The specific function of microglia, the tissue resident macrophages of the brain and spinal cord, has been difficult to ascertain because of a lack of tools to distinguish microglia from other immune cells, thereby limiting specific immunostaining, purification, and manipulation. Because of their unique developmental origins and predicted functions, the distinction of microglia from other myeloid cells is critically important for understanding brain development and disease; better tools would greatly facilitate studies of microglia function in the developing, adult, and injured CNS. Here, we identify transmembrane protein 119 (Tmem119), a cell-surface protein of unknown function, as a highly expressed microglia-specific marker in both mouse and human. We developed monoclonal antibodies to its intracellular and extracellular domains that enable the immunostaining of microglia in histological sections in healthy and diseased brains, as well as isolation of pure nonactivated microglia by FACS. Using our antibodies, we provide, to our knowledge, the first RNAseq profiles of highly pure mouse microglia during development and after an immune challenge. We used these to demonstrate that mouse microglia mature by the second postnatal week and to predict novel microglial functions. Together, we anticipate these resources will be valuable for the future study and understanding of microglia in health and disease.

Summary Microglia, the resident macrophages of the central nervous system (CNS), engage in various CNS-specific functions that are critical for development and health. To better study microglia and the properties that distinguish them from other tissue macrophage populations, we have optimized serum-free culture conditions to permit robust survival of highly ramified adult microglia under defined-medium conditions. We find that astrocyte-derived factors prevent microglial death ex vivo and that this activity results from three primary components, CSF-1/IL-34, TGF-β2, and cholesterol. Using microglial cultures that have never been exposed to serum, we demonstrate a dramatic and lasting change in phagocytic capacity after serum exposure. Finally, we find that mature microglia rapidly lose signature gene expression after isolation, and that this loss can be reversed by engrafting cells back into an intact CNS environment. These data indicate that the specialized gene expression profile of mature microglia requires continuous instructive signaling from the intact CNS.

Summary Phagocytosis is required for a broad range of physiological functions, from pathogen defense to tissue homeostasis, but mechanisms required for phagocytosis of diverse substrates remain incompletely understood. Here, we develop a rapid magnet-based phenotypic screening strategy, and perform eight genome-wide CRISPR screens in human cells to identify genes regulating phagocytosis of distinct substrates. After validating select hits in focused mini-screens, orthogonal assays and primary human macrophages, we demonstrate that 1) the previously-uncharacterized gene NHLRC2 is a central player in phagocytosis, regulating RhoA-Rac1 signaling cascades that control actin polymerization and filopodia formation, 2) very long chain fatty acids are essential for efficient phagocytosis of certain substrates, and 3) the previously-uncharacterized Alzheimer’s disease-associated gene TM2D3 can preferentially influence uptake of amyloid-β aggregates. These findings illuminate new regulators and core principles of phagocytosis, and more generally establish an efficient method for unbiased identification of cellular uptake mechanisms across diverse physiological and pathological contexts.

Neurons receive signals through dendrites that vary widely in number and organization, ranging from one primary dendrite to multiple complex dendritic trees. For example, retinal amacrine cells (ACs) project primary dendrites into a discrete synaptic layer called the inner plexiform layer (IPL) and only rarely extend processes into other retinal layers. Here, we show that the atypical cadherin Fat3 ensures that ACs develop this unipolar morphology. AC precursors are initially multipolar, but lose neurites as they migrate through the neuroblastic layer. In fat3 mutants, pruning is unreliable and ACs elaborate two dendritic trees: one in the IPL and a second projecting away from the IPL that stratifies to form an additional synaptic layer. Since complex nervous systems are characterized by the addition of layers, these results demonstrate that mutations in a single gene can cause fundamental changes in circuit organization that may drive nervous system evolution.

SUMMARY The sense of balance depends on the intricate architecture of the inner ear, which contains three semicircular canals used to detect motion of the head in space. Changes in the shape of even one canal cause drastic behavioral deficits, highlighting the need to understand the cellular and molecular events that ensure perfect formation of this precise structure. During development, the canals are sculpted from pouches that grow out of a simple ball of epithelium, the otic vesicle. A key event is the fusion of two opposing epithelial walls in the center of each pouch, thereby creating a hollow canal. During the course of a gene trap mutagenesis screen to find new genes required for canal morphogenesis, we discovered that the Ig superfamily protein Lrig3 is necessary for lateral canal development. We show that this phenotype is due to ectopic expression of the axon guidance molecule Netrin1 (Ntn1), which regulates basal lamina integrity in the fusion plate. Through a series of genetic experiments, we show that mutually antagonistic interactions between Lrig3 and Ntn1 create complementary expression domains that define the future shape of the lateral canal. Remarkably, removal of one copy of Ntn1 from Lrig3 mutants rescues both the circling behavior and the canal malformation. Thus, the Lrig3/Ntn1 feedback loop dictates when and where basement membrane breakdown occurs during canal development, revealing a new mechanism of complex tissue morphogenesis.