Circulating monocytes can compete for virtually any tissue macrophage niche and become long-lived replacements that are phenotypically indistinguishable from their embryonic counterparts. As the factors regulating this process are incompletely understood, we studied niche competition in the brain by depleting microglia with >95% efficiency using Cx3cr1CreER/+R26DTA/+ mice and monitored long-term repopulation. Here we show that the microglial niche is repopulated within weeks by a combination of local proliferation of CX3CR1+F4/80lowClec12a– microglia and infiltration of CX3CR1+F4/80hiClec12a+ macrophages that arise directly from Ly6Chi monocytes. This colonization is independent of blood brain barrier breakdown, paralleled by vascular activation, and regulated by type I interferon. Ly6Chi monocytes upregulate microglia gene expression and adopt microglia DNA methylation signatures, but retain a distinct gene signature from proliferating microglia, displaying altered surface marker expression, phagocytic capacity and cytokine production. Our results demonstrate that monocytes are imprinted by the CNS microenvironment but remain transcriptionally, epigenetically and functionally distinct.

The cytokine transforming growth factor-β (TGF-β) regulates the development and homeostasis of several tissue-resident macrophage populations, including microglia. TGF-β is not critical for microglia survival but is required for the maintenance of the microglia-specific homeostatic gene signature. Under defined host conditions, circulating monocytes can compete for the microglial niche and give rise to long-lived monocyte-derived macrophages residing in the central nervous system (CNS). Whether monocytes require TGF-β for colonization of the microglial niche and maintenance of CNS integrity is unknown. We found that abrogation of TGF-β signaling in CX3CR1 monocyte-derived macrophages led to rapid onset of a progressive and fatal demyelinating motor disease characterized by myelin-laden giant macrophages throughout the spinal cord. Tgfbr2-deficient macrophages were characterized by high expression of genes encoding proteins involved in antigen presentation, inflammation and phagocytosis. TGF-β is thus crucial for the functional integration of monocytes into the CNS microenvironment.

Harris and collaborators show that neutropenia results in increased formation of plasma cells and elevated antibody production.

Epithelial stem and progenitor cells contribute to the formation and maintenance of the epithelial barrier throughout life. Most stem and progenitor cell populations are tucked away in anatomically distinct locations, enabling exclusive interactions with niche signals that maintain stemness. While the development of epithelial organoid cultures provides a powerful tool for understanding the role of stem and progenitor cells in homeostasis and disease, the interaction within the niche environment is largely absent, thereby hindering the identification of factors influencing stem cell behavior. Fibroblasts play a key role in directing epithelial stem and progenitor fate. Here, a comprehensive organoid-fibroblast co-culture protocol enabling the delineation of fibroblast subpopulations in esophageal progenitor cell renewal and differentiation is presented. In this protocol, a method to isolate both epithelial cells and fibroblasts in parallel from the esophagus is described. Distinct fluorescenceactivated cell sorting strategies to isolate both the esophageal progenitor cells as well as the fibroblast subpopulations from either transgenic reporter or wild-type mice are outlined. This protocol provides a versatile approach that can be adapted to accommodate the isolation of specific fibroblast subpopulations. Establishing and passaging esophageal epithelial organoid mono-cultures is included in this protocol, enabling a direct comparison with the co-culture system. In addition, a 3D clearing approach allowing for detailed image analysis of epithelial-fibroblast interactions is described. Collectively, this protocol describes a comparative and relatively highthroughput method for identifying and understanding esophageal stem cell niche components in vitro.

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here we report that the transcriptional regulator ID1 is enriched in basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and established TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1 and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.

The regulatory circuits that coordinate epidermal differentiation during development are still not fully understood. Here we report that the transcriptional regulator ID1 is enriched in basal epidermal progenitor cells and find ID1 expression to be diminished upon differentiation. In utero silencing of Id1 impairs progenitor cell proliferation, leads to precocious delamination of targeted progenitor cells and enables differentiated keratinocytes to retain progenitor markers and characteristics. Transcriptional profiling suggests ID1 acts by mediating adhesion to the basement membrane while inhibiting spinous layer differentiation. Co-immunoprecipitation reveals ID1 binding to transcriptional regulators of the class I bHLH family. We localize bHLH Tcf3, Tcf4 and Tcf12 to epidermal progenitor cells during epidermal stratification and established TCF3 as a downstream effector of ID1-mediated epidermal proliferation. Finally, we identify crosstalk between CEBPA, a known mediator of epidermal differentiation, and Id1 and demonstrate that CEBPA antagonizes BMP-induced activation of Id1. Our work establishes ID1 as a key coordinator of epidermal development, acting to balance progenitor proliferation with differentiation and unveils how functional crosstalk between CEBPA and Id1 orchestrates epidermal lineage progression.