Abstract
Microglia are yolk sac derived immune cells that invade the mouse embryonic brain around mid-gestation during a time that coincides with early development of the nervous system. Previous research indicates that microglia can influence neurodevelopmental processes such as progenitor maintenance and cell differentiation. This notion led me to ask whether microglia play a functional role(s) in the developing hypothalamus?
In this study, I have thus systematically identified the spatiotemporal timing of microglia invasion into the developing embryonic tuberal hypothalamus and their activation states using marker analyses and fluorescent imaging approaches. The results showed that microglia invaded the hypothalamus starting at E11.5 and that a significant portion of their population aligned with ventricular neural progenitors at E15.5. The microglia that aligned at the ventricle were shown to have a non-ramified structure and co-labeled with various activation markers, which suggested that they were in a highly activated state. This data led me to wonder whether microglia were influencing a developmental process happening at the ventricle at E15.5. Previous studies of hypothalamic ventricle progenitors have shown that neurogenesis occurs prior to E15.5, after which gliogenesis was predicted to commence but this process had not yet been elucidated in the tuberal hypothalamus. Here, I characterized gliogenesis in the tuberal hypothalamus and found that the timing in which microglia congregated at the ventricle aligned with when ventricle progenitors were concluding neurogenesis and undergoing gliogenesis. These data led me to determine whether microglia play a role in influencing glial cell development in the embryonic tuberal hypothalamus.
To test this hypothesis, I employed a pharmacological knock-down approach to eliminate microglia from the embryonic brain. I found that the elimination of microglia did, indeed, lead to an alteration in glial cell development. Specifically, I uncovered a reduction of the late born glioblasts and astrocytes, as well as, an alteration in the phenotype of oligodendrocyte precursor populations and postnatal α-tanycytes. I also detected an alteration in the phenotype of neural progenitor cells, known as radial glia cells (RGCs).
To investigate the mechanism behind how microglia were influencing RGCs and late born glial populations I looked at whether microglial secrete signals to influence glial development or if they partook in physical interactions with progenitor cells since activated microglia have well known roles in both secretion of cytokines and growth factors and in phagocytosis. To look at phagocytosis, I employed the use of a unique live cell imaging approach to visualize the interaction between microglial and RGCs. By using in utero electroporation to label distinct RGC populations in a mouse line with fluorescently labeled microglia, I revealed a fascinating interplay between progenitors and microglia. Through live cell imaging I observed microglia wrapping around extended RGC processes and severing them in a unique and novel interaction. I also examined secreted signals by culturing embryonic brain slices of control and microglia knock-down samples and analyzed secreted proteins within each. Here I found several candidate cytokines and growth factors that may be involved in contributing to the development of embryonic glial populations. Combined, I uncovered that microglia influence the development of embryonic glial cell populations by interacting with RGCs along the ventricular zone during early glial cell development. I propose a mechanism whereby microglia influence these developing populations through both physical and cytokine mediated interactions. Overall, I show a unique and novel influence of microglia in the development of the tuberal hypothalamus.
