PROJECT SUMMARY
Pruning neuronal connections and eliminating superfluous neurons is required to generate optimized neuronal
circuits in the mature brain. Although it is well established that neurons and glia coordinate the refinement of
neural circuits, the molecular mechanisms underlying this process remain poorly defined. This K99/R00 proposal
will help me advance my career as I investigate the cellular mechanisms by which glia help refine neuronal
circuits during development. To generate a deep molecular understanding of neuronal remodeling, I will use the
Drosophila larval nervous system, which goes through extensive neuronal remodeling during metamorphosis. In
a preliminary screen for new model cells to study neuronal remodeling in vivo, I discovered several new markers
for cells that exhibit novel types of remodeling events. In addition, I conducted two large-scale screens to identify
new glial pathways that assist in pruning and the elimination of neuronal debris. First, using a single neuronal
lineage, I performed an in vivo RNAi screen for glial genes required for glial pruning of neurons. Second, I
transcriptionally profiled glia during pruning, identifying upregulated genes in glia, and also screened them for
regulators of neuronal remodeling. These genes will serve as a molecular entry point for me to define how glia
refine neural networks during development. During the mentored award phase, I will build a system that will allow
me to monitor the dynamic cell biological changes that occur during glial phagocytosis and will use this new
system to test novel molecules for their role remodeling. In Aim1, I will study the specific process of glial
phagocytosis using genetically encoded cellular markers and explant live imaging. These assays will serve as
the foundation for understanding how the molecules I identified play a role in glial phagocytosis. In Aim 2, I will
use this new system to understand how Tweek, a highly conserved molecule, functions during glial phagocytosis
of pruned neurons. Surprisingly, Tweek has no known protein domains or molecular function. I will use
CRISPR/Cas9 genomic engineering to create human disease-associated mutations in the fly. This will potentially
allow me to understand how mutations in Tweek's human homolog KIAA1109 cause a rare autosomal
neurological disease. Finally, in Aim 3, which will be mostly carried out in my own lab, I will use the tools I build
in this proposal to examine how a collection of newly identified phagocytic receptors drive neuronal remodeling
of synapses, neurites, and in multiple types of lineages. I outlined a series of research and career development
milestones that will be met during this award and allow me to grow as a scientist. To strengthen areas needed
for a successful research career, the aims are combined with neuroscience coursework and training in areas
such as scientific writing, mentoring, and project design. I have a committee made up of an excellent group of
scientists who are dedicated to my success and eager to assist me in my professional development. My long-
term career goal as an independent investigator is to understand the molecular mechanisms of neuronal
remodeling and how abnormalities in this process are associated with neurodevelopmental disorders.