Efficient hemogenic endothelial cell specification by RUNX1 is dependent on baseline chromatin accessibility of RUNX1-regulated TGFß target genes.

Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo from hemogenic endothelial cells (HECs) via an endothelial-to-hematopoietic transition (EHT) that requires the transcription factor RUNX1. Ectopic expression of RUNX1 alone can efficiently promote EHT and HSPC formation from embryonic endothelial cells (ECs), but less efficiently from fetal or adult ECs. Efficiency correlated with baseline accessibility of TGFß-related genes associated with endothelial-to-mesenchymal transition (EndoMT) and participation of AP-1 and SMAD2/3 to initiate further chromatin remodeling along with RUNX1 at these sites. Activation of TGFß signaling improved the efficiency with which RUNX1 specified fetal ECs as HECs. Thus, the ability of RUNX1 to promote EHT depends on its ability to recruit the TGFß signaling effectors AP-1 and SMAD2/3, which in turn is determined by the changing chromatin landscape in embryonic versus fetal ECs. This work provides insight into regulation of EndoMT and EHT that will guide reprogramming efforts for clinical applications.

Runx1 is sufficient for blood cell formation from non-hemogenic endothelial cells in vivo only during early embryogenesis.

Hematopoietic cells differentiate during embryogenesis from a population of endothelial cells called hemogenic endothelium (HE) in a process called the endothelial-to-hematopoietic transition (EHT). The transcription factor Runx1 is required for EHT, but for how long and which endothelial cells are competent to respond to Runx1 are not known. Here, we show that the ability of Runx1 to induce EHT in non-hemogenic endothelial cells depends on the anatomical location of the cell and the developmental age of the conceptus. Ectopic expression of Runx1 in non-hemogenic endothelial cells between embryonic day (E) 7.5 and E8.5 promoted the formation of erythro-myeloid progenitors (EMPs) specifically in the yolk sac, the dorsal aorta and the heart. The increase in EMPs was accompanied by a higher frequency of HE cells able to differentiate into EMPs in vitro Expression of Runx1 just 1 day later (E8.5-E9.5) failed to induce the ectopic formation of EMPs. Therefore, endothelial cells, located in specific sites in the conceptus, have a short developmental window of competency during which they can respond to Runx1 and differentiate into blood cells.

The Role of Runx1 in Embryonic Blood Cell Formation.

The de novo generation of hematopoietic stem and progenitor cells (HSPC) occurs solely during embryogenesis from a population of epithelial cells called hemogenic endothelium (HE). During midgestation HE cells in multiple intra- and extraembryonic vascular beds leave the vessel wall as they transition into HSPCs in a process termed the endothelial to hematopoietic transition (EHT). Runx1 expression in HE cells orchestrates the transcriptional switch necessary for the transdifferentiation of endothelial cells into functional HSPCs. Runx1 is widely considered the master regulator of developmental hematopoiesis because it plays an essential function during specification of the hematopoietic lineage during embryogenesis. Here we review the role of Runx1 in embryonic HSPC formation, with a particular focus on its role in hemogenic endothelium.

Insights into blood cell formation from hemogenic endothelium in lesser-known anatomic sites.

BACKGROUND: Hematopoietic stem and progenitor cells (HSPCs) are generated de novo in the embryo in a process termed the endothelial to hematopoietic transition (EHT). EHT is most extensively studied in the yolk sac and dorsal aorta. Recently new sites of hematopoiesis have been described, including the heart, somites, head, and venous plexus of the yolk sac. RESULTS: We examined sites of HSPC formation in well-studied and in less well-known sites by mapping the expression of the key EHT factor Runx1 along with several other markers by means of confocal microscopy. We identified sites of HSPC formation in the head, heart and somites. We also identified sites of HSPC formation in both the arterial and venous plexuses of the yolk sac, and show that progenitors with lymphoid potential are enriched in hematopoietic clusters in close proximity to arteries. Furthermore, we demonstrate that many of the cells in hematopoietic clusters resemble monocytes or granulocytes based on nuclear shape. CONCLUSIONS: We identified sites of HSPC formation in the head, heart, and somites, confirming that embryonic hematopoiesis is less spatially restricted than previously thought. Furthermore, we show that HSPCs in the yolk sac with lymphoid potential are located in closer proximity to arteries than to veins. Developmental Dynamics 245:1011-1028, 2016. © 2016 Wiley Periodicals, Inc.

Extravascular endothelial and hematopoietic islands form through multiple pathways in midgestation mouse embryos.

The de novo generation of hematopoietic cells occurs during midgestation when a population of endothelial cells called hemogenic endothelium transitions into hematopoietic progenitors and stem cells. In mammalian embryos, the newly formed hematopoietic cells form clusters in the lumens of the major arteries in the embryo proper and in the vascular plexus of the yolk sac. Small clusters of hematopoietic cells that are independent of the vasculature (referred to here as extravascular islands) were shown to form in the mesentery during vascular remodeling of the vitelline artery. Using three-dimensional imaging of whole mouse embryos we demonstrate that extravascular budding of hematopoietic clusters is a more widespread phenomenon that occurs from the vitelline and the umbilical arteries both proximal to the embryo proper and distal in the extraembryonic yolk sac and placenta. Furthermore, we show that there are several mechanisms by which hematopoietic clusters leave the arteries, including vascular remodeling and extrusion. Lastly, we provide static images suggesting that extravascular islands contribute to the formation of new blood vessels. Thus, extravascular islands may represent a novel mechanism of vasculogenesis whereby established vessels contribute endothelial and hematopoietic cells to developing vascular beds.

Loss of neurofibromin Ras-GAP activity enhances the formation of cardiac blood islands in murine embryos.

Type I neurofibromatosis (NF1) is caused by mutations in the NF1 gene encoding neurofibromin. Neurofibromin exhibits Ras GTPase activating protein (Ras-GAP) activity that is thought to mediate cellular functions relevant to disease phenotypes. Loss of murine Nf1 results in embryonic lethality due to heart defects, while mice with monoallelic loss of function mutations or with tissue-specific inactivation have been used to model NF1. Here, we characterize previously unappreciated phenotypes in Nf1-/- embryos, which are inhibition of hemogenic endothelial specification in the dorsal aorta, enhanced yolk sac hematopoiesis, and exuberant cardiac blood island formation. We show that a missense mutation engineered into the active site of the Ras-GAP domain is sufficient to reproduce ectopic blood island formation, cardiac defects, and overgrowth of neural crest-derived structures seen in Nf1-/-embryos. These findings demonstrate a role for Ras-GAP activity in suppressing the hemogenic potential of the heart and restricting growth of neural crest-derived tissues.

Human definitive haemogenic endothelium and arterial vascular endothelium represent distinct lineages.

The generation of haematopoietic stem cells (HSCs) from human pluripotent stem cells (hPSCs) will depend on the accurate recapitulation of embryonic haematopoiesis. In the early embryo, HSCs develop from the haemogenic endothelium (HE) and are specified in a Notch-dependent manner through a process named endothelial-to-haematopoietic transition (EHT). As HE is associated with arteries, it is assumed that it represents a subpopulation of arterial vascular endothelium (VE). Here we demonstrate at a clonal level that hPSC-derived HE and VE represent separate lineages. HE is restricted to the CD34(+)CD73(-)CD184(-) fraction of day 8 embryoid bodies and it undergoes a NOTCH-dependent EHT to generate RUNX1C(+) cells with multilineage potential. Arterial and venous VE progenitors, in contrast, segregate to the CD34(+)CD73(med)CD184(+) and CD34(+)CD73(hi)CD184(-) fractions, respectively. Together, these findings identify HE as distinct from VE and provide a platform for defining the signalling pathways that regulate their specification to functional HSCs.

Hematopoietic stem cell arrival triggers dynamic remodeling of the perivascular niche.

Hematopoietic stem and progenitor cells (HSPCs) can reconstitute and sustain the entire blood system. We generated a highly specific transgenic reporter of HSPCs in zebrafish. This allowed us to perform high-resolution live imaging on endogenous HSPCs not currently possible in mammalian bone marrow. Using this system, we have uncovered distinct interactions between single HSPCs and their niche. When an HSPC arrives in the perivascular niche, a group of endothelial cells remodel to form a surrounding pocket. This structure appears conserved in mouse fetal liver. Correlative light and electron microscopy revealed that endothelial cells surround a single HSPC attached to a single mesenchymal stromal cell. Live imaging showed that mesenchymal stromal cells anchor HSPCs and orient their divisions. A chemical genetic screen found that the compound lycorine promotes HSPC-niche interactions during development and ultimately expands the stem cell pool into adulthood. Our studies provide evidence for dynamic niche interactions upon stem cell colonization. PAPERFLICK:

Inflammatory signaling regulates embryonic hematopoietic stem and progenitor cell production.

Identifying signaling pathways that regulate hematopoietic stem and progenitor cell (HSPC) formation in the embryo will guide efforts to produce and expand HSPCs ex vivo. Here we show that sterile tonic inflammatory signaling regulates embryonic HSPC formation. Expression profiling of progenitors with lymphoid potential and hematopoietic stem cells (HSCs) from aorta/gonad/mesonephros (AGM) regions of midgestation mouse embryos revealed a robust innate immune/inflammatory signature. Mouse embryos lacking interferon γ (IFN-γ) or IFN-α signaling and zebrafish morphants lacking IFN-γ and IFN-ϕ activity had significantly fewer AGM HSPCs. Conversely, knockdown of IFN regulatory factor 2 (IRF2), a negative regulator of IFN signaling, increased expression of IFN target genes and HSPC production in zebrafish. Chromatin immunoprecipitation (ChIP) combined with sequencing (ChIP-seq) and expression analyses demonstrated that IRF2-occupied genes identified in human fetal liver CD34(+) HSPCs are actively transcribed in human and mouse HSPCs. Furthermore, we demonstrate that the primitive myeloid population contributes to the local inflammatory response to impact the scale of HSPC production in the AGM region. Thus, sterile inflammatory signaling is an evolutionarily conserved pathway regulating the production of HSPCs during embryonic development.

Distinct temporal requirements for Runx1 in hematopoietic progenitors and stem cells.

The transcription factor Runx1 is essential for the formation of yolk sac-derived erythroid/myeloid progenitors (EMPs) and hematopoietic stem cells (HSCs) from hemogenic endothelium during embryogenesis. However, long-term repopulating HSCs (LT-HSCs) persist when Runx1 is conditionally deleted in fetal liver cells, demonstrating that the requirement for Runx1 changes over time. To define more precisely when Runx1 transitions from an essential factor to a homeostatic regulator of EMPs and HSCs, and whether that transition requires fetal liver colonization, we performed conditional, timed deletions of Runx1 between E7.5 and E13.5. We determined that Runx1 loss reduces the formation or function of EMPs up through E10.5. The Runx1 requirement in HSCs ends later, as deletion up to E11.5 eliminates HSCs. At E11.5, there is an abrupt transition to Runx1 independence in at least a subset of HSCs that does not require fetal liver colonization. The transition to Runx1 independence in EMPs is not mediated by other core binding factors (Runx2 and/or Runx3); however, deleting the common non-DNA-binding ß subunit (CBFß) severely compromises LT-HSC function. Hence, the requirements for Runx1 in EMP and HSC formation are temporally distinct, and LT-HSC function is highly reliant on continued core binding factor activity.

Semaphorin 3d signaling defects are associated with anomalous pulmonary venous connections.

Total anomalous pulmonary venous connection (TAPVC) is a potentially lethal congenital disorder that occurs when the pulmonary veins do not connect normally to the left atrium, allowing mixing of pulmonary and systemic blood. In contrast to the extensive knowledge of arterial vascular patterning, little is known about the patterning of veins. Here we show that the secreted guidance molecule semaphorin 3d (Sema3d) is crucial for the normal patterning of pulmonary veins. Prevailing models suggest that TAPVC occurs when the midpharyngeal endothelial strand (MES), the precursor of the common pulmonary vein, does not form at the proper location on the dorsal surface of the embryonic common atrium. However, we found that TAPVC occurs in Sema3d mutant mice despite normal formation of the MES. In these embryos, the maturing pulmonary venous plexus does not anastomose uniquely with the properly formed MES. In the absence of Sema3d, endothelial tubes form in a region that is normally avascular, resulting in aberrant connections. Normally, Sema3d provides a repulsive cue to endothelial cells in this area, establishing a boundary. Sequencing of SEMA3D in individuals with anomalous pulmonary veins identified a phenylalanine-to-leucine substitution that adversely affects SEMA3D function. These results identify Sema3d as a crucial pulmonary venous patterning cue and provide experimental evidence for an alternate developmental model to explain abnormal pulmonary venous connections.

The expression of Sox17 identifies and regulates haemogenic endothelium.

Although it is well recognized that haematopoietic stem cells (HSCs) develop from a specialized population of endothelial cells known as haemogenic endothelium, the regulatory pathways that control this transition are not well defined. Here we identify Sox17 as a key regulator of haemogenic endothelial development. Analysis of Sox17-GFP reporter mice revealed that Sox17 is expressed in haemogenic endothelium and emerging HSCs and that it is required for HSC development. Using the mouse embryonic stem cell differentiation model, we show that Sox17 is also expressed in haemogenic endothelium generated in vitro and that it plays a pivotal role in the development and/or expansion of haemogenic endothelium through the Notch signalling pathway. Taken together, these findings position Sox17 as a key regulator of haemogenic endothelial and haematopoietic development.

Whole-mount three-dimensional imaging of internally localized immunostained cells within mouse embryos.

We describe a three-dimensional (3D) confocal imaging technique to characterize and enumerate rare, newly emerging hematopoietic cells located within the vasculature of whole-mount preparations of mouse embryos. However, the methodology is broadly applicable for examining the development and 3D architecture of other tissues. Previously, direct whole-mount imaging has been limited to external tissue layers owing to poor laser penetration of dense, opaque tissue. Our whole-embryo imaging method enables detailed quantitative and qualitative analysis of cells within the dorsal aorta of embryonic day (E) 10.5-11.5 embryos after the removal of only the head and body walls. In this protocol we describe the whole-mount fixation and multimarker staining procedure, the tissue transparency treatment, microscopy and the analysis of resulting images. A typical two-color staining experiment can be performed and analyzed in ∼6 d.

Erythroid/myeloid progenitors and hematopoietic stem cells originate from distinct populations of endothelial cells.

Hematopoietic stem cells (HSCs) and an earlier wave of definitive erythroid/myeloid progenitors (EMPs) differentiate from hemogenic endothelial cells in the conceptus. EMPs can be generated in vitro from embryonic or induced pluripotent stem cells, but efforts to produce HSCs have largely failed. The formation of both EMPs and HSCs requires the transcription factor Runx1 and its non-DNA binding partner core binding factor ß (CBFß). Here we show that the requirements for CBFß in EMP and HSC formation in the conceptus are temporally and spatially distinct. Panendothelial expression of CBFß in Tek-expressing cells was sufficient for EMP formation, but was not adequate for HSC formation. Expression of CBFß in Ly6a-expressing cells, on the other hand, was sufficient for HSC, but not EMP, formation. The data indicate that EMPs and HSCs differentiate from distinct populations of hemogenic endothelial cells, with Ly6a expression specifically marking the HSC-generating hemogenic endothelium.