Microglia and Myelin: Improved Tools and Molecular Interactions

Abstract

Myelination of axons evolved in vertebrates to promote signal transduction and the development of a compact and energy-efficient nervous system. Myelin development and homeostasis are critical for normal function and impairment of myelin is associated with sensory, motor, and cognitive dysfunction. While it has long been understood that oligodendrocytes produce myelin in the CNS, more recent studies also implicate additional cell types including microglia as providers of important molecular cues in myelination. Studies dissecting these cues have made great strides, yet the functions of many candidate molecular mediators remain incompletely defined, at least in part due to the scarcity of suitable tools. The goal of my thesis was threefold: (1) creating and supplying the research community with much needed improved genetic tools to study the role of microglia, (2) developing analysis tools to facilitate the analysis of white-matter ultrastructure, and (3) defining the role of a candidate molecular mediator, IgG, as well as its receptor, in white-matter development. In Chapter 1, I report the generation and characterization of novel transgenic mouse lines for the labeling and the inducible genetic manipulation of microglia. Harnessing the microglia-specific Tmem119 locus and CRISPR/Cas, I engineered knock-in mice expressing EGFP or CreERT2 and show that these lines are highly specific in discerning microglia from other closely related myeloid cells. Extending this work, in Chapter 2, I present evidence for the generation of a highly efficient and specific constitutively active Cre line leveraging the Fcrlslocus. Most notably, flow cytometric analysis shows that this line completely spares monocytes and other white blood cells. Complementing Tmem119- EGFP and Tmem119-CreERT2 mice that have already been adopted by hundreds of labs, Fcrls-Cre mice will enable genetic studies of microglia with improved specificity. In Chapter 3, I report the development and characterization of MyelTracer, an easy-to-install software suite made available to the research community for the quantification of myelin g-ratio, a key metric in studies of myelin morphology. Finally, in Chapter 4, I present evidence for the occurrence of maternally derived IgG on microglia in the postnatal brain. Using a genetic mouse model to study its functional relevance, I show that brains of mice lacking IgG postnatally harbor fewer myelinating oligodendrocytes and thinner axons in the corpus callosum. Further, using both newly generated conditional Fcer1g (part of IgG receptor) and existing constitutive Fcer1g knockout models, I show that the effect of IgG is not mediated through the canonical IgG-Fc receptor pathway. Independent of Fc receptors, IgG appears to be required for the function of a subset of microglia that occurs in white-matter tracts postnatally and is required for normal white-matter development. Overall, the work presented in this thesis resulted in the generation of several much-needed tools for the research community, and it reveals molecular insights into the role of IgG in the developing brain.