Summary Cardiac macrophages are crucial for tissue repair after cardiac injury but have not been well characterized. Here we identify four populations of cardiac macrophages. At steady state, resident macrophages were primarily maintained through local proliferation. However, after macrophage depletion or during cardiac inflammation, Ly6chi monocytes contributed to all four macrophage populations, whereas resident macrophages also expanded numerically through proliferation. Genetic fate mapping revealed that yolk-sac and fetal monocyte progenitors gave rise to the majority of cardiac macrophages, and the heart was among a minority of organs in which substantial numbers of yolk-sac macrophages persisted in adulthood. CCR2 expression and dependence distinguished cardiac macrophages of adult monocyte versus embryonic origin. Transcriptional and functional data revealed that monocyte-derived macrophages coordinate cardiac inflammation, while playing redundant but lesser roles in antigen sampling and efferocytosis. These data highlight the presence of multiple cardiac macrophage subsets, with different functions, origins and strategies to regulate compartment.

Summary Although classified as hematopoietic cells, tissue- resident macrophages (MFs) arise from embryonic precursors that seed the tissues prior to birth to generate a self-renewing population, which is maintained independently of adult hematopoiesis. Here we reveal the identity of these embryonic precursors using an in utero MF-depletion strategy and fate-mapping of yolk sac (YS) and fetal liver (FL) hematopoiesis. We show that YS MFs are the main precursors of microglia, while most other MFs derive from fetal monocytes (MOs). Both YS MFs and fetal MOs arise from erythro-myeloid progenitors (EMPs) generated in the YS. In the YS, EMPs gave rise to MFs without monocytic intermediates, while EMP seeding the FL upon the establishment of blood circulation acquired c-Myb expression and gave rise to fetal MOs that then seeded embryonic tissues and differentiated into MFs. Thus, adult tissue- resident MFs established from hematopoietic stem cell-independent embryonic precursors arise from two distinct developmental programs.

Understanding gene regulation and function requires a genome-wide method capable of capturing both gene expression levels and isoform diversity at the single-cell level. Short-read RNAseq is limited in its ability to resolve complex isoforms because it fails to sequence full-length cDNA copies of RNA molecules. Here, we investigate whether RNAseq using the long-read single-molecule Oxford Nanopore MinION sequencer is able to identify and quantify complex isoforms without sacrificing accurate gene expression quantification. After benchmarking our approach, we analyse individual murine B1a cells using a custom multiplexing strategy. We identify thousands of unannotated transcription start and end sites, as well as hundreds of alternative splicing events in these B1a cells. We also identify hundreds of genes expressed across B1a cells that display multiple complex isoforms, including several B cell-specific surface receptors. Our results show that we can identify and quantify complex isoforms at the single cell level.

SUMMARY The generation of distinct hematopoietic cell types, including tissue-resident immune cells, distinguishes fetal from adult hematopoiesis. However, the mechanisms underlying differential cell production to generate a layered immune system during hematopoietic development are unclear. Using an irreversible lineage tracing model, we identify a definitive hematopoietic stem cell (HSC) that supports long-term multilineage reconstitution upon transplantation into adult recipients, but does not persist into adulthood in situ. These HSCs are fully multipotent, yet display both higher lymphoid cell production and greater capacity to generate innate-like B and T lymphocytes as compared to coexisting fetal HSCs and adult HSCs. Thus, these developmentally restricted HSCs define the origin and generation of early lymphoid cells that play essential roles in establishing self-recognition and tolerance, with important implications for understanding autoimmune disease, allergy, and rejection of transplanted organs.