Pro-infammatory Aorta-Gonad-Mesonephros-associated macrophages are involved in embryonic development of hematopoietic stem cells

Hematopoietic stem cells (HSC) are generated from specialized endothelial cells of the embryonic aorta. Previously, inflammatory factors have been implicated in regulating mouse HSC development, but it is unclear what cells in the embryonic aorta-gonad- mesonephros (AGM) microenvironment produce these factors. In the adult, macrophages play both pro- and anti-inflammatory roles. We sought to examine whether macrophages or other hematopoietic cells found in the embryo prior to HSC generation are involved in the AGM HSC-generative microenvironment. Our CyTOF results indicate two abundant myeloid cell types - mannose-receptor positive AGM- associated macrophages (AGM-aM) and mannose-receptor negative macrophages/progenitors. We show that the appearance of macrophages in the AGM is dependent on CX3CR1. AGM-aM express a pro-inflammatory signature, localize to the embryonic aorta and dynamically interact with nascent and emerging intra-aortic hematopoietic cells (IAHC). Importantly, upon macrophage depletion, no adult- repopulating HSCs are detected, thus implicating unique pro-inflammatory AGM- associated macrophages in regulating the embryonic development of HSCs. Mannose receptor negative and mannose receptor positive macrophages were extracted from 3 independent litters of E10.5 MacGreen embryos. Each sample was representative of 3-5 AGM. The AGM were dissected and dissociated by collagenese digestion and then mannose receptor negative and positive macrophages were extracted by FACS sorting.