Hemogenic endothelium: Origins, regulation, and implications for vascular biology

Seminars in Cell & Developmental Biology 22 (2011) 1036–1047

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Seminars in Cell & Developmental Biology journal homepage: www.elsevier.com/locate/semcdb

Review

Hemogenic endothelium: Origins, regulation, and implications for vascular biology Joan P. Zape a , Ann C. Zovein a,b,∗ a b

Cardiovascular Research Institute, University of California San Francisco, San Francisco, CA 94143, USA Division of Neonatology, Department of Pediatrics, UCSF School of Medicine, San Francisco, CA 94143, USA

a r t i c l e

i n f o

Article history: Available online 6 October 2011 Keywords: Endothelium VE-cadherin Hemogenic endothelium Cre lox Hematopoiesis Hemangioblast

a b s t r a c t The study of endothelial development has been intertwined with hematopoiesis since the early 20th century when a bi-potential cell (hemangioblast) was noted to produce both endothelial and hematopoietic cells. Since then, ideas regarding the nature of connection between the vascular and hematopoietic systems have ranged from a tenuous association to direct lineage origination. In this review, historical data that spans hematopoietic development is examined within the context of hemogenic endothelium. Hemogenic endothelium, a specialized endothelial population capable of hematopoiesis, is an emerging theory that has recently gained momentum. Evidence across species and decades are reviewed, as are the possible modulators of the phenomenon, which include pathways that specify definitive hematopoiesis (Runx1), arterial identity (Notch1), as well as physiological and developmental factors. © 2011 Elsevier Ltd. All rights reserved.

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Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1036 Commonalities between endothelial and hematopoietic cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037 2.1. Historical perspectives on the hemangioblast, and a common origin for ECs and HSCs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037 2.2. The history of conflicting evidence between avian, amphibian, and murine data . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1037 2.3. New technology reconciles the conflicting evidence among species . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1039 Specification of hemogenic endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1039 3.1. The mammalian vascular beds implicated in the process . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1039 3.2. Distinct mesodermal origins of hemogenic endothelium . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1041 3.3. The possible requisite of endothelial sub-type (artery versus vein) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1041 Regulation and mechanics of endothelial HSC emergence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042 4.1. Notch – Runx1 pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042 4.2. BMP and Wnt pathways . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042 4.2.1. BMP signaling . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042 4.2.2. The Wnt pathway . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1042 4.3. The Hox genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1043 4.4. Mechanics of HSC emergence . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1043 4.4.1. Flow . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1043 4.4.2. Endothelial to HSC transitions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1043 Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044 5.1. Endothelial-derived blood – an all or none phenomenon? . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044 5.2. Implications for translational applications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044 5.3. Implications for vascular diversity in function and form . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044 References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1044

1. Introduction ∗ Corresponding author at: 555 Mission Bay Blvd, Rm 352X/Box 3120, University of California San Francisco, San Francisco, CA 94143 – 3120, USA. Tel.: +1 415 476 8547. E-mail address: [email protected] (A.C. Zovein). 1084-9521/$ – see front matter © 2011 Elsevier Ltd. All rights reserved. doi:10.1016/j.semcdb.2011.10.003

A functional vascular system requires two key components: the endothelial cells that form the vessel walls and the blood cells that traverse and occupy the lumen. Much like the old adage of

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the chicken and the egg, it has been difficult to distinguish what comes first: blood vessels or blood cells. Historically the initial answer was “both”: a bi-potential mesodermal precursor called the “hemangioblast” [1–4] was thought to give rise to both the vascular and hematopoietic systems. However, through a large body of recent work [5–13] there is growing acceptance of the concept that hematopoietic stem cells (HSCs) arise from a subset of endothelial cells during embryonic development (a phenomenon termed hemogenic endothelium). The implications of this include the therapeutic possibility of reverting mature endothelium (which is more easily maintained in culture than HSCs) to produce HSCs in a patient specific manner. However, many obstacles remain. Only certain vascular beds are capable of producing blood in the embryo, and their propensity to create HSCs is short lived during a small developmental window. In addition, it is not known how endothelial cells produce HSCs, and whether the process can be expanded to large-scale production in vitro. In the following review we will detail the historical debate of the hemangioblast versus hemogenic endothelium (HE), the developmental models and data that supported both sides of the debate, the new technologies that gave momentum to the pendulum swing of endothelial derived blood, and the remaining implications for vascular biology. 2. Commonalities between endothelial and hematopoietic cells 2.1. Historical perspectives on the hemangioblast, and a common origin for ECs and HSCs Hematopoietic and endothelial lineages appear to emerge simultaneously from the mesodermal layer of the mammalian embryo [14]. Through observations of yolk sac blood islands, depicted as densely packed identical groups of cells that later differentiate into an endothelial outer layer and hematopoietic center, it was believed that these two components of circulation originate from one stem cell: the hemangioblast [1–4]. Hemangioblast specification is noted to take place in the primitive streak as early as E7.0 [15,16] (in the mouse), and its bi-potentiality is thought to remain for a brief period. The ability of one cell to actively produce both a hematopoietic cell and an endothelial cell was initially demonstrated by clonal analysis in embryoid bodies (EBs) [17–19], and by in vivo cell fate tracing [20–22]; albeit in low percentages. Fate tracing in the chick suggests only approximately 7% of dividing mesodermal cells fit the criteria of a true hemangioblast [22], which is similar to the ∼12% of hemangioblast-like cells fate mapped in the zebrafish [21]. Large scale clonal analysis of entire embryos suggested a heterogeneous origin of the two lineages, with bi-potentiality occurring as a rare event [23]. The fact that endothelial and hematopoietic lineages share a number of common surface markers and transcription factors has contributed greatly to the bi-potential hypothesis. Genetic pathways and markers implicated throughout hemangioblast development first begin with those of mesodermal differentiation such as Brachyury [16,17], BMP4 [24–26], and Flk-1 [18,27–30], then encompass surface markers sometimes shared by both cell types such as CD34 (mucosialin) [31–33], VE-cadherin [30,34] and CD31 (PECAM) [30,31], to those universally restricted to hematopoiesis such as CD45 surface antigen [30]. Runx1, a transcription factor critical to hematopoietic programs [35], is also expressed in endothelium, as is GATA-2 [36]. While other critical factors that include GATA-1 (important for erythroid differentiation [37,38]), and PU.1 (important for myeloid and lymphoid development [39,40]), are restricted to hematopoietic lineages without endothelial cross-over. In addition, when conditional transgenic animals are used to fate trace endothelium (via VE-cadherin Cre [41], Tie-2 Cre [42,43], Tie-1 Cre [44]), hematopoietic cells are

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labeled, to a varying degree, in conjunction with the endothelium. The shared gene and surface marker expression between the two lineages, combined with earlier anatomical observations, provided credence to the theory that a mesodermal precursor was capable of bi-potentiality, e.g. the hemangioblast. Although the extra-embryonic yolk sac is considered the first site of hemangioblast emergence and blood formation [14], its contribution to hematopoiesis was initially described as “primitive”, largely due to studies delineating its limited hematopoietic repertoire [15,45,46]. Traditionally, the presence of “definitive” hematopoiesis had been relegated to the fetal liver, which peaks in blood production from E13 to E14 in the mouse [47–49]. Historically, yolk sac progenitors were thought to migrate to the fetal liver for differentiation into definitive hematopoietic cells [47]. This may still prove to be true, as it has been demonstrated that the yolk sac is capable of definitive hematopoietic activity as assessed by colony assays [15,50], erythroid progenitor maturation [17,51,52], long term reconstitution of both neonatal ablated mice [53] and adult irradiated mice after HoxB4 transcription factor upregulation [54]. These studies taken together suggest the yolk sac is likely capable of producing definitive hematopoietic cells, but that the cells may require conditioning or maturation prior to becoming “definitive”. The definitive hematopoietic program in the yolk sac appears late after the initial hemangioblast stage (around E8–9). Thus it’s been suggested that as functional circulation is established during this period, the cells capable of definitive hematopoiesis do not arise in situ within the yolk sac [55]. However, fate tracing strategies [56], as well as circulation deficient mutant mouse strains [57], have since provided additional evidence that early yolk sac precursors are capable of definitive hematopoiesis. 2.2. The history of conflicting evidence between avian, amphibian, and murine data The initial death knell for the yolk sac as the sole generator of HSCs came from seminal work done in the avian species during the mid 1970s [58,59] by Franc¸oise Dieterlen-Lièvre and her associates. Once more the endothelium became collaterally involved in the debate. By grafting quail embryos to chick yolk sacs the authors were able to demonstrate an intra-embryonic source of definitive hematopoiesis [58,59] (Fig. 1A). The intra-embryonic site encompassed the avian aorta and exhibited hematopoietic cells associated with the ventral wall (or “floor”) of the aortic endothelium [60,61]. Similar data then followed in Xenopus (and Rana pipiens), where a series of transplantation [62–64] and cytogenetic fate tracing [63–66] experiments demonstrated that while early hematopoiesis occurs in the ventral blood islands (VBI) of the embryo [67] (analogous to the yolk sac in the avian and mammalian species), a mesodermal area in close proximity to the developing aorta (called the dorsal lateral plate mesoderm, DLP) [67] was responsible for adult hematopoiesis in the species. However, the Xenopus data also suggested a possible VBI contribution to thymic colonization in the species [67], i.e. a yolk sac contribution to definitive hematopoiesis, which differed from earlier data in the avian species [58] and R. pipiens [62] (Fig. 1B). This was later refuted in fate tracing experiments that demonstrated the separation of the two compartments DLP and VBI to definitive and primitive hematopoiesis, respectively [68,69]. Studies then arose re-evaluating the process in the mammalian species, specifically in murine [55,70,71] and human [48] embryos (Fig. 1C). It was noted that the mammalian intra-embryonic hematopoietic site also consists of the dorsal aorta and surrounding splanchnic mesoderm. Initially this area is called the para-aortic splanchnopleure (P-Sp) at E8.5–9.5 (in mouse), and then develops into the AGM (aorta-gonad-mesonephros region) at E10.5–11.5 [55,72–74]. It is within this AGM region that blood cell clusters

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Fig. 1. Grafting and transplant experiments across species depict hematopoietic contributions. (A) In the avian model, quail-chick chimeras demonstrate an intra-embryonic source of all hematopoietic compartments. (B) In amphibians, transplantation of cytogenetically distinct anlagen reveals the relative contributions of the ventral blood islands (VBI) and dorsal lateral plate (DLP). Asterisks denote areas of differing data in the literature, e.g. whether there is VBI contribution to definitive hematopoiesis in the thymus or adult post-metamorphosis circulation. (C) In the murine model, assays that compare the yolk sac to the para-aortic splanchnopleure (P-Sp) or AGM demonstrate the intra-embryonic compartments are capable of reconstituting adult irradiated hosts prior to the yolk sac. The asterisk denotes the ability of the yolk sac to transplant adult hosts later in development, or with manipulation of the gene expression background.

can be seen “budding” from the aortic wall, appearing as though the aortic endothelium was “hemogenic” [31,48,75–77]. There also exists evidence for this mechanism in the yolk sac [78–80], placenta [81–86], as well as other major vessels that include vitelline and umbilical arteries [33,48,71,87], and the allantois [33,88,89] (Fig. 2). The murine AGM region has been shown in numerous assays to generate hematopoietic stem cells with colony forming ability [70], multi-lineage differentiation [74,90], and long term reconstitution of adult irradiated hosts [55,73,74,91]. Runx1, a critical transcription factor for definitive hematopoiesis [35,92] is highly expressed in the region and spans the underlying mesoderm, the endothelium, and associated blood cells [71,93]. Because Runx1 expression is found within the underlying mesoderm and endothelium in the murine species, similar to other hematopoietic transcription factors found in what is termed sub-aortic patches (SAP) [94–96], it

was hypothesized that the underlying mesoderm was the source of hematopoietic cells in the region. While initial data in the avian species did not necessarily refute this model [97,98], a fate tracing approach that used DiI-Acetylated LDL (which is taken up by endothelium) [76], demonstrated that when endothelium is tagged prior to circulation, later circulating blood cells also contain the tagged dye, suggesting that the hematopoietic source is endothelial. And therein began a debate as to whether the true source of HSCs was mesodermal (as the yolk sac hemangioblast paradigm had always suggested) or endothelial. The DiI-Ac-LDL study was then followed by an avian retroviral tagging experiment [99] that demonstrated the para-aortic foci (or SAPs in mouse) were also derived from the endothelium. Yet again, innovative avian experiments seemed to suggest a different paradigm than what was established based on the murine data. That is not to say there was

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Fig. 2. Hemogenic endothelial sites are depicted. In the murine model at E10.5 the various hemogenic vascular beds are noted to give rise to definitive hematopoiesis: the dorsal aorta, vitelline artery, umbilical arteries, yolk sac and placenta. The relative contribution of each vascular bed to later hematopoiesis at mid- to late gestation (fetal liver, where HSCs migrate for expansion and differentiation, but do not arise in situ), and in the adult (bone marrow, thymus, spleen), remains to be elucidated.

not evidence in the murine species that supported the hypothesis of hemogenic endothelium. A body of work conducted by Nishikawa and colleagues [34,80,100,101], had initially framed hematopoiesis as occurring through an endothelial intermediate via a murine endothelial cell (EC) culture experiment as early as 1998 [80]. In addition, the DiI-Ac-LDL labeling study was later adopted in the murine system [102] and demonstrated similar results to the previous avian experiments, lending credence to the idea of mammalian hemogenic endothelium. Yet, the complexities of the mammalian system created a significant road-block to full acceptance of the hypothesis. 2.3. New technology reconciles the conflicting evidence among species Refined experimental tools were required to circumvent the following obstacles: (1) mammalian vascular beds implicated in hemogenic endothelium are established after functional circulation making it difficult to trace origin and egress, (2) the windows of HSC emergence are limited and overlapping within various vascular beds, and (3) a multitude of shared transcription factors and signaling molecules are expressed between the mesoderm, endothelium, and HSCs within these areas. The advent of conditional gene targeting in mice (and temporal control of tissue specific gene induction), ex vivo embryo and organ culture techniques, and live cell and tissue imaging, contributed to a wave of data that allowed the pendulum to swing in favor of endothelial derived hematopoiesis within the mammalian species, and thus reconciling the multispecies data. Our group took advantage of temporal conditional gene labeling in mice to label the endothelium at E9.5 using a tamoxifen inducible VE-cadherin Cre mouse line crossed to a LacZ Cre reporter line, to follow the progeny of hemogenic endothelium throughout the life span of the animal; demonstrating definitive HSC emergence from the endothelium [5]. As VE-cadherin is canonically expressed in all vasculature throughout the animal [41,103], in vitro endothelial fate tracing was conducted in separate organ cultures, and confirmed that while the dorsal aorta, placenta, and yolk sac were capable of endothelial HSC emergence between E10.5 and E12.5, the embryonic lung and liver endothelia were not [5] (Fig. 3A). Thus, HSC emergence is specific to previously described vascular beds. However, VE-cadherin protein had been noted to be expressed as a cell surface marker on early HSCs [104], which could be resultant of carry over from their endothelial origin, or alternatively HSCs may exhibit VE-cadherin promoter activity and hence be

labeled in addition to, but not as a consequence of, endothelial labeling. By fate tracing circulating cells ex vivo using the temporal VE-cadherin Cre, as well as evaluating VE-cadherin mRNA levels, we demonstrated that indeed HSCs were labeled only in the presence of labeled endothelium [5]. A conditional gene targeting approach by Speck and colleagues demonstrated that not only is the endothelium responsible for HSC emergence, but furthermore the critical hematopoietic transcription factor Runx1 is required in the endothelium, and not in the hematopoietic compartment [6]. By genetic deletion of Runx1 in the endothelium and separately in hematopoietic cells using a VE-cadherin Cre and VAV-Cre, respectively [6], the group demonstrated that the requirement for Runx1 (and hence definitive hematopoiesis) was relegated entirely to the endothelial compartment, thus giving functional relevance to the hypothesis of endothelial derived hematopoiesis (Fig. 3B). In the same journal issue [6–8], two other groups presented data that further advanced the concept of hemogenic endothelium, both using an ES culture system (Fig. 3C). The work of Lancrin et al. demonstrated that embryonic stem cell blast colony formation produces hematopoietic cells through an endothelial intermediate, with a requirement of Runx1 and Scl transcription factors [8]; suggesting the early yolk sac hemangioblast displays properties more akin to hemogenic endothelium. Continuous live imaging of murine ES cells by Eilken et al. demonstrated real time hematopoietic cell emergence from mesodermally derived differentiated endothelial cell colonies [7]. Soon thereafter live imaging of murine AGM explants captured HSC production by the endothelium [10]. The videos of both the ES culture and the AGM explants captured the appearance of rounded cell clusters with HSC surface marker expression develop from phenotypic endothelium [7,10]. Again in the same issue that reported the AGM imaging, in vivo live whole organism imaging demonstrated the phenomenon in zebrafish [9,11]. Thus, technological advances have allowed for mechanistic and physiological evidence on a cellular, organ, and organismal level demonstrating that indeed in the mammalian system, much like the avian, amphibian, and zebrafish models; definitive HSCs emerge from the endothelium.

3. Specification of hemogenic endothelium 3.1. The mammalian vascular beds implicated in the process As previously mentioned, there exists a subset of developmental vascular beds in which hemogenic endothelium is thought to take

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Fig. 3. An overview of recent studies in the mouse system that contributed to the hemogenic endothelial hypothesis. (A) Fate tracing studies in vivo and in vitro that specifically label in the endothelium (as delineated by VE-cadherin expression, VEC+) demonstrate the ability of hemogenic vascular sites to give rise to hematopoietic cells. The studies also demonstrate the inability of non-hemogenic endothelial sites to produce hematopoietic cells (fetal liver and lung). (B) Conditional deletion of the critical hematopoietic transcription factor Runx1 in endothelium, and separately in hematopoietic cells, demonstrates the requirement of Runx1 in hematopoiesis is solely regulated to the endothelium. (C) In vitro live imaging demonstrates that mouse embryonic stem cells (ESCs) can differentiate into endothelial intermediates before giving rise to hematopoietic cells. The steps involve (1) endothelial surface marker expression (VE-cadherin, or Tie-2), with the acquisition of stem cell marker c-kit in one study, (2) hemogenic endothelial specification exhibited by CD41 expression, which is then also expressed in budding hematopoietic cells, and (3) final hematopoietic cell expression of the specific hematopoietic marker CD45.

place: the dorsal aorta, umbilical and vitelline arteries, the placenta and yolk sac. Historically the aorta and the vitelline and umbilical arteries were first demonstrated to be capable of: large HSC cluster formation [35,76], transplantation of irradiated hosts [71,73], and hematopoietic emergence via endothelial labeling/imaging in the avian [76] and murine species [5,10,102]. The placenta was postulated later to be an organ for HSC emergence and expansion [81,82,84], with the eventual re-introduction of the yolk sac to the discussion as well [57]. Again, the issue of circulation made it difficult to surmise whether the placenta and/or yolk sac possessed hemogenic capacity on their own. By using the Ncx1−/− circulation deficient mouse mutants two groups were able to demonstrate HSC emergence, one in the placenta [84], and the other in the yolk

sac [57]; and data from both organs implicated an endothelial origin of HSCs. The yolk sac, as mentioned previously, has been a bit of a chameleon, where it was once thought of as the site of the early hemangioblast, is now instead believed to be a site of hemogenic endothelium (with the term hemangioblast a possible misnomer). Early yolk sac fate tracing had already suggested that the yolk sac was capable of generating definitive HSCs [56], but one criticism of that particular fate tracing strategy was that at early embryonic stages it was theoretically possible to also label the allantois [105] – a precursor to the placenta and an area that has also been shown to harbor HSCs (also via quail-chick chimeras [88], DiI-Ac-LDL tracing [89], and other experiments including colony evaluation of the precirculation [106] and pre-chorioallantoic fusion [107] allantois).

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A recent publication utilizing a CM-DiI derivative for chorionic membrane labeling in the mouse suggests that the allantois is the source of placental HSCs, and that the placental vasculature functions as a signaling niche for HSC expansion in the organ [108]. Much like the confounding factor of circulation, many of the hemogenic vascular beds have developmental precursor structures that may mask the exact timing and location of when and where hemogenic endothelium appears, and when it ceases to be hemogenic. A similar story emerges with regard to the mesenteric blood islands [109]. Described as large clusters of cells morphologically resembling the earlier described yolk sac islands, located in the mid-gut mesentery between E9.5 and E11.5, they seemed to outsize other sites of hematopoiesis by shear number of morphologic and phenotypic [110] hematopoietic cells [109]. While the mesenteric blood islands appear during a similar window as other hemogenic vascular beds, it was unclear whether they originated de novo in the mid-gut mesenchyme or whether they were derived from another structure. Conditional fate mapping with a VE-cadherin mouse line clarified the origin of the mesenteric blood island structures to be the vitelline artery [111]. The vitelline artery, through its branching morphogenesis and remodeling events (in close proximity to the mid-gut loop), gives rise to collections of hematopoietic cells surrounded by endothelium, as traced in the conditional fate mapping system, that appear to become extravascular [111]. While the extravascular location of these hematopoietic clusters has been called into question due to the lack of c-kit and Runx1 expression in the extravascular compartment [112], the genetic fate tracing studies demonstrate a vitelline arterial contribution to the mesodermal blood islands [111]. Thus, just as the allantois may be responsible for the later structure’s (placenta) contribution to hematopoiesis [108], the varied hemogenic vascular beds may derive from a smaller subset of developmental precursors prior to each separate vascular bed expansion and differentiation.

3.2. Distinct mesodermal origins of hemogenic endothelium Along the same lines of multiple hemogenic vascular beds arising from a smaller subsets of earlier embryonic beds e.g. the allantois → placenta/umbilical arteries, yolk sac vasculature → vitelline artery/mesenteric blood islands; there may exist specific mesodermal subsets that give rise to hemogenic endothelium. Early avian work by Dieterlen-Lièvre’s group yet again set the stage for a body of work delineating specific mesodermal origins of the aortic endothelium, and later all hemogenic vascular beds. When trying to understand the nature of the hemogenic aortic floor, Pardanaud et al. [113] used chick-quail grafting techniques to delineate the contributions of the paraxial mesoderm and the splanchnopleure from the lateral plate. The former was restricted to the endothelium of the surrounding tissues and the aortic roof, while only the splanchnopleural mesoderm was capable of populating the endothelial layer of aortic floor. This subject was revisited by Jaffredo and colleagues who undertook somite grafting experiments with DiI lineage tracing in the avian species [114]. By grafting larger areas of the segmental plate and analyzing the aorta after cessation of aortic hematopoiesis, the group noted that the vascular smooth muscle cells (VSMCs) of the dorsal aorta were of somite origin, and that the aortic endothelial floor, initially splanchnopleural derived, is replaced by somitic mesodermal derived endothelium [114]. The timing of the endothelial replacement coincided with the cessation of hematopoiesis in the area. Lineage tracing of myotome derivatives on a clonal level in mice also demonstrated contributions of somite populations to the aortic endothelium [115]. As more restricted grafts were assayed it was noted that the initial aortic VSMC population is later replaced by sclerotomal mesoderm

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[116]. This was also demonstrated using conditional fate mapping in the mouse [117]. However, the mouse data appears to differ in some respects with the avian data. By fate tracing both paraxial somite derived mesoderm using a Meox-1 Cre mouse line, and lateral plate mesoderm via a Hoxb6 (containing a lateral plate mesoderm enhancer) Cre line, Per Lindahl’s group noted that the murine VSMC precursors of the aortic wall are initially composed of lateral plate mesoderm (LPM) that is later replaced by somite derived mesoderm, which remains as the VSM population in the adult aorta [117]. However, the endothelium of the adult aorta appears to not be derived (or replaced) from somitic mesoderm and remains untraced in the Meox-1 Cre line, while the Hoxb6 LPM fate mapping faithfully traces the aortic endothelium (dorsal and ventral walls) from E9.5 to adulthood [117]. Fate tracing hematopoietic contributions using the Hoxb6 Cre line demonstrates that the endothelium of the dorsal aorta, placenta, yolk sac, vitelline artery and its derivatives (mesenteric blood islands) are all faithfully traced back to the early LPM population [111]. In addition, the entirety of the adult bone marrow is also labeled, suggesting that all hematopoietic contributions from the endothelium can be traced back to the lateral plate mesodermal population [111]. If one evaluates a second Cre line that traces somite derived mesoderm (Myocardin Cre), the endothelium of the adult aorta remains unlabeled, and thus un-replaced by the population labeled in that line [5]. The lack of hemogenic endothelial replacement within the murine dorsal aorta may demonstrate inter-species differences from the replacement seen in the chick-quail transplantations [114]. However, past historical events in the field instructs us to proceed with caution when ascribing contradictory evidence to species variations. Further avian data suggests that of the somitic mesoderm compartments the sclerotome is responsible for the adult VSM population and the dermatomyotome (dm) is responsible for the endothelial contribution, although the dm grafts demonstrated very low endothelial incorporation [118]. One explanation for the differences in species could be aberrant Cre expression in the mouse system, however upon review of the Hoxb6 Cre line there is clear exclusion of the dermatomyotome [119]. In addition, within the Meox-1 Cre [120] and Myocardin Cre [121] lines there is clear inclusion of the dermatomyotome. Another issue is that the adult murine aorta spans a great distance from which to evaluate thin histological sections throughout, making it difficult to discern the initial embryonic area of aortic hemogenic endothelium, its adult size and span, and its possible replacement by somite derived mesoderm. As with earlier discrepancies between species, perhaps new tools will evolve to allow exclusive mapping of hemogenic endothelial populations and better address the question of hemogenic endothelial fate in the mammalian system. 3.3. The possible requisite of endothelial sub-type (artery versus vein) Hemogenic endothelium within the embryo is relegated to arterial vessels [70,77,109], as opposed to venous or lymphatic vessels. One case of non-arterial intra-embryonic HSC emergence was evidenced after conditional deletion of the orphan nuclear factor COUP-TFII in the endothelium of transgenic mice [122]. The loss of COUP-TFII in venous endothelium resulted in venous de-repression of Notch signaling and the adoption of an arterial phenotype, complete with HSC budding from the veins [122]. The venous adoption of arterial identity was demonstrated by expression of arterial specific markers including: Notch1, Jagged1, ephrinB2 and neuropilin-1 [122]. However, in zebrafish the requirement of arterial identity for HSC emergence appears less stringent as Notch activation can result in venous HSC emergence without concomitant expression of the arterial marker ephrinB2 [123]. Venous

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HSC emergence is also accomplished by expansion Vegf signaling [124] in the zebrafish, which is known to act upstream of Notch [125]. While Notch signaling is a requisite for endothelial arterial identity [126,127], and Notch1 mouse mutants demonstrate loss of hemogenic endothelial function [128], HSC emergence does not seem to always require an arterial identity (as identified by ephrinB2 expression in zebrafish) [123]. Hence, one could surmise that while intra-embryonic hemogenic vascular sites have clear delineation of venous and arterial endothelium (the latter from which HSCs emerge), early yolk sac and ES models of hemangioblast and/or hemogenic endothelium, which appear prior to endothelial specification, do not require an arterial identity [7,8]. Thus, the prerequisite of arterial identity for endothelial-derived hematopoiesis may only be necessary in mid-gestation, or alternatively the HSCs derived from early un-specified endothelium [7,8] may differ from HSCs born of endothelium with a true arterial phenotype. 4. Regulation and mechanics of endothelial HSC emergence 4.1. Notch – Runx1 pathways As previously mentioned, Notch signaling is a critical factor in arterial identity [127,129,130], and was later found to be critical for HSC emergence from the endothelium [96,128,131]. Kumano et al. [128] demonstrated that aorta-gonad-mesonephros (AGM) explants of Notch1−/− embryos have significantly decreased hematopoietic and endothelial activity. However, this model used a global deletion of Notch1, making it difficult to infer the true role of Notch1 in hemogenic endothelium. When the ubiquitin ligase Mindbomb (responsible for Notch ligand endocytosis and subsequent Notch signaling [132]) was ablated in the AGM endothelium, there was a noted decrease in HSC production, but not abrogation [96]. The Notch signaling molecule consists of a transmembrane receptor that upon binding to ligands of the classical DSL ligand family, is cleaved by ␥-secretase, releasing its active form: intracellular domain of Notch (ICN) [133]. The ICN translocates to the nucleus and can associate with a transcriptional mediator CSL/RPJ␬ that allows transcription of downstream target genes [133,134]. The classical Notch ligands delta-like 1 (Dll-1) [96] and Jagged-1 [135] have been shown to play a role in AGM hematopoiesis, where overexpression of either in stromal co-cultures were able to rescue Mindbomb and Jagged1 mutants, respectively. Notch1 has also been implicated in controlling another transcription factor critical to definitive hematopoiesis, GATA-2, via RPJ␬ binding to its promoter [131]. Indeed, GATA-2 upregulation could rescue impaired AGM hematopoiesis in Jagged-1 mutants [135]. Zebrafish models of Notch activation demonstrate that active Notch signaling is capable of HSC expansion in the AGM but can be abolished by blocking Runx1 expression [123]; suggesting that Runx1 is downstream of Notch signaling in the HE → HSC pathway. Mouse models also demonstrate this as retroviral delivery of Runx1 rescues Notch1 null mutant hematopoiesis in early P-Sp explants [136]. While Notch signaling is key in cell fate specification in many contexts, it is yet unclear how Notch regulates downstream Runx1 expression as no known Notch intracellular domain (NICD)/RPJ␬ binding sites have been found [123] on the proximal or distal Runx1 promoter [137]. While both Runx1 promoters are active throughout development and in hemogenic endothelium [138,139], the P2 promoter has recently been shown to rescue hematopoiesis from Runx1 null mutant aortic endothelium [139]. In addition to the two promoter sites, the Runx1 gene contains a hematopoietic specific +23 enhancer element regulated by GATA2, Runx, and Ets transcription factors, as well as the SCL complex [140]. While these transcriptional enhancers have all been associated with definitive hematopoiesis, a direct link to Notch regulation is still missing; although there exists the potential of Notch regulation of GATA-2

[131], which then may regulate Runx1 through the +23 enhancer element. Very recent data suggests that Runx1 interacts with GATA2 as part of a large heptad of transcription factors allowing DNA binding and activation of downstream hematopoietic programs [141]. Furthermore double heterozygous mice of Runx1+/− and GATA-2+/−, each separately exhibiting mild phenotypes, together recapitulate the definitive hematopoietic defect evidenced by each null mutant [141]. A recent genetic screen in zebrafish has implicated histone deacetylase 1 (HDAC1) as a hematopoietic regulator downstream of Notch, and possibly regulating Runx1 [124]. HDAC1 mutants demonstrate absence of HSCs similar to Notch signaling mutants, and can be rescued by Runx1, but not active Notch. Recent biochemical evidence demonstrates that HDAC1 binds to Runx1, and the binding is abrogated by Runx1 phosphorylation via cyclin dependent kinases (Cdks) [142]. As Notch and Wnt pathways can activate Cdks, this may be a possible avenue for their regulation of Runx1 [142]. However, the HDAC zebrafish mutants exhibit decreased Runx1 expression suggesting an alternate mechanism, or a system exquisitely sensitive to HDAC1 levels. It is likely that the role of Notch is of a global cell fate switch [143] that activates the hemogenic endothelial programme given the correct contextual background, without direct regulation of Runx1. Notch pathways also cross-talk with BMP and Wnt pathways that have been shown to directly interact with Runx1 among other transcription factors. 4.2. BMP and Wnt pathways 4.2.1. BMP signaling Of the TGFbeta superfamily secreted growth factors, BMP-4 has been demonstrated to play an important role in hematopoiesis. Early in development BMP signaling is required for ventral mesoderm formation [144], but is again later active in lateral mesoderm to restrict hemato-vascular lineages [145]. Suppression of BMP signaling within lateral mesoderm demonstrates an increase in hematopoietic and vascular markers in zebrafish [145]. Mouse and human AGM mesenchyme highly expresses BMP-4 [146–148], and AGM cultures increase hematopoietic capacity with the addition of BMP-4 [148,149]. The initial suppression and later expansion of hematopoiesis due to BMP signaling may be a matter of timing as distinct roles have been delineated for BMP regulation [150]. Interestingly enough, like Notch, BMP has also been shown to induce GATA-2 [151] and Runx1 [152] expression in hematopoietic organs. BMP-4 ligand binds receptor dimers to phosphorylate downstream Smad proteins that regulate GATA-2 [151,153] and Runx1 [152]. BMP effects on blood and endothelial lineage (BC/EC) differentiation from early extra-embryonic mesoderm has recently been shown to be modulated by Notch activity, where high Notch activity in the context of BMP signaling resulted in shifts to smooth muscle (SMC) populations over BC/EC populations [154]. Wnt activation in the same context resulted in similar SMC differentiation, but when investigated earlier in mesodermal development, Wnt activity upregulated Notch signaling (but also partially required Notch activity) [154]. Overall the data suggest that Wnt activity may act in concert with Notch in early mesoderm specification. Whether this hierarchy is intact in later hemogenic endothelial specification, and can be associated with Runx1 and GATA-2 regulation, remains to be seen. 4.2.2. The Wnt pathway The Wnt pathway consists of multiple Wnt ligands that bind Frizzled receptors activating Dishevelled proteins (Dvl) that then inhibit ␤-catenin degradation. ␤-catenin can then interact with lymphoid enhancer-binding factor 1/T cell-specific transcription factor (LEF/TCF) to activate downstream target genes. Members of the Wnt pathway including Wnt5, Wnt3a, ␤-catenin, Dvl-3 and

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Dvl-1, are all expressed in the AGM region during hematopoiesis [155,156]. Upregulation of Wnt pathway members, or alternatively the addition of soluble recombinant Wnt proteins, have been shown to increase hematopoietic capacity [157–159]. Specifically, Wnt3a has a role in HSC renewal [159]. Wnt3a mutants exhibit decreased numbers of fetal liver HSCs, suggesting decreased production in earlier hematopoietic sites such as the AGM, or decreased expansion in the fetal liver [160] (as HSCs migrate to the fetal liver from hemogenic endothelial sites for expansion and differentiation). BMP-4 has been shown to induce Wnt signaling in hematopoietic formation [150], and Wnt has also been implicated in cross-talk with Notch [154,157]. Thus, there exists complex circuitry involved in the specification of hemogenic endothelium with context dependent roles of Wnt, BMP, and Notch pathways in multiple steps of the process that remains to be fully delineated. 4.3. The Hox genes Hox genes were first identified as coordinators of growth and elongation of the embryonic body axis (reviewed in [161]). Moreover, their role in normal hematopoiesis has also been extensively studied. Analysis of bone marrow samples from both murine and human show that hematopoietic cells express genes in the Hox A, B, C and D clusters [162–164]. Studies have also shown that HoxB4 is a potent stimulator of HSC expansion [165–168]. Thus, the role of Hox genes in the hemogenic endothelium and/or possibly early hematopoietic development further warranted an investigation. A study done by Iacovino and colleagues [169] reveal a dichotomous expression between HoxA3 and Runx1 at E8.5 in the mouse, where HoxA3 expression is restricted in the developing embryonic aorta and Runx1 is expressed in the extra-embryonic yolk sac. In later stages, HoxA3 disappears from the aortic endothelium prior to Runx1 emergence and the hemogenic program. HoxA3 induction during hemogenesis led to a diminished capacity to form hematopoietic colonies in vitro and ex vivo, and was reversible upon Runx1 over-expression [169]. The results of this study suggest that HoxA3 plays a critical role in early specification of hemogenic endothelium, while prolonged endothelial HoxA3 expression results in repression of HSC emergence through decreased Runx1. Whether genes paralogous to HoxA3 have a similar regulatory and/or fine tuning role is still unknown. Finally, the precise mechanism as to how HoxA3 establishes the hemogenic endothelial program while repressing the hematopoietic program is still unclear. CHIP analysis from the study revealed that Runx1 contains putative HoxA3 binding sites [169], which may then hold some important clues as to how HoxA3 might repress the hematopoietic program.

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4.4. Mechanics of HSC emergence 4.4.1. Flow While not a strict requirement for HSC emergence (as evidenced by in vitro cultures [7,8] and circulation mutants [57,84]), circulatory flow and shear stress have certainly been implicated in augmentation of HSC emergence from the endothelium [170,171]. The endothelial response to shear stress is orchestrated by similar pathways in hemogenic endothelial specification, where PECAM1, VE-cadherin, and VEGFR2 work in concert as a mechanosensory complex in mature endothelium [172]. In the developing embryo high levels of shear stress are noted between E8.5 and E10.5, which correlates well with the timing of HE specification [173]. In a wall shear stress (WSS) model in ES cultures and E9.5 P-Sp cultures, it was shown that WSS could increase nitric oxide (NO) production and Runx1 expression, in addition to increased HSC emergence, although the latter was inhibited with NO blockade while Runx1 expression was unchanged [171]. Thus flow may function to specify HE and elicit hematopoietic cell emergence, but in a uncoupled fashion. In zebrafish, a lack of a heartbeat (and thus complete absence of circulation) resulted in abnormal vascular development and complete absence of definitive hematopoiesis [170]. This is a more pronounced effect than previously described circulation deficient mouse models in which decreased numbers of HSCs in the P-Sp/AGM [57] and abnormal hematopoietic budding was seen [84]. The NO dependency on flow and its regulation of HSC emergence downstream was also evidenced in the zebrafish model [170], and conserved in a mouse model of NO inhibition [170]. Lastly, to come full circle in HE regulation, laminar shear stress has recently been shown to increase the arterial marker EphrinB2 via Notch1 signaling in ES differentiation [174] suggesting part of the function of flow is to “arterialize” the endothelium as a step towards HE specification.

4.4.2. Endothelial to HSC transitions Thus far we have reviewed the wealth of data that suggests definitive HSCs emerge from specialized hemogenic endothelium, and the factors that regulate the process which include signaling pathways, transcriptional regulators, and physiologic flow patterns. However, the mechanics behind how exactly endothelial cells give rise to hematopoietic cells still eludes us. A few recent studies have provided some clues. Live imaging of ES cultures depict a HE cell as an adherent cell that then becomes semi-adherent and from that appears a rounded hematopoietic cell [7] (Fig. 4A). In vivo zebrafish imaging was able to capture aortic endothelial cells that round up and delaminate from the aortic wall, undergoing what

Fig. 4. Mechanisms of hematopoietic emergence from the endothelium. (A) In vivo zebrafish imaging suggests that the endothelium enters a hematopoietic transition (EHT), where an endothelial cell will round off the vessel wall and become a hematopoietic cell in the circulation. (B) In the mouse model, hematopoietic cells appear to be in direct contact, and possible continuance, with the underlying endothelium, which suggests a possible asymmetric divisional process.

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the authors describe as an endothelial to hematopoietic transition (EHT) [11]. This is a very intriguing concept as the hemogenic endothelium of the dorsal aorta (and elsewhere) is transient in its hematopoietic capacity, and at least in the avian species the HE of the aorta appears to be replaced after hemogenic capacity has ceased [114]. However it does not explain images captured within the mammalian system that depict HSCs attached to endothelial cells [76,109], and in some cases appearing to share cytoplasm [109,111] akin to an asymmetric division (Fig. 4B). Asymmetric division is a well-known mechanism in stem cell niches where a parent cell divides to give rise to a differentiated daughter cell while maintaining parental stemness. It is an attractive theory as the subaortic mesenchyme [95], and other HE environments [108], contain rich signaling centers that could theoretically help orchestrate an asymmetric divisional process. However this has not as yet been captured in HE, and thus may not necessarily be a modality of HSC emergence. Alternatively, vascular beds may employ unique methods of HSC emergence as their vascular and flow patterns may differ.

fibroblasts, where Oct4 expression and cytokine treatment was able to elicit multi-lineage blood production in vitro [178]. It remains to be seen whether this occurs through an endothelial intermediate, but as it did not require a mesodermal transition evidenced by lack of Brachyury and GATA-2 expression, the results are quite provocative in the context of hematopoietic development [178].

5.3. Implications for vascular diversity in function and form Hemogenic endothelial vascular beds not only share the unique function of hemogenic capacity, but also share a common mesodermal origin. This may suggest that vascular diversity and origins may be linked to phenotypic differences in form and function. Different subsets of endothelia that comprise a vascular bed may be pre-determined to not only a certain anatomic location but also a unique phenotypic signature which may, in the context of flow or genetic alterations, respond differently due to its developmental origins. Thus, the specification of hemogenic endothelium may teach us as much about vascular biology as it does hematopoiesis.

5. Conclusions 5.1. Endothelial-derived blood – an all or none phenomenon? Now that the pendulum has swung in favor of endothelial derived hematopoiesis, is it the only method of definitive blood production? While primitive waves of hematopoiesis originate in the yolk sac [57] which may or may not be endothelial derived, definitive hematopoiesis has been demonstrated to emanate from the endothelium. However there exist a few pieces of data that may suggest the embryo is capable of non-endothelial blood production. For example, VE-cadherin null mice demonstrate hematopoietic cells within the yolk sac that exhibit definitive characteristics [175]. However, while the endothelia in these mice are delayed in their differentiation [103], they are present and hence VE-cadherin expression may not be a complete requirement of hemogenic endothelium. Fate tracing of VE-cadherin descendants also show varying BM contributions [6,41], which may be due to differences in Cre expression within the conditional mouse lines. Alternatively it may suggest a possible non-endothelial hematopoietic contribution, or when considering the VE-cadherin null data, the ability of primitive endothelium (not fully differentiated) to give rise to hematopoietic cells. Chimera analyses of the mouse yolk sac suggests that while HE is likely a major contributor to hematopoiesis in this organ [23], there also appears a smaller non-endothelial contribution to yolk sac blood island populations [23,176]. In total, the multitude of recent data employing state of the art technologies presented in this review suggests that the endothelium is responsible for the vast majority of, if not all, definitive hematopoiesis. 5.2. Implications for translational applications The therapeutic attractiveness of the endothelium as a precursor to HSC emergence/expansion is the ease of its maintenance in culture as compared to hematopoietic cells. However, the availability of HE vascular beds occurs at a time when the endothelium is thought to no longer have hemogenic capacity, with the possible exception of the placenta [177]. Hemogenic endothelium is a transient phenomenon that dissipates as the endothelium matures. Yet, if a specific endothelium was once hemogenic in utero, it may be possible to manipulate it back to a more primitive state to acquire hemogenic capacity. Ideally to reprogram these endothelial cells in the sole presence of soluble factors would allow future endothelial banking for patient specific derived HSCs. The groundwork for this possibility has been demonstrated in human dermal

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