Identification Of The Cellular And Molecular Mechanisms Involved In The Generation Of Neural Stem Cells In The Hippocampus

The adult mammalian brain is an exceptionally complex organ which in humans contains around 160 billion cells comprising hundreds of different cell types. The majority of these cells are made during embryonic development by neural stem cells found lining the ventricular walls of the brain. Remarkably, the generation of neurons continues throughout life in a specific location of the brain, the dentate gyrus of the hippocampus, which is a V-shaped structure situated away from the ventricular system, involved in learning and memory. In the dentate gyrus, adult neural stem cells can divide to generate neurons that integrate into the existing hippocampal network in a process called adult neurogenesis. The dentate gyrus is frequently involved in neurodevelopmental disorders such as epilepsy and schizophrenia which may result from abnormal development of the dentate gyrus and the dentate neural stem cells. Yet, little is known about how the dentate gyrus develops and how the embryonic precursors of the adult neural stem cells in the dentate gyrus behave during embryonic development.

New techniques where one looks at single neural stem cells have revealed that neural stem cells in both the adult and developing brain are a very heterogenous population of cells and the different sub-populations have distinct behaviours. The embryonic neural stem cells that generate the neural stem cells in the adult dentate gyrus are found in an area of the brain called the dentate neuroepithelium. During development, these precursor cells migrate away from the ventricle to the area of the brain that is going to become the dentate gyrus. It is currently not known how the neural stem cells that are destined to become adult stem cells are different from the stem cells in other parts of the brain. Our pilot data indicate that the cells that give rise to the adult neural stem cells have different properties compared to cells in other areas of the developing brain, such as cell division dynamics and the ability to migrate.
The "mechanistic target of rapamycin" (mTOR) pathway is important for many cellular functions such as cell growth and migration. Our pilot data indicate that the neural stem cells in the developing dentate gyrus have high levels of mTOR signalling. In this project, we aim to explore the molecular and cellular mechanisms that control neural stem cell generation and migration in the developing mouse dentate gyrus. The main objective of this proposal is to:

1) Examine the cell cycle dynamics and migration of the neural stem cells in the developing dentate gyrus and determine how their fate potential changes with time.
2) Determine the functional role of mTOR signalling in the neural stem cells that generate the dentate gyrus.
3) Identify new molecular regulators of the neural stem cells in the developing dentate gyrus.

Adult neurogenesis in the dentate gyrus has garnered significant attention over the past twenty years as a form of cellular plasticity important for cognitive function. Little attention has however been paid to embryonic development and the molecular mechanisms regulating the generation of adult neural stem cells. Here we propose an experimental program to understand the origin and generation of adult neural stem cells and to identify novel targets to enhance regeneration after injury or prevent developmental disorders.

Unlike other parts of the brain, the DG contains neural stem cells (NSCs) that continue to generate neurons in adulthood in a process called adult neurogenesis. Studies have shown that the DG is involved in neurodevelopmental disorders such as schizophrenia and epilepsy, yet little is known about how the DG develops and how the NSCs in the adult DG go through development without losing their capacity to generate neurons. This project will make use of the expertise on the neural stem cells in the dentate gyrus, clonal lineage tracing, fate mapping, and RNA sequencing that I have gained during my postdoc at Johns Hopkins University and the University of Pennsylvania. In my previous work, I identified that the protein Hopx is enriched in a subset of NSCs in the developing mouse brain. Using the Hopx-CreERT2:mTmG mouse line, I was able to show that these cells not only contribute to neurogenesis during development but also give rise to the NSCs in the adult DG. In this project, we will examine the cellular behaviour of DG NSCs during development and identify molecular regulators of DG development.

Specifically, we will (1) examine the mechanisms of cell division of the NSCs in the developing brain that generate the adult NSCs, (2) determine the role of mTOR signalling on the NSCs in the developing DG, and (3), perform single-cell RNA sequencing of these developmental progenitors to reveal novel signalling and transcriptional regulators important for DG development and functionally validate our findings using in utero electroporation.

This project will not only generate a valuable and comprehensive database of genes involved in the development of the DG but also reveal novel concepts on how NSCs can retain their stem cell capacity after development and away from the ventricular system. Findings from this project will be used to develop potential therapeutics to regenerate the brain after injury or disease.