Most haematopoietic cells renew from adult haematopoietic stem cells (HSCs), however, macrophages in adult tissues can self-maintain independently of HSCs. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSCs, and fetal macrophages can develop independently of Myb, a transcription factor required for HSC, and can persist in adult tissues. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC-derived progenitors are still unclear. Here we show in mice that the vast majority of adult tissue-resident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells) and lung (alveolar macrophages) originate from a Tie2(+) (also known as Tek) cellular pathway generating Csf1r(+) erythro-myeloid progenitors (EMPs) distinct from HSCs. EMPs develop in the yolk sac at embryonic day (E) 8.5, migrate and colonize the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes. Kupffer cells, microglia and Langerhans cells are only marginally replaced in one-year-old mice, whereas alveolar macrophages may be progressively replaced in ageing mice. Our fate-mapping experiments identify, in the fetal liver, a sequence of yolk sac EMP-derived and HSC-derived haematopoiesis, and identify yolk sac EMPs as a common origin for tissue macrophages.

Haematopoietic stem cells (HSCs) are widely studied by HSC transplantation into immune- and blood-cell-depleted recipients. Single HSCs can rebuild the system after transplantation. Chromosomal marking, viral integration and barcoding of transplanted HSCs suggest that very low numbers of HSCs perpetuate a continuous stream of differentiating cells. However, the numbers of productive HSCs during normal haematopoiesis, and the flux of differentiating progeny remain unknown. Here we devise a mouse model allowing inducible genetic labelling of the most primitive Tie2(+) HSCs in bone marrow, and quantify label progression along haematopoietic development by limiting dilution analysis and data-driven modelling. During maintenance of the haematopoietic system, at least 30% or ∼5,000 HSCs are productive in the adult mouse after label induction. However, the time to approach equilibrium between labelled HSCs and their progeny is surprisingly long, a time scale that would exceed the mouse's life. Indeed, we find that adult haematopoiesis is largely sustained by previously designated 'short-term' stem cells downstream of HSCs that nearly fully self-renew, and receive rare but polyclonal HSC input. By contrast, in fetal and early postnatal life, HSCs are rapidly used to establish the immune and blood system. In the adult mouse, 5-fluoruracil-induced leukopenia enhances the output of HSCs and of downstream compartments, thus accelerating haematopoietic flux. Label tracing also identifies a strong lineage bias in adult mice, with several-hundred-fold larger myeloid than lymphoid output, which is only marginally accentuated with age. Finally, we show that transplantation imposes severe constraints on HSC engraftment, consistent with the previously observed oligoclonal HSC activity under these conditions. Thus, we uncover fundamental differences between the normal maintenance of the haematopoietic system, its regulation by challenge, and its re-establishment after transplantation. HSC fate mapping and its linked modelling provide a quantitative framework for studying in situ the regulation of haematopoiesis in health and disease.

Most haematopoietic cells renew from adult hematopoietic stem cells (HSC)1-3, however, macrophages in adult tissues can self-maintain independently of HSC4-7. Progenitors with macrophage potential in vitro have been described in the yolk sac before emergence of HSC8-13, and fetal macrophages13-15 can develop independently of Myb 4 , a transcription factor required for HSC16, and can persist in adult tissues4,17,18. Nevertheless, the origin of adult macrophages and the qualitative and quantitative contributions of HSC and putative non-HSC progenitors are still unclear19. Here we show that the vast majority of adult tissueresident macrophages in liver (Kupffer cells), brain (microglia), epidermis (Langerhans cells), and lung (alveolar macrophages) originate from a Tie2 + cellular pathway generating Csf1r + erythro-myeloid progenitors (EMP) distinct from HSC. EMP develop in the yolk sac at embryonic day (E) 8.5, migrate and colonise the nascent fetal liver before E10.5, and give rise to fetal erythrocytes, macrophages, granulocytes and monocytes until at least E16.5. Subsequently, HSC-derived cells replace erythrocytes, granulocytes and monocytes, whereas Kupffer cells, microglia, and Langerhans cells are not replaced in 1-year old animals, while alveolar macrophages may be progressively replaced in aging mice. Our fate mapping 6

Developmental deconvolution of complex organs and tissues at the level of individual cells remains challenging. Non-invasive genetic fate mapping1 has been widely used, but the low number of distinct fluorescent marker proteins limits its resolution. Much higher numbers of cell markers have been generated using viral integration sites2, viral barcodes3, and strategies based on transposons4 and CRISPR/Cas9 genome editing5; however, temporal and tissue-specific induction of barcodes in situ has not been achieved. Here we report the development of an artificial DNA recombination locus (termed Polylox) that enables broadly applicable endogenous barcoding based on the Cre-loxP recombination system6,7. Polylox recombination in situ reaches a practical diversity of several hundred thousand barcodes, allowing tagging of single cells. We have used this experimental system, combined with fate mapping, to assess haematopoietic stem cell (HSC) fates in vivo. Classical models of haematopoietic lineage specification assume a tree with few major branches. More recently, driven in part by the development of more efficient single-cell assays and improved transplantation efficiencies, different models have been proposed, in which unilineage priming may occur in mice and humans at the level of HSCs8–10. We have introduced barcodes into HSC progenitors in embryonic mice, and found that the adult HSC compartment is a mosaic of embryo-derived HSC clones, some of which are unexpectedly large. Most HSC clones gave rise to multilineage or oligolineage fates, arguing against unilineage priming, and suggesting coherent usage of the potential of cells in a clone. The spreading of barcodes, both after induction in embryos and in adult mice, revealed a basic split between common myeloid-erythroid development and common lymphocyte development, supporting the long-held but contested view of a tree-like haematopoietic structure.

The cellular differentiation pathway originating from the bone marrow leading to early T lymphocytes remains poorly understood. The view that T cells branch off from a lymphoid-restricted pathway has recently been challenged by a model proposing a common progenitor for T cell and myeloid lineages. We generated interleukin-7 receptor alpha (Il7r) Cre recombinase knockin mice and traced lymphocyte development by visualizing the history of Il7r expression. Il7r fate mapping labeled all T cells but few myeloid cells. More than 85% of T cell progenitors were Il7r reporter(+) and, hence, had arisen from an Il7r-expressing pathway. In contrast, the overwhelming majority of myeloid cells in the thymus were derived from Il7r reporter(-) cells. Thus, lymphoid-restricted progenitors are the major route to T cells, and distinct origins of lymphoid and myeloid lineages represent a fundamental hallmark of hematopoiesis.

Cell competition is an emerging principle underlying selection for cellular fitness during development and disease. Competition may be relevant for cancer, but an experimental link between defects in competition and tumorigenesis is elusive. In the thymus, T lymphocytes develop from precursors that are constantly replaced by bone-marrow-derived progenitors. Here we show that in mice this turnover is regulated by natural cell competition between 'young' bone-marrow-derived and 'old' thymus-resident progenitors that, although genetically identical, execute differential gene expression programs. Disruption of cell competition leads to progenitor self-renewal, upregulation of Hmga1, transformation, and T-cell acute lymphoblastic leukaemia (T-ALL) resembling the human disease in pathology, genomic lesions, leukaemia-associated transcripts, and activating mutations in Notch1. Hence, cell competition is a tumour suppressor mechanism in the thymus. Failure to select fit progenitors through cell competition may explain leukaemia in X-linked severe combined immune deficiency patients who showed thymus-autonomous T-cell development after therapy with gene-corrected autologous progenitors.

Tie1 is a mechanistically poorly characterized endothelial cell (EC)-specific orphan receptor. Yet, Tie1 deletion is embryonic lethal and Tie1 has been implicated in critical vascular pathologies, including atherosclerosis and tumor angiogenesis. Here, we show that Tie1 does not function independently but exerts context-dependent effects on the related receptor Tie2. Tie1 was identified as an EC activation marker that is expressed during angiogenesis by a subset of angiogenic tip and remodeling stalk cells and downregulated in the adult quiescent vasculature. Functionally, Tie1 expression by angiogenic EC contributes to shaping the tip cell phenotype by negatively regulating Tie2 surface presentation. In contrast, Tie1 acts in remodeling stalk cells cooperatively to sustain Tie2 signaling. Collectively, our data support an interactive model of Tie1 and Tie2 function, in which dynamically regulated Tie1 versus Tie2 expression determines the net positive or negative effect of Tie1 on Tie2 signaling.

Recently we have identified the novel mitochondrial peptidase responsible for degrading presequences and other short unstructured peptides in mitochondria, the presequence peptidase, which we named PreP peptidasome. In the present study we have identified and characterized the human PreP homologue, hPreP, in brain mitochondria, and we show its capacity to degrade the amyloid ␤-protein (A␤). PreP belongs to the pitrilysin oligopeptidase family M16C containing an inverted zincbinding motif. We show that hPreP is localized to the mitochondrial matrix. In situ immuno-inactivation studies in human brain mitochondria using anti-hPreP antibodies showed complete inhibition of proteolytic activity against A␤. We have cloned, overexpressed, and purified recombinant hPreP and its mutant with catalytic base Glu 78 in the inverted zinc-binding motif replaced by Gln. In vitro studies using recombinant hPreP and liquid chromatography nanospray tandem mass spectrometry revealed novel cleavage specificities against A␤-(1-42), A␤-(1-40), and A␤ Arctic, a protein that causes increased protofibril formation an early onset familial variant of Alzheimer disease. In contrast to insulin degrading enzyme, which is a functional analogue of hPreP, hPreP does not degrade insulin but does degrade insulin B-chain. Molecular modeling of hPreP based on the crystal structure at 2.1 Å resolution of AtPreP allowed us to identify Cys 90 and Cys 527 that form disulfide bridges under oxidized conditions and might be involved in redox regulation of the enzyme. Degradation of the mitochondrial A␤ by hPreP may potentially be of importance in the pathology of Alzheimer disease.Several human disorders are associated with the deposition of aggregated peptides. One of them is Alzheimer disease (AD) 4 in which the polymerization of amyloid ␤-protein (A␤) into insoluble fibrils in brain seems to be a pathological event. An extracellular accumulation of A␤ has been the main focus of molecular studies associated with AD (1). However, increasing attention is directed toward intracellular events including the mitochondrial role in AD (2). There are many links between mitochondrial dysfunctions and AD (3, 4). Impairment of mitochondrial energy metabolism and altered cytochrome c oxidase activity are among the earliest detectable defects in AD (5, 6). It has been shown that Alzheimer amyloid precursor protein (APP) 695 is not only targeted to the plasma membrane but also to mitochondria (5). Accumulation of APP in the outer mitochondrial membrane caused dysfunctions and impaired energy metabolism. The active ␥-secretase complex including presenilin, nicastrin, APH-1, and PEN-2, which cleave APP to generate A␤, has been shown to be present in the mitochondrial outer membrane (7). Furthermore, the occurrence of A␤ in mitochondria of AD patients and its direct binding to ABAD (A␤-binding alcohol dehydrogenase, also called ERAB) induces apoptosis and free radical generation in neurons (8). A recent study demonstrated that A␤ is present in the mitochondrial matrix in A...