Independent origins of fetal liver haematopoietic stem and progenitor cells.

Self-renewal and differentiation are tightly controlled to maintain haematopoietic stem cell (HSC) homeostasis in the adult bone marrow1,2. During fetal development, expansion of HSCs (self-renewal) and production of differentiated haematopoietic cells (differentiation) are both required to sustain the haematopoietic system for body growth3,4. However, it remains unclear how these two seemingly opposing tasks are accomplished within the short embryonic period. Here we used in vivo genetic tracing in mice to analyse the formation of HSCs and progenitors from intra-arterial haematopoietic clusters, which contain HSC precursors and express the transcription factor hepatic leukaemia factor (HLF). Through kinetic study, we observed the simultaneous formation of HSCs and defined progenitors-previously regarded as descendants of HSCs5-from the HLF+ precursor population, followed by prompt formation of the hierarchical haematopoietic population structure in the fetal liver in an HSC-independent manner. The transcription factor EVI1 is heterogeneously expressed within the precursor population, with EVI1hi cells being predominantly localized to intra-embryonic arteries and preferentially giving rise to HSCs. By genetically manipulating EVI1 expression, we were able to alter HSC and progenitor output from precursors in vivo. Using fate tracking, we also demonstrated that fetal HSCs are slowly used to produce short-term HSCs at late gestation. These data suggest that fetal HSCs minimally contribute to the generation of progenitors and functional blood cells before birth. Stem cell-independent pathways during development thus offer a rational strategy for the rapid and simultaneous growth of tissues and stem cell pools.

[Hematopoietic system formation during embryogenesis].

Hematopoietic cells are a group of cells that first appear in the midembryonic stage of the mouse and are essential for body growth and maintenance. For a long time, their development was widely assumed to be divided into primitive and definitive hematopoiesis. However, erythromyeloid progenitors were identified as the wave between primitive and definitive hematopoiesis, and at least three waves were recognized. An even more multilayered structure of hematopoietic development is becoming evident in recent years, with the progress and spread of lineage tracing experiments. This review will focus on recent advances in the behavior of hematopoietic stem and progenitor cells in the embryo revealed by cell lineage-tracking experiments.

Functional and molecular characterization of mouse Gata2-independent hematopoietic progenitors.

The Gata2 transcription factor is a pivotal regulator of hematopoietic cell development and maintenance, highlighted by the fact that Gata2 haploinsufficiency has been identified as the cause of some familial cases of acute myelogenous leukemia/myelodysplastic syndrome and in MonoMac syndrome. Genetic deletion in mice has shown that Gata2 is pivotal to the embryonic generation of hematopoietic stem cells (HSCs) and hematopoietic progenitor cells (HPCs). It functions in the embryo during endothelial cell to hematopoietic cell transition to affect hematopoietic cluster, HPC, and HSC formation. Gata2 conditional deletion and overexpression studies show the importance of Gata2 levels in hematopoiesis, during all developmental stages. Although previous studies of cell populations phenotypically enriched in HPCs and HSCs show expression of Gata2, there has been no direct study of Gata2 expressing cells during normal hematopoiesis. In this study, we generate a Gata2Venus reporter mouse model with unperturbed Gata2 expression to examine the hematopoietic function and transcriptome of Gata2 expressing and nonexpressing cells. We show that all the HSCs are Gata2 expressing. However, not all HPCs in the aorta, vitelline and umbilical arteries, and fetal liver require or express Gata2. These Gata2-independent HPCs exhibit a different functional output and genetic program, including Ras and cyclic AMP response element-binding protein pathways and other Gata factors, compared with Gata2-dependent HPCs. Our results, indicating that Gata2 is of major importance in programming toward HSC fate but not in all cells with HPC fate, have implications for current reprogramming strategies.

Unique N-terminal sequences in two Runx1 isoforms are dispensable for Runx1 function.

BACKGROUND: The Runt-related transcription factors (Runx) are a family of evolutionarily conserved transcriptional regulators that play multiple roles in the developmental control of various cell types. Among the three mammalian Runx proteins, Runx1 is essential for definitive hematopoiesis and its dysfunction leads to human leukemogenesis. There are two promoters, distal (P1) and proximal (P2), in the Runx1 gene, which produce two Runx1 isoforms with distinct N-terminal amino acid sequences, P1-Runx1 and P2-Runx1. However, it remains unclear whether P2-Runx specific N-terminal sequence have any specific function for Runx1 protein. RESULTS: To address the function of the P2-Runx1 isoform, we established novel mutant mouse models in which the translational initiation AUG (+1) codon for P2-Runx1 isoform was modulated. We found that a truncated P2-Runx1 isoform is translated from a downstream non-canonical AUG codon. Importantly, the truncated P2-Runx1 isoform is sufficient to support primary hematopoiesis, even in the absence of the P1-Runx1 isoform. Furthermore, the truncated P2-Runx1 isoform was able to restore defect in basophil development caused by loss of the P1-Runx1 isoform. The truncated P2-Runx1 isoform was more stable than the canonical P2-Runx1 isoform. CONCLUSIONS: Our results demonstrate that the N-terminal sequences specific for P2-Runx1 are dispensable for Runx1 function, and likely serve as a de-stabilization module to regulate Runx1 production.

Runx Family Genes in Tissue Stem Cell Dynamics.

The Runx family genes play important roles in development and cancer, largely via their regulation of tissue stem cell behavior. Their involvement in two organs, blood and skin, is well documented. This review summarizes currently known Runx functions in the stem cells of these tissues. The fundamental core mechanism(s) mediated by Runx proteins has been sought; however, it appears that there does not exist one single common machinery that governs both tissue stem cells. Instead, Runx family genes employ multiple spatiotemporal mechanisms in regulating individual tissue stem cell populations. Such specific Runx requirements have been unveiled by a series of cell type-, developmental stage- or age-specific gene targeting studies in mice. Observations from these experiments revealed that the regulation of stem cells by Runx family genes turned out to be far more complex than previously thought. For instance, although it has been reported that Runx1 is required for the endothelial-to-hematopoietic cell transition (EHT) but not thereafter, recent studies clearly demonstrated that Runx1 is also needed during the period subsequent to EHT, namely at perinatal stage. In addition, Runx1 ablation in the embryonic skin mesenchyme eventually leads to complete loss of hair follicle stem cells (HFSCs) in the adult epithelium, suggesting that Runx1 facilitates the specification of skin epithelial stem cells in a cell extrinsic manner. Further in-depth investigation into how Runx family genes are involved in stem cell regulation is warranted.

Runx1 is required for the endothelial to haematopoietic cell transition but not thereafter.

Haematopoietic stem cells (HSCs) are the founder cells of the adult haematopoietic system, and thus knowledge of the molecular program directing their generation during development is important for regenerative haematopoietic strategies. Runx1 is a pivotal transcription factor required for HSC generation in the vascular regions of the mouse conceptus-the aorta, vitelline and umbilical arteries, yolk sac and placenta. It is thought that HSCs emerge from vascular endothelial cells through the formation of intra-arterial clusters and that Runx1 functions during the transition from 'haemogenic endothelium' to HSCs. Here we show by conditional deletion that Runx1 activity in vascular-endothelial-cadherin-positive endothelial cells is indeed essential for intra-arterial cluster, haematopoietic progenitor and HSC formation in mice. In contrast, Runx1 is not required in cells expressing Vav1, one of the first pan-haematopoietic genes expressed in HSCs. Collectively these data show that Runx1 function is essential in endothelial cells for haematopoietic progenitor and HSC formation from the vasculature, but its requirement ends once or before Vav is expressed.

Development of the hematopoietic system: expanding the concept of hematopoietic stem cell-independent hematopoiesis.

Hematopoietic stem cells (HSCs) give rise to nearly all blood cell types and play a central role in blood cell production in adulthood. For many years it was assumed that these roles were similarly responsible for driving the formation of the hematopoietic system during the embryonic period. However, detailed analysis of embryonic hematopoiesis has revealed the presence of hematopoietic cells that develop independently of HSCs both before and after HSC generation. Furthermore, it is becoming increasingly clear that HSCs are less involved in the production of functioning blood cells during the embryonic period when there is a much higher contribution from HSC-independent hematopoietic processes. We outline the current understanding and arguments for HSC-dependent and -independent hematopoiesis, mainly focusing on mouse ontogeny.

Metabolic regulation in erythroid differentiation by systemic ketogenesis in fasted mice.

Erythroid terminal differentiation and maturation depend on an enormous energy supply. During periods of fasting, ketone bodies from the liver are transported into circulation and utilized as crucial fuel for peripheral tissues. However, the effects of fasting or ketogenesis on erythroid behavior remain unknown. Here, we generated a mouse model with insufficient ketogenesis by conditionally knocking out the gene encoding the hepatocyte-specific ketogenic enzyme hydroxymethylglutary-CoA synthase 2 (Hmgcs2 KO). Intriguingly, erythroid maturation was enhanced with boosted fatty acid synthesis in the bone marrow of a hepatic Hmgcs2 KO mouse under fasting conditions, suggesting that systemic ketogenesis has a profound effect on erythropoiesis. Moreover, we observed significantly activated fatty acid synthesis and mevalonate pathways along with reduced histone acetylation in immature erythrocytes under a less systemic ketogenesis condition. Our findings revealed a new insight into erythroid differentiation, in which metabolic homeostasis and histone acetylation mediated by ketone bodies are essential factors in adaptation toward nutrient deprivation and stressed erythropoiesis.

A newly identified gene Ahed plays essential roles in murine haematopoiesis.

The development of haematopoiesis involves the coordinated action of numerous genes, some of which are implicated in haematological malignancies. However, the biological function of many genes remains elusive and unknown functional genes are likely to remain to be uncovered. Here, we report a previously uncharacterised gene in haematopoiesis, identified by screening mutant embryonic stem cells. The gene, 'attenuated haematopoietic development (Ahed)', encodes a nuclear protein. Conditional knockout (cKO) of Ahed results in anaemia from embryonic day 14.5 onward, leading to prenatal demise. Transplantation experiments demonstrate the incapacity of Ahed-deficient haematopoietic cells to reconstitute haematopoiesis in vivo. Employing a tamoxifen-inducible cKO model, we further reveal that Ahed deletion impairs the intrinsic capacity of haematopoietic cells in adult mice. Ahed deletion affects various pathways, and published databases present cancer patients with somatic mutations in Ahed. Collectively, our findings underscore the fundamental roles of Ahed in lifelong haematopoiesis, implicating its association with malignancies.

Activation of NOTCH signaling impedes cell proliferation and survival in acute megakaryoblastic leukemia.

The genetic lesions that drive acute megakaryoblastic leukemia (AMKL) have not been fully elucidated. To search for genetic alterations in AMKL, we performed targeted deep sequencing in 34 AMKL patient samples and 8 AMKL cell lines and detected frequent genetic mutations in the NOTCH pathway in addition to previously reported alterations in GATA-1 and the JAK-STAT pathway. Pharmacological and genetic NOTCH activation, but not inhibition, significantly suppressed AMKL cell proliferation in both in vitro and in vivo assays employing a patient-derived xenograft model. These results suggest that NOTCH inactivation underlies AMKL leukemogenesis. and NOTCH activation holds the potential for therapeutic application in AMKL.

Three-dimensional cartography of hematopoietic clusters in the vasculature of whole mouse embryos.

Hematopoietic cell clusters in the aorta of vertebrate embryos play a pivotal role in the formation of the adult blood system. Despite their importance, hematopoietic clusters have not been systematically quantitated or mapped because of technical limitations posed by the opaqueness of whole mouse embryos. Here, we combine an approach to make whole mouse embryos transparent, with multicolor marking, to allow observation of hematopoietic clusters using high-resolution 3-dimensional confocal microscopy. Our method provides the first complete map and temporal quantitation of all hematopoietic clusters in the mouse embryonic vasculature. We show that clusters peak in number at embryonic day 10.5, localize to specific vascular subregions and are heterogeneous, indicating a basal endothelial to non-basal (outer cluster) hematopoietic cell transition. Clusters enriched with the c-Kit(+)CD31(+)SSEA1(-) cell population contain functional hematopoietic progenitors and stem cells. Thus, three-dimensional cartography of transparent mouse embryos provides novel insight into the vascular subregions instrumental in hematopoietic progenitor/stem cell development, and represents an important technological advancement for comprehensive in situ hematopoietic cluster analysis.

Three-dimensional imaging of whole midgestation murine embryos shows an intravascular localization for all hematopoietic clusters.

Hematopoietic cell clusters associated with the midgestation mouse aorta, umbilical and vitelline arteries play a pivotal role in the formation of the adult blood system. Both genetic and live-imaging data indicate that definitive hematopoietic progenitor/stem cells (visualized as clusters) are generated from hemogenic endothelium. A 3-dimensional (3-D) whole embryo immunostaining and imaging technique has allowed quantitation and cartographic mapping of intravascular hematopoietic clusters. During this period the vitelline artery is most extensively remodeled, and several reports have suggested that vitelline remodeling leads to extravascular hematopoietic cluster emergence. Whether the earliest definitive progenitors/stem cells are intra or extra vascular could influence the process by which these cells migrate to the next hematopoietic territory, the fetal liver. Hence, by 3-D imaging we more closely examined hematopoietic clusters in the vitelline and associated connected small vessels and show here that hematopoietic clusters (particularly large clusters) are intravascular during the period of vascular remodeling.

The EMT regulator Zeb2/Sip1 is essential for murine embryonic hematopoietic stem/progenitor cell differentiation and mobilization.

Zeb2 (Sip1/Zfhx1b) is a member of the zinc-finger E-box-binding (ZEB) family of transcriptional repressors previously demonstrated to regulate epithelial-to-mesenchymal transition (EMT) processes during embryogenesis and tumor progression. We found high Zeb2 mRNA expression levels in HSCs and hematopoietic progenitor cells (HPCs), and examined Zeb2 function in hematopoiesis through a conditional deletion approach using the Tie2-Cre and Vav-iCre recombination mouse lines. Detailed cellular analysis demonstrated that Zeb2 is dispensable for hematopoietic cluster and HSC formation in the aorta-gonadomesonephros region of the embryo, but is essential for normal HSC/HPC differentiation. In addition, Zeb2-deficient HSCs/HPCs fail to properly colonize the fetal liver and/or bone marrow and show enhanced adhesive properties associated with increased ß1 integrin and Cxcr4 expression. Moreover, deletion of Zeb2 resulted in embryonic (Tie2-Cre) and perinatal (Vav-icre) lethality due to severe cephalic hemorrhaging and decreased levels of angiopoietin-1 and, subsequently, improper pericyte coverage of the cephalic vasculature. These results reveal essential roles for Zeb2 in embryonic hematopoiesis and are suggestive of a role for Zeb2 in hematopoietic-related pathologies in the adult.

Childhood hematopoietic stem cells constitute the permissive window for RUNX1-ETO leukemogenesis.

Cancer is a very rare event at the cellular level, although it is a common disease at the body level as one third of humans die of cancer. A small subset of cells in the body harbor the cellular features that constitute a permissive window for a particular genetic change to induce cancer. The significance of a permissive window is ironically best shown by a large number of failures in generating the animal model for acute myeloid leukemia (AML) with t(8;21). Over the decades, the RUNX1-ETO fusion gene created by t(8;21) has been introduced into various types of hematopoietic cells, largely at adult stage, in mice; however, all the previous attempts failed to generate tractable AML models. In stark contrast, we recently succeeded in inducing AML with the clinical features seen in human patients by specifically introducing RUNX1-ETO in childhood hematopoietic stem cells (HSCs). This result in mice is consistent with adolescent and young adult (AYA) onset in human t(8;21) patients, and suggests that childhood HSCs constitute the permissive window for RUNX1-ETO leukemogenesis. If loss of a permissive window is induced pharmacologically, cancer cells might be selectively targeted. Such a permissive window modifier may serve as a novel therapeutic drug.

Highly efficient Runx1 enhancer eR1-mediated genetic engineering for fetal, child and adult hematopoietic stem cells.

A cis-regulatory genetic element which targets gene expression to stem cells, termed stem cell enhancer, serves as a molecular handle for stem cell-specific genetic engineering. Here we show the generation and characterization of a tamoxifen-inducible CreERT2 transgenic (Tg) mouse employing previously identified hematopoietic stem cell (HSC) enhancer for Runx1, eR1 (+24 m). Kinetic analysis of labeled cells after tamoxifen injection and transplantation assays revealed that eR1-driven CreERT2 activity marks dormant adult HSCs which slowly but steadily contribute to unperturbed hematopoiesis. Fetal and child HSCs that are uniformly or intermediately active were also efficiently targeted. Notably, a gene ablation at distinct developmental stages, enabled by this system, resulted in different phenotypes. Similarly, an oncogenic Kras induction at distinct ages caused different spectrums of malignant diseases. These results demonstrate that the eR1-CreERT2 Tg mouse serves as a powerful resource for the analyses of both normal and malignant HSCs at all developmental stages.

Hepatic leukemia factor-expressing paraxial mesoderm cells contribute to the developing brain vasculature.

Recent genetic lineage tracing studies reveal heterogeneous origins of vascular endothelial cells and pericytes in the developing brain vasculature, despite classical experimental evidence for a mesodermal origin. Here we provide evidence through a genetic lineage tracing experiment that cephalic paraxial mesodermal cells give rise to endothelial cells and pericytes in the developing mouse brain. We show that Hepatic leukemia factor (Hlf) is transiently expressed by cephalic paraxial mesenchyme at embryonic day (E) 8.0-9.0 and the genetically marked E8.0 Hlf-expressing cells mainly contribute to the developing brain vasculature. Interestingly, the genetically marked E10.5 Hlf-expressing cells, which have been previously reported to contain embryonic hematopoietic stem cells, fail to contribute to the vascular cells. Combined, our genetic lineage tracing data demonstrate that a transient expression of Hlf marks a cephalic paraxial mesenchyme contributing to the developing brain vasculature. This article has an associated First Person interview with the first author of the paper.

Bone marrow imaging reveals the migration dynamics of neonatal hematopoietic stem cells.

Hematopoietic stem cells (HSCs) are produced from the blood vessel walls and circulate in the blood during the perinatal period. However, the migration dynamics of how HSCs enter the bone marrow remain elusive. To observe the dynamics of HSCs over time, the present study develops an intravital imaging method to visualize bone marrow in neonatal long bones formed by endochondral ossification which is essential for HSC niche formation. Endogenous HSCs are labeled with tdTomato under the control of an HSC marker gene Hlf, and a customized imaging system with a bone penetrating laser is developed for intravital imaging of tdTomato-labeled neonatal HSCs in undrilled tibia, which is essential to avoid bleeding from fragile neonatal tibia by bone drilling. The migration speed of neonatal HSCs is higher than that of adult HSCs. Neonatal HSCs migrate from outside to inside the tibia via the blood vessels that penetrate the bone, which is a transient structure during the neonatal period, and settle on the blood vessel wall in the bone marrow. The results obtained from direct observations in vivo reveal the motile dynamics and colonization process of neonatal HSCs during bone marrow formation.

A Runx1 intronic enhancer marks hemogenic endothelial cells and hematopoietic stem cells.

Runx1 is essential for the generation of hematopoietic stem cells (HSCs) and is frequently mutated in human leukemias. However, the cis-regulatory mechanisms modulating the Runx1 gene expression remain to be elucidated. Herewith, we report the identification of an intronic Runx1 enhancer, Runx1 +24 mouse conserved noncoding element (mCNE), using a combinatorial in silico approach involving comparative genomics and retroviral integration sites mapping. The Runx1 +24 mCNE was found to possess hematopoietic-specific enhancer activity in both zebrafish and mouse models. Significantly, this enhancer is active specifically in hemogenic endothelial cells (ECs) at sites where the de novo generation of HSCs occurs. The activity of this enhancer is also strictly restricted to HSCs within the hematopoietic compartment of the adult bone marrow. We anticipate that Runx1 +24 mCNE HSC enhancer will serve as a molecular handle for tracing and/or manipulating hemogenic ECs/HSCs behavior in vivo, and consequently become an invaluable tool for research on stem cell and cancer biology.

Generation of reconstituted hemato-lymphoid murine embryos by placental transplantation into embryos lacking HSCs.

In order to increase the contribution of donor HSC cells, irradiation and DNA alkylating agents have been commonly used as experimental methods to eliminate HSCs for adult mice. But a technique of HSC deletion for mouse embryo for increase contribution of donor cells has not been published. Here, we established for the first time a procedure for placental HSC transplantation into E11.5 Runx1-deficient mice mated with G1-HRD-Runx1 transgenic mice (Runx1-/-::Tg mice) that have no HSCs in the fetal liver. Following the transplantation of fetal liver cells from mice (allogeneic) or rats (xenogeneic), high donor cell chimerism was observed in Runx1-/-::Tg embryos. Furthermore, chimerism analysis and colony assay data showed that donor fetal liver hematopoietic cells contributed to both white blood cells and red blood cells. Moreover, secondary transplantation into adult recipient mice indicated that the HSCs in rescued Runx1-/-::Tg embryos had normal abilities. These results suggest that mice lacking fetal liver HSCs are a powerful tool for hematopoiesis reconstruction during the embryonic stage and can potentially be used in basic research on HSCs or xenograft models.

Autophagy is dispensable for the maintenance of hematopoietic stem cells in neonates.

Hematopoietic stem cells (HSCs) undergo self-renewal or differentiation to sustain lifelong hematopoiesis. HSCs are preserved in quiescence with low mitochondrial activity. Recent studies indicate that autophagy contributes to HSC quiescence through suppressing mitochondrial metabolism. However, it remains unclear whether autophagy is involved in the regulation of neonatal HSCs, which proliferate actively. In this study, we clarified the role of autophagy in neonatal HSCs using 2 types of autophagy-related gene 7 (Atg7)-conditional knockout mice: Mx1-Cre inducible system and Vav-Cre system. Atg7-deficient HSCs exhibited excess cell divisions with enhanced mitochondrial metabolism, leading to bone marrow failure at adult stage. However, Atg7 deficiency minimally affected hematopoiesis and metabolic state in HSCs at neonatal stage. In addition, Atg7-deficient neonatal HSCs exhibited long-term reconstructing activity, equivalent to wild-type neonatal HSCs. Taken together, autophagy is dispensable for stem cell function and hematopoietic homeostasis in neonates and provide a novel aspect into the role of autophagy in the HSC regulation.