Molecular Mechanisms Regulating Gliogenesis in the Neocortex

Neurons and glia are the cells that make up our brain. In the cortex, the brain area that enables us to think, speak and be conscious, neurons and most glia are produced by a type of neural stem cell, called radial glia progenitors (RGPs). It is vital that no errors occur in this process as disruptions can lead to neurodevelopmental disorders such as microcephaly, a condition in which a baby`s head and cortex are significantly smaller than that of other babies. But how is this production of neurons and glia cells controlled? Neurons and glia build a complex network that is necessary for the correct function of the cortex. Astrocytes are a type of glia cell and play a critical role in the development and function of neural circuits of the cortex, with astrocyte dysfunction comprising a component of nearly all neurological disorders. The goal of this study is to determine the cellular and molecular mechanisms of astrocyte generation. To visualize the production of astrocytes from individual RGPs and to manipulate genes in these cells at the same time, I will use MADM (Mosaic Analysis with Double Markers) technology. I hypothesize that intracellular trafficking of signaling molecules is one key mechanism involved in astrocyte lineage progression. Previous studies have suggested a crucial and dose-dependent regulatory function of EGF receptor (EGFR) signaling in gliogenesis. I demonstrated in my previous work that Lgl1 and Egfr are two genes involved in regulating astrocyte proliferation. I hypothesize that LGL1 regulates the trafficking of active EGFR, thereby controlling the rate of astrocyte proliferation. To test this hypothesis I will develop a MADM-based astrocyte cell culture system which will provide sub-cellular resolution of intracellular trafficking. Furthermore, I will test the function of intracellular trafficking in astrocyte production by disrupting a gene which is a critical component of both the endosomal and lysosomal pathways. I am confident the findings will have widespread and lasting impact in both the cortical development and gliogenesis fields, as well as the broader neuroscience community as a whole. This study promises to elucidate the molecular mechanisms underlying glia stem cell behavior, while providing novel insight into the complexity of neural stem cell regulation. Ultimately, such advances can result in a deeper understanding of brain function and why human brain development is so sensitive to disruptions in glia production and regulation. Perhaps even more important, our findings may also be of relevance for many fields in regenerative medicine relying on cellular reprogramming and stem cell-based therapies.

Neurons and glia are the cells that make up our brain. In the cortex, the brain area that enables us to think, speak and be conscious, neurons and most glia are produced by a type of neural stem cell, called radial glia progenitors (RGPs). No errors must occur in this process as disruptions can lead to neurodevelopmental disorders such as microcephaly, a condition in which a baby's head and cortex are significantly smaller than that of other babies. But how is this production of neurons and glia cells controlled? Neurons and glia build a complex network that is necessary for the correct function of the cortex. Astrocytes are a type of glia cell and play a critical role in the development and function of neural circuits of the cortex, with astrocyte dysfunction comprising a component of nearly all neurological disorders. The goal of this study was to determine the cellular and molecular mechanisms of astrocyte generation. Using a genetic technique called Mosaic Analysis with Double Markers (MADM) we could knockout a gene in single astrocyte stem cells and at the same time, make visible and track what happens in these cells. I demonstrated in my previous work that Lgl1 and Egfr are two genes involved in regulating astrocyte proliferation. In this study I have now dissected at the transcriptional level how Lgl1 is regulating astrocyte output using a novel approach by combing 10x Genomics and single-cell RNA-sequencing of MADM labelled cells. This data has revealed novel insights into molecular pathways that are uniquely required for astrocyte production. One notable study supported by this fellowship looked at the function of the gene Cdkn1c in cells in a region of the brain called the the cortex. When Cdkn1c was removed from the progenitor cells in this region, the whole cortex was smaller and there was massive death of neurons and astrocytes by apoptosis. Using MADM in the cortex, we could remove Cdkn1c from single cells and then observe their fate. We found that having two copies of the intact DNA, the intact instruction manual, was enough to protect a cell from death. Cdkn1c also plays a role in the development of human tumours and these new findings likely have important implications for this field. These findings may potentially be translated to other brain regions and glia types. Ultimately, such advances can result in a deeper understanding of brain function and why human brain development is so sensitive to disruptions in glia production and regulation. Perhaps even more important, these findings may be of relevance for many fields in regenerative medicine relying on cellular reprogramming and stem cell-based therapies.