mediated manipulation of cellular activity with high spatiotemporal control, in a reversible manner.

molecular biology↗

bioRxiv2018

Cerebellar nuclei neurons dictate growth of the cortex through developmental scaling of presynaptic Purkinje cells

Efficient function of neural systems requires the production of specific cell types in the correct proportions. Here we report that reduction of the earliest born neurons of the cerebellum, excitatory cerebellar nuclei neurons (eCN), results in a subsequent reduction in growth of the cerebellar cortex due to an accompanying loss of their presynaptic target Purkinje cells. Conditional knockout of the homeobox genes En1 and En2 (En1/2) in the rhombic lip-derived eCN and granule cell precursors leads to embryonic loss of a subset of medial eCN and cell non-autonomous and location specific loss of Purkinje cells, with subsequent proportional scaling down of cortex growth. We propose that subsets of eCN dictate the survival of their specific Purkinje cell partners, and in turn sonic hedgehog secreted by Purkinje cells scales the expansion of granule cells and interneurons to produce functional local circuits and the proper folded morphology of the cerebellum.

developmental biology↗