Notch ligand Dll1 mediates cross-talk between mammary stem cells and the macrophageal niche

INTRODUCTION The stem cell niche plays a crucial role in regulating key stem cell properties, including self-renewal, differentiation, and cell fate change. Although stem cell niches in several organs have been well described, the cellular and molecular characteristics of the mammary gland stem cell (MaSC) niche remain largely underexplored. Stromal cell populations—including fibroblasts, macrophages, and other immune cells—are important for mammary gland development and have been implicated in MaSC niche function. However, the signaling mechanisms driving mammary stromal cell–dependent regulation of MaSC activity remain elusive. Insight into the cross-talk between MaSCs and the niche cells is important for understanding both normal tissue homeostasis and disease conditions such as breast cancer. RATIONALE Notch signaling is broadly involved in cell fate regulation during development. Although Notch receptors have been implicated in various aspects of mammary gland development, the role of Notch ligands in MaSC regulation is less clear. In this study, we focus on the Notch ligand Dll1, which is highly expressed in MaSC-enriched mammary epithelial cell (MEC) populations. Conditional knockout (cKO) of Dll1 in MECs resulted in a significant delay in branching morphogenesis during mammary gland development and a deficiency in alveoli formation during pregnancy and lactation, suggesting a key role of Dll1-mediated pathways in mammary gland development. RESULTS We found that Dll1 cKO mice have a reduced number of MaSCs at different stages of mammary gland development in virgin and pregnant animals. Furthermore, using Dll1 reporter mice, we found that Dll1 expression is enriched in MaSCs, and Dll1 + MaSCs have a greater regenerative potential than Dll1 − MaSCs. Lineage tracing with Dll1-Cre-ERT2;dTomato reporter mice revealed that Dll1 + cells can produce both basal and luminal cells. Dll1 cKO mice exhibit a significant reduction in the number of mammary gland macrophages. The mammary macrophages have molecular features, including enrichment of Wnt and Notch signaling pathway components, that are distinct from those of macrophages in other tissue. Dll1 binds to Notch2 and Notch3 on mammary macrophages to activate Notch signaling, which is necessary to sustain macrophage numbers in the niche and their MaSC-promoting activity. Using a MaSC-macrophage coculture system, we also showed that MaSC-derived Dll1 induces expression of Wnt ligands—such as Wnt3, Wnt10, and Wnt16—from macrophages, and these ligands feed back to MaSCs to promote their stem cell activities. In vivo experiments involving genetic and pharmacological depletion of macrophages, as well as macrophage-specific deficiency of Notch signaling, further validated the crucial role of mammary macrophages in sustaining MaSC activity. CONCLUSION We identified Notch ligand Dll1 as a marker that is enriched in MaSCs and demonstrated that Dll1 + MaSCs can generate both basal and luminal cells. Our study establishes macrophages as important cellular components of the MaSC niche through intercellular coupling of Notch and Wnt signaling. Dll1 produced from MaSCs activates Notch signaling in macrophages to sustain their numbers and enhance the expression of Wnt ligands, which in turn supports Wnt signaling in MaSCs to maintain stem cell activity. Our study defines a Dll1-mediated MaSC niche in which the survival and function of MaSCs and stromal macrophages are mutually regulated by cross-talk between the two cell types through Notch and Wnt signaling.