Role of the homeobox gene Meis 1 in hematopoietic stem cells generation in the vertebrate embryo: a functional analysis

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El trabajo experimental presentado en esta memoria ha sido

realizado en el Departamento de Inmunología y Oncología del

Centro Nacional de Biotecnología (CSIC) y en el Departamento de

Biología del Desarrollo Cardiovascular de la Fundación Centro

Nacional de Investigaciones Cardiovasculares (CNIC) Carlos III, bajo

la dirección del Dr. Miguel Torres Sánchez.

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A mi hermana, a mi madre,

a Joan.

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ABBREVIATIONS

AGM: Aorta-gonads-mesonephros ALL: acute lymphoid leukemia AML: acute myeloid leukemia BI: blood islands

BM: bone marrow

cDNA: complementary DNA CFU: colony-forming-unit

ChIP-chip: chromatin immunoprecipitation coupled to microarray/chip hybridization CML: chronic myeloid leukemia

DA: dorsal aorta DLP: dorsal lateral plate dpc: days post-coitum dpf: days post-fertilization E: embryonic day ECs: endothelial cells Ery: erythrocyte ES: embryonic stem cells FL: fetal liver

GO: gene ontology GOF: gain-of-function

GFP: green fluorescent protein GRN: gene regulatory network HD: homeodomain

hpe: hours post-electroporation HCs: hematopoietic cells

HPCs: hematopoietic progenitors cells HSCs: hematopoietic stem cells IP: immunoprecipitation ISH: in situ hybridization K.o.: knock-out LOF: loss-of-function LPM: lateral plate mesoderm LTR: long-term reconstitution Mk: megakaryocyte

PCR: polymerase chain reaction PSp: para-aortic splanchnopleura RA: retinoic acid

SCID: Severe Combined Immunodeficiency sp: somite-pair

SP: side population

TALE: three amino-acid loop extension TF: transcription factor

YS: yolk sac

VBI: ventral blood islands

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1. Introduction

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1.1 Embryology of hematopoiesis in vertebrates

Hematopoiesis is the dynamic process whereby blood cells are continuously produced in an organism. Blood cell production is sustained throughout the life of an organism by a population of self-renewing multipotent hematopoietic stem cells (HSCs). There are three hallmark characteristics of the HSCs: ability for self-renew, potential to differentiate into all hematopoietic lineages, and capacity for long-term reconstitution (LTR) of the hematopoietic system when transplanted into lethally irradiated recipients.

Vertebrate hematopoiesis is thought to occur in two successive waves, primitive and definitive, which differ in the cell types produced and their anatomic location. Whereas the primitive wave occurs transiently and produces only certain blood lineages, the definitive program continues throughout the whole lifespan and produces HSCs capable of giving rise to all blood lineages (rev. in Galloway et al., 2003).

Fig.1 Gastrulation in the mammalian embryo. (A) Human embryo and uterine connections during human gastrulation: sagital section (small box) and cutaway view looking down onto the dorsal surface. A single epithelial cell layer (the epiblast) is transformed into the three germ layers of the embryo (ectoderm, mesoderm, endoderm) and the basic body plan of the animal is established. (B) Surface ectoderm cells undergo an epithelial-mesenchymal transition, delaminate, and migrate through the primitive streak. Fate maps indicate that different mesodermal derivatives arise from specific regions along the posterior-anterior axis of the streak. A temporal pattern can also be recognized as, early in gastrulation, cells leaving the streak contribute mainly to the mesoderm of extraembryonic tissues: the yolk sac, amnion, and allantois.

Ingression of progenitors for the embryonic mesoderm begins around the midstreak stage, with the formation of progenitors that will give rise to lateral plate mesoderm (LPM) of the upper body as well as cardiac and cranial mesoderm.

These are followed, at the late-streak stage, by progenitors for paraxial and LPM of the trunk (rev. in Baron et al., 2005).

EM: extraembryonic mesoderm, ST: syncytiotrophoblast, A: amniotic cavity; YS: yolk sac; Epi: epiblast; Hypo: hypoblast; PS:

primitive streak.

1.1.1 Primitive hematopoiesis

Primitive hematopoiesis takes place in the so-called “blood islands” (BI) of the yolk sac (YS) and only generates a restricted range of blood cell types, including primitive red blood cells, granulocytes, and macrophages, to satisfy the immediate needs of the embryo (rev. in Hartenstein, 2006).

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Fig. 2 Primitive hematopoiesis in the vertebrate embryo. (A) Scheme of YS BI formation in the mouse embryo. (1) Early streak stage embryo. (II) Mid-late streak stage embryo. (III) Zoom in box in II. (IV) E8 (V). Zoom in box in IV. (B) Extraembryonic BI in the vertebrate embryo displayed by in situ hybridization (ISH) against Lmo2 (I, III, IV, V) or Scl (II) expression. (I) Zebrafish: 5-somite stage embryo (Davidson et al., 2004). (II) Frog: 26.25hpf Xenopus embryo (Walmsley et al., 2002). (III) Chick: 4-somite stage, HH 8 embryo, Bar: 1.5 mm (Minko et al., 2003). (IV and V) Mouse: 8-day postcoitum (dpc) late head-fold stage mouse embryo. (IV) Lateral view: YS-BI arise in a belt all around the embryo (black arrowheads).

Bar: 100 µm (Manaia et al., 2000). (V) Dorsal view. Bar: 100 µm (Manaia et al., 2000). Al: allantois, BI: blood islands, Ch:

chorion, Em: embryo, ICM: inner cell mass, NF: neural folds PS: primitive streak, PSp: para-aortic splanchnopleura, RBI:

rostral blood island, Sp: splanchnopleura, YS-yolk sac.

BI are extraembryonic mesoderm-derived structures; consequently it is only shortly after gastrulation when the first blood cells can be observed. In the mouse embryo, gastrulation begins around E6.5, as mesoderm cells ingress through the primitive streak to place themselves between the primitive ectoderm and visceral endoderm germ layers.

Mesoderm cells that exit the posterior primitive streak migrate proximally and appose visceral endoderm, extraembryonic ectoderm, and embryonic ectoderm to form the YS, the chorion, and the amnion, respectively (fig. 1, fig. 2). The extraembryonic mesoderm giving rise to BI is thus the earliest type of mesoderm laid down during gastrulation and is the one that will give rise to the YS (Kinder et al., 1999, Psychoyos et al., 1996, Schoenwolf et al., 1992).

The YS is a two-layer structure of mesoderm-derived and of visceral endoderm- derived cell layers. YS BI develop in the mesoderm-derived layer and consist of an outer layer of endothelial cells (ECs) surrounding a core of hematopoietic cells (HCs): at E7.5, in the YS mesoderm layer, solid aggregates of cells become apparent. Afterwards, most peripheral cells flatten and turn into ECs, while the inner cells progressively loose their connections, leading to the formation of a lumen, and will finally differentiate further into primitive erythrocytes, which are nucleated and six-fold larger than adult red cells. Strikingly,

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the first BI and the earliest vascular network form in an extraembryonic ring in the mouse embryo (fig.2, B.IV).

The close apposition of mesoderm and visceral endoderm in the gastrulating embryo and in the mature yolk sac (Baron et al., 2001, Farrington et al., 1997) suggests that interactions between primitive endoderm and mesoderm might play a role in the initiation of embryonic hematopoiesis and vasculogenesis. Though some in vitro approaches do not support this view (Palis et al., 1995, Bielinska et al., 1996, Belaoussoff et al., 1998, Dyer et al., 2001), results from in vitro and in vivo experiments prove the requirement of the endoderm for the emergence of hematopoietic progenitors (Wilt et al., 1965, Miura et al., 1969, Kessel et al., 1987, Gordon-Thomson et al., 1994, Pardanaud et al., 1999).

The vascular system develops concomitantly with YS BI , and once the heart starts to beat at the four- to six-somite-pair (sp) stage (approximately at E8.25-E8.5) vitelline circulation is established, connecting the dorsal aorta (DA), the YS and the heart (Downs et al., 1998). Accordingly, primitive erythrocytes will be distributed along the embryo and will remain in the circulation until E15.5-16.5, when hematopoiesis is sustained by fetal liver- derived non-nucleated erythrocytes (Steiner et al., 1973). It is worth mentioning that complete establishment of flow between the embryo and YS is not finalized until E10.0 (McGrath et al., 2003). The second major vascular route, the umbilical circuit, develops shortly after vitelline circulation is established. Umbilical vessels arise from the allantoic mesoderm after chorioallantoic fusion, and connect the DA to the placental labyrinth vascular network and subsequently to the fetal liver.

The YS BI of mammals and birds have their counterparts in other vertebrates, in the ventral BI (VBI) of amphibians (Durand et al., 2005) and in the inner cell mas (ICM) of teleost fish (rev. in Galloway et al., 2003) (fig. 2).

1.1.2 Definitive hematopoiesis

Definitive hematopoiesis results from the establishment of a new type of definitive HSC capable of long-term self-renewal and of differentiating into all blood cell lineages. The predominant anatomic site of hematopoiesis changes several times during vertebrate ontogeny, appearing sequentially at various intra- and extraembryonic locations (rev. in Galloway et al., 2003):

- in mammals: AGM (Aorta-Gonads-Mesonephros), YS, allantois, chorion, placenta, liver, spleen and bone marrow (BM);

- in birds: AGM, spleen, thymus, bursa of Fabricius, allantois and BM;

- in amphibians: DLP (dorsal lateral plate, AGM equivalent), liver and thymus;

- in teleost fishes: AGM equivalent and kidney.

Current knowledge on the emergence of definitive hematopoiesis is reviewed below, including comparison of this process in different species.

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Fig. 3 Definitive hematopoiesis. (A) Hematopoietic sites during mouse embryogenesis. When primitive hematopoiesis decreases, the AGM has started the production of LTR-HSCs that will subsequently appear in liver, YS and placenta by E11.

Spleen will be colonized at E14 and BM at E16. (B) AGM area in the mouse embryo. Sections at E8.5 (I), E9.5 (II) and E10.5 (III and IV). Splanchonpleural mesoderm will develop into a two-branched aorta, which will fuse in an only vessel by E10.5.

At each side of the vessel paired mesonephric and genital structures also develop. (V) Distribution of candidate hemogenic sites in the arteries at the AGM stage (boxed area in E10.5 whole mount). The DA, now a single unpaired vessel, forks into two branches at the level of the hind limb buds. These branches are the two umbilical arteries. The umbilical arteries fuse ventrally to the gut tube at the base of the umbilical ring to form an unpaired vessel. The vitelline artery branches off from the aorta as a conspicuous vessel in the middle region of the trunk. (C) HSC clusters in vertebrate species (rev. in Hartenstein, 2006). (I, II) Mouse embryo (Azcoitia et al., 2005) (III) Chick embryo (Jaffredo et al., 2005) (IV) Xenopus (Ciau- Uitz, 2000) (V) Zebrafish (Kalev-Zylinska et al., 2002). Ao, DA: Dorsal aorta; Ect: ectoderm; End: endoderm; FL: fetal liver;

G: gut; GR: genital ridge; LPM: lateral plate mesoderm; Mn: mesonephros; n, NT: neural tube; PGCs: primordial germ cells;

S: somite; SoP: somatopleura; SpP: splanchnopleura; Ua: umbilical artery; Va: vitelline artery.

1.1.2.1 Para-aortic splanchnopleura/AGM

The first functional definitive HSCs, capable of long-term repopulation of the adult BM, emerge in the early embryo from a region that surrounds the developing aorta in close association with the mesonephros, mesentery and gonads. This region called the AGM (Aorta-Gonads-Mesonephros) derives from the para-aortic splanchnopleura (PSp) (Cumano et al., 1996, Medvinsky and Dzierzak, 1996, Muller et al., 1994, de Bruijn et al., 2000). The AGM region is thought to give competence to, maintain, or expand HSCs (de Bruijn et al., 2000).

The lateral plate mesoderm (LPM) is continuous with the extraembryonic mesoderm.

The intraembryonic LPM (adjacent to the two bands of intermediate mesoderm) is subdivided by the coelomic cavity into two layers. The dorsal layer, called the somatic (parietal) mesoderm, underlies the ectoderm and, together with it, forms the somatopleura; the ventral

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layer, called the splanchnic (visceral) mesoderm, overlies the endoderm and, together with it, forms the splanchnopleura (Sp) (fig. 3B.I).

The AGM generally extends from the forelimbs to the hindlimbs of the E9.5-E12.5 mouse embryo. This region derives from the E8 Sp, where the first intraembryonic vessels, the paired DA, appear (Kaufman et al., 1992), and at this stage is therefore called para-aortic splanchnopleura. At E8.5, the aortas are linked to the YS vasculature through the vitelline artery (called omphalomesenteric inside the embryo trunk), and at E9 the paired aortas fuse to from a single midline DA (Garcia-Porrero et al., 1995). The umbilical artery forms the connection between the DA and the placenta (fig. 3).

Hematopoietic progenitors appear at two locations in the AGM: intra-aortic clusters in close association with the endothelium at the floor of the aorta, and sub-aortic patches. Intra- aortic clusters have been found in all vertebrate species studied so far (fig. 3C). HSCs appear as dense ‘mushroom-shaped’ clusters of cells budding from the floor of the aorta, and the vitelline and umbilical arteries (de Bruijn et al., 2000). The aortic endothelium associated with these clusters is referred to as ‘hemogenic endothelium’ since early l900’s (Jordan, 1916).

Sub-aortic patches have been described in murine and human embryos (Godin et al., 1999, Petrenko et al., 1999, Marshall et al., 1999 et al., 2000). A direct relationship between these sub-aortic patches and hematopoietic development is indicated by their location below the intra-aortic hematopoietic clusters for the whole duration of intra-embryonic HSC generation and by their disappearance when the AGM no longer produces hematopoietic precursors (Godin et al., 1999, Manaia et al., 2000). However, the function of these sub-aortic patches, if any, remains to be determined. In chick embryos, sub-aortic patches have also been described as an intermediate hematopoietic site (see below) (Dieterlen-Lièvre et al., 1981).

Fig. 4 Distribution of hemogenic potential within the AGM region. (A) Schematic drawing. Analysis of Runx1-lacZ targeted mouse embryos revealed that hematopoietic potential within the AGM is restricted to three discrete cell types:

ventral para-aortic mesenchymal cells, ECs from aortic floor (hemogenic endothelium), and small numbers of HCs in the aortic lumen (North et al., 2002). (B-F) Transverse sections of the AGM region from an E11.5 Runx1^lz/+ embryo (North et al., 2002) incubated with X-gal to visualize Runx1 expression (blue) and double-stained (brown) for (B) CD45 to detect hematopoietic cells, (D) CD31 (PECAM), (E) VEGF-R2 (Flk-1), or (F) VE-cadherin. Arrows indicate hematopoietic clusters. Ao: aortic lumen.

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Fig. 5 Hematopoiesis scheme.

LTR-HSCs give rise to multipotent precursors that divide actively and then generate commited precursors which in turn will differentiate into mature blood cells. In the mouse, a phenotypic profile of cell surface markers and transcription factors has been useful for the selection of LTR- HSC populations by cell sorting. All HSCs in the AGM are CD45⁺, Ly6A (Sca1)-GFP⁺, c-kit⁺ and CD34⁺ (rev. in Dzierzak et al., 2008). HSCs are included in the LSK (lineage^-, Sca-1⁺, c-Kit⁺) fraction of AGM, FL and BM cells. It should be noted that aortic hematopoietic clusters are not homogeneous (e.g. VE- cadh rev. in Dzierzak et al., 2008). Moreover, recently SLAM family receptors have been used to enrich for HSCs in cell sorts of BM samples (Kiel et al., 2005, Kim et al., 2006).

1.1.2.2 Other intraembryonic hematopoietic sites

Other secondary tissues, such as the kidney, liver and bone marrow, are thought to be reservoirs for the long-term maintenance of the adult hematopoietic system and are not considered to be the founding source of HSCs, since stem cell emergence and hematopoietic activity seem not to take place simultaneously in mouse intraembryonic sites (Godin et al., 1999).

The hematopoietic rudiments that become successively active during mid-gestation only provide the environment in which extrinsic HSCs are maintained and give rise to a differentiated progeny. When the activity of one site recedes, new migrants colonize the next site, presumably via the blood stream (Delassus et al., 1996), although this has not been definitively documented.

The fetal liver is the first rudiment to be colonised and predominates as the primary site of hematopoiesis from E12.0 through to birth. Seeding of the fetal liver in the mouse

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embryo begins at 10 dpc (Houssaint et al., 1981) and includes two exogenous sources: first, committed HPCs from the YS and, second, HSCs of presumed AGM origin.

The thymus becomes colonized at 11 dpc and is active by 12 dpc, whereas the spleen is colonized at 13 dpc and the BM at 15 dpc, and both become active sites of hematopoiesis prior to birth (E16 and E18.0, respectively) (rev. in Godin et al., 2005, Galloway et al., 2003).

In avian embryos, the liver is not a hematopoietic organ. Instead, para-aortic foci arise between E6 and E8 in the ventral mesenchyme of the aorta and display diffuse hematopoietic activity. In addition, thymus colonization starts by E7-E8.5 and follows a cyclic pattern until hatching. The bursa of Fabricius, which develops as an appendage of the cloaca and is the site of B-lymphocyte differentiation in birds, is colonized between E8-E14. Finally chick BM colonization occurs from E10 onwards (rev. Dieterlen-Lièvre et al., 2004). By the time of hatching, the BM has become the primary source of HCs, and it continues to produce blood cells throughout the life of the adult.

In Xenopus, as in mammalian embryos, two distinct waves of cells from the VBI and DLP colonize the liver, which will serve as the definitive site of hematopoiesis at both larval and adult stages. At 26 days of development, a substantial amount of the hematopoietic activity in the liver is the result of the differentiation of stem cells derived from the DLP stem cell source (Chen et al., 1995). During metamorphosis at 35 dpf, DLP-derived erythropoiesis decreases and is surpassed by VBI-derived erythropoiesis. In addition, definitive HCs originating from the VBI undergo lymphopoiesis in the thymus, and this activity reaches a peak at 35 dpf. It is not until 42 dpf that DLP-derived definitive red blood cells enter the circulation. Thymus and spleen also become hematopoietic at this stage (rev. in Ciau-Uitz et al., 2006, Galloway et al., 2003).

In the zebrafish embryo, colonization of the thymus begins by 3 dpf, and by 4 dpf that of the pancreas and the kidney. Recently a new transitional niche was identified: the caudal hematopoietic tissue (CHT), which is colonized by 3 dpf (Murayama et al., 2006).

1.1.2.3 Extraembryonic hematopoietic sites

In the midgestation mouse embryo the placenta and YS serve as sites of definitive HSC expansion at E11-13 (Kumaravelu et al., 2002, Gekas et al., 2005, Ottersbach et al., 2005); and chorion and allantois (which fuse to form the chorio-allantoic placenta), have been shown also to have hematopoietic potential (Zeigler et al., 2006, Corbel et al., 2007). Murine placenta contains hematopoietic stem cells within the vascular labyrinth region (Gekas et al., 2005, Ottersbach et al., 2005). The placental labyrinth is composed of maternal blood sinus and fetal capillaries, thus serving as a site for the exchange of oxygen and nutrients. Starting at E10.5-11, there is a parallel appearance of HSCs in both the placenta and the AGM region.

The expansion of the HSC pool size in placenta is much greater (>15-fold) than that in AGM.

Furthermore, the expansion of the HSC pool size in placenta occurs prior to and during the expansion of the HSC pool in the fetal liver but declines toward the end of gestation (E15), presumably reflecting mobilization of placental HSCs to colonize the fetal liver. Therefore,

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the placental HSCs appear to be the more potent resource, compared to the yolk sac and AGM regions, for expansion of the HSC pool in the fetal liver.

In the chick embryo, an additional site of HSC production is the allantois: the allantoic bud retrieved from the quail at a prevascular stage and grafted heterotypically in the chick coelom produces progenitors that colonize the host BM, giving rise to both HCs and ECs (Caprioli et al., 1998, 2001).

1.1.3 Ontogeny of definitive HSCs

As outlined above, definitive HSCs appear sequentially at different embryonic and extraembryonic structures; however, the ontogenic relationships between these HSC niches and their relationship with primitive hematopoietic precursors is not completely understood and remains a matter of controversy (rev. in Dzierzak et al., 2008, Yoshimoto et al., 2008).

Till and McCulloch (1961) showed that the hematopoietic system of an irradiated mouse could be reconstituted by intravenous injection of bone marrow cells from a healthy mouse. This experiment uncovered the existence of pluripotent, self-renewable HSC and established a standard test for the presence of LTR-HSC in a cell sample. Nowadays, this test is the most stringent assay for identifying HSCs.

A set of experiments performed in the 60s and 70s put forth the hypothesis that all HSCs originate from the YS. Studies using twin and parabiosed (joined by their circulation) chick embryos demonstrated that there is an extensive traffic of HCs within the chick embryo and that the hematopoietic organs are mostly populated by blood-borne cells (Moore et al., 1965). Furthermore, injection of a 7-day chick YS cell suspension into irradiated 13-day-old chick embryos repopulated the spleen and bone marrow (Moore et al., 1967). In mouse embryo culture experiments, a 7 dpc embryo grown in vitro without YS developed normally, but lacked HCs (Moore et al., 1970). Further support for this view came from the observation that hematopoietic colonies could be derived from the yolk sac starting from 7.5 dpc, but could only be derived from the embryo proper two days later, well after the establishment of circulation. These results were also supported by transplantation experiments in which YS cells isolated from 8 to 10 dpc embryos were transplanted into the YS cavity of isochronic hosts; these experiments detected donor derived thymocytes, albeit at low frequency, in the recipient mice (Weissmann et al., 1978).

In striking contrast, similar transplantation experiments performed in the avian embryo before the onset of circulation showed that stem cells for the definitive blood cell series do not originate from the YS BI and instead must arise within the embryo proper (Dieterlen-Lièvre et al., 1975, Lassila et al. 1978, 1982). These intra-embryonic HSCs were probably located in a region neighbouring the DA, according to histological and in vitro analysis (Dieterlen-Lièvre et al., 1981).

Transplantation experiments in frogs showed that their embryonic blood cells have a different source than their adult blood cells (Chen et al., 1995). Whether those two sources ultimately are formed from a common precursor is still a matter of debate (Walmsley et al.,

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2002, Lane et al., 2002). Lineage tracing experiments in Xenopus and Danio rerio have established clearly that primitive and definitive hematopoiesis have a different clonal origin (Ciau-Uitz et al., 2000, Kimmel et al., 1990, Warga et al., 1999). However, these are examples of anamniote species.

Several reports published in the 1990s pointed to an identical situation for the mouse embryo. In vivo studies of colony-forming-units spleen (CFU-S) showed that the AGM region contains a higher frequency of CFU-S activity than the YS and that the morphology of AGM- derived colonies closely resembles that of FL-derived colonies (Medvinsky et al., 1993, 1996).

In parallel, Godin et al. (1993) dissected the Sp region from day 8.5-9dpc embryos (10-18sp) and grafted it under the kidney capsule of SCID mice, achieving lymphoid cell reconstitution in those mice. However, grafts were obtained from embryos shortly after completion of vascular circulation, which occurs at the 7-somite stage, and they might have been contaminated by progenitors from the YS BI.

Transplantation of cells from various regions of the E8-E12 mouse conceptus showed that HSCs conferring complete, long-term, multilineage, substantial hematopoietic repopulation of irradiated adult recipient mice first appear only at E10.5 in the AGM region of the embryo body and in the vitelline and umbilical arteries (Muller et al., 1994). Shortly after this, HSC activity also appears in the 11dpc liver and YS, but always at a lower frequency than in the AGM region, suggesting that LTR-HSCs appearing in the YS and in other tissues of the embryo at 11 dpc are the result of dissemination of LTR-HSCs from the AGM region.

In addition, proof that HSCs initiate autonomously and exclusively in the AGM region was obtained by means of an in vitro 10 dpc-AGM organ culture system followed by CFU-S assay (Medvinsky and Dzierzak, 1996).

At the same time, Runx1 transcription factor (see below) was found to be essential for the formation of intra-aortic hematopoietic clusters (Okuda et al., 1996, North et al., 1999).

Parallel transplantation experiments in birds demonstrated that the aortic endothelium has a dual origin –roof and sides being contributed by somite-derived ECs and floor by splanchnopleura-derived ECs (Pardanaud et al., 1996). Thus, Runx1 may specifically mark ECs and HCs derived from putative hemangioblasts in the splanchnopleural mesoderm.

Most of these experiments, however, do not address the lineage relationship between YS and embryo. It is possible that embryonic definitive HSCs might derive from YS precursors that need to mature into functional HSCs in the AGM region. Experiments that assess the potential of explanted tissues before the onset of circulation are of limited significance because, first the in vitro environment might alter the potential of the cells and, second, they would only be meaningful regarding HSC ontogeny if it is assumed that the circulation is the only way for HSCs to migrate, which has not been formally proven.

Indeed, several studies have suggested a contribution of the YS to definitive HSCs.

For instance, the E8-9 murine YS harbours HSCs that engraft in embryonic or fetal mice and then contribute to adult hematopoiesis (Weissman et al., 1978, Toles et al., 1989). Yoder

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(2001) argues that the inability to demonstrate engraftment of YS cells isolated earlier than E11 in lethally irradiated adult mice may be due to a failure of the YS HSCs to home and engraft in an adult microenvironment, hence transplantation models where the recipient subjects more closely mirror the stage of development of the donor cells should be employed.

Based on the observation that the liver of the newborn mouse continues as an active hematopoietic organ for 1 to 2 weeks after birth, sublethally myeloablated newborn mice were transplanted with E9 or E10 YS cells (Wolf et al., 1995, Harrison et al., 1997, Ema et al., 2000). In multiple experiments, YS cells were found to give rise to all blood cell lineages over the long-term (more than 11 months), and marrow from the primary E9 and E10 YS-engrafted recipients also reconstituted B- and T-lymphocyte, granulocyte, and erythroid lineages in secondary lethally irradiated adult recipient mice (Yoder and Kiatt, 1997, Yoder et al., 1997, Yoder, Hiatt and Mukherjee, 1997). These results suggested that YS HSCs capable of repopulating lymphoid and myeloid lineages in adult animals were present prior to E11 but were not identifiable upon direct transplantation into adult recipients. In other words, the murine YS may in fact contain cells with LTR-HSC potential, but their ability to repopulate a host would depend on a supportive environment determined by the age of the recipient. Since the liver normally serves as the site for YS and PSp/AGM HPCs maturation, YS cells have the ability to engraft in newborn recipient mice.

Accordingly, precursors for adult repopulating HSCs were found in both YS and PSp regions at day 8.0 and 8.5, when YS and PSp cells were isolated from the precirculation embryo and cocultured for 4 days on an AGM-derived stromal line (Matsuoka et al., 2001). It is noteworthy, however, that these experiments only address the potential of different cell populations when removed from their normal environment and exposed to a challenging test, but they do not address the actual contribution of these populations during ontogeny in vivo.

Taking into account this possible YS origin of HSCs, three general models describing the origin of HSCs in the fetal liver have been put forward (fig. 6).

In fact, while most reports identify the AGM as the primary source of pluripotential HSCs, none specifies their origin. In other words, the cell lineage history of HSCs from gastrulation to the AGM remains unclear. Furthermore, the lineage relationship between the AGM HSCs and the subsequent HSC populations in liver, YS, placenta, etc. remains undemonstrated. There are two possibilities:

- There is a single event of determination to hematopoietic fate within the conceptus.

There may be, at an undetermined site, a single or small cohort of hematopoietically fated cells (pre-LTR-HSC) with multilineage potential that for some reason cannot repopulate adult recipients to high levels. Various events, such as a progressive increase in expansion potential, maturation to an adult phenotype, or both, may occur in the AGM region to generate high level LTR-HSC activity. Within the YS microenvironment, such cells would differentiate to primitive erythrocytes.

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- Alternatively, hematopoiesis might arise many times within the embryo, from precursors that are fated to the hematopoietic lineage but which have varying hematopoietic potential. These cells could emerge independently of each other, from several cohorts within spatially distinct areas of the embryo. Intraembryonic HSCs might arise from precursor mesoderm cells that migrate through the primitive streak to reach para-aortic sites.

Fig. 6 Models of HSC-colonization of fetal liver. (A) YS and AGM HSCs arise independently, whereupon they both colonize the fetal liver and produce definitive HCs.

(B) All hematopoietic stem cells are derived from the YS.

The AGM cells provide the correct environmental cues to enable YS cells to mature into HSCs and contribute to adult hematopoiesis. (C) AGM is the sole source of HSCs, and any definitive activity seen in the YS is derived from AGM cells that travel through the circulation to the YS.

1.1.3.1 Hemangioblast and hemogenic endothelium

The YS was the focus of attention for many years and was thought to be the source of adult hematopoiesis because it is the earliest tissue in the embryo that shows hematopoietic activity. Early in the last century, Sabin described the close spatial and temporal relationship between blood cells and vascular cells when emerging in the chick YS BI (Sabin, 1920). In 1932, Murray coined the term “hemangioblast” to refer to the components of the cell aggregates appearing in YS and precursors of BI. According to Murray, the primitive streak contains mesenchymal cells strongly biased towards the development of hemangioblasts, which in turn give rise to ECs and red blood cells (Murray, 1932).

The existence of such hemangioblast understood as a common precursor for both endothelial and hematopoietic lineages is still controversial. Evidences in favour are: (1) a common timing for HC and EC generation: the induction and generation of HCs in the three hematopoietic sites, yolk sac, AGM, and placenta coincides with that of ECs, (2) a collection of markers shared by both cell types: coexpression of EC markers CD31/CD34 and hematopoietic stem cell markers Kit/CD34 (and Sca-1, partially) is found in the HCs in the YS (Yoder et al., 1997), AGM (North et al., 2002), and placenta (Ottersbach et al., 2005), (3) several gene mutations and deletions in zebrafish and mouse embryos that affect both cell types (Flk-1, Shh –Chung et al., 2002, Gering et al., 2005), and (4) in vitro clonal approaches in which a single VEGF-R2-positive progenitor cell gives rise to both ECs and blood cells (Eichmann et al., 1997, Choi et al., 1998, Fraser et al., 2002, Huber, 2004, Furuta et al., 2006). However, individual hemangioblast progenitor contribution to both endothelial and hematopoietic lineages within a single BI is a rare event, as demonstrated by an in vivo lineage experiment undertaken in the mouse embryo (Ueno et al., 2006).

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Another example of the close relationship between HCs and ECs is the previously introduced concept of ‘hemogenic endothelium’(fig. 7). Transplantation experiments and in vivo tracing of ECs in avian embryos have clearly proved the lineage relationship between ECs and nascent intra-aortic clusters and para-aortic foci (Pardanaud et al., 1999, Jaffredo et al., 1998, 2000). As hematopoiesis proceeds, the hemogenic endothelium (made of splanchnopleura-born ECs) disappears from the aortic floor and is replaced by somitic ECs (Pouget et al., 2006). Thus, the aortic hemogenic floor appears as a transitory structure spent out and replaced.

In the mouse embryo, in vitro approaches also favor the hypothesis that hemogenesis requires an endothelial transition: a VE-cadherin⁺ single-EC isolated from a E9.5 mouse embryo is able to give rise to both ECs and blood cells (Nishikawa et al., 1998); moreover, the phenotypic (morphologic and genetic) changes undergone by ECs when giving rise to HCs have been described accurately using VEGF-R2⁺, E-cadherin^-endothelial populations derived from ESC cultures (Eilken et al., 2009, Lancrin et al., 2009).

The use of transgenic mice supports too the hemogenic endothelium alternative: A transgenic mice carrying the GFP gene under the control of the Ly6-A (Sca1) promoter showed that definitive HSCs appear in the endothelial layer (de Bruijn et al., 2002). Also, Runx1 function in hematopoiesis was dissected by ablating that gene using Cre-recombinase- mediated excision: use of the Tie2 promoter and enhancer to control Cre expression revealed that Runx1 function is required in Tie2-expressing cells (Li et al., 2006), whereas use of the VE-cadherin promoter showed that Runx1 is required for EC to HC transition (Chen et al., 2009).

In vivo lineage tracing attempts to label or to genetically modify aortic floor are scarce because the regulatory sequences controlling the expression of HSC-specific TFs (transcription factors) are not fully understood (rev. in Yoshimoto et al., 2008). However, two recent publications use transgenic animals carrying an inducible Cre gene crossed with a ROSA26R Cre reporter line. In the first one, inducible Cre gene was under the control of a 2.5-kb fragment of the VE-cadherin mouse promoter (Zovein et al., 2008): a short pulse of tamoxifen, delivered at the stage of AGM hemogenesis, resulted in abundant labeled HCs in the fetal liver, the bone marrow, and the thymus as development progressed. In the second one, inducible Cre gene was under the control the proximal P2 element of Runx1 promoter (Samokhvalov et al. et al., 2007): a single pulse of tamoxifen given at E7.5 (when Runx1 expression is restricted to YS BI) resulted in labeling of a large amount of the HSC fraction in adult BM. Those are key contributions, since the first one proves the contribution of ECs to adult hematopoiesis and the second does the same with yolk sac BIs. However, half-life of tamoxifen could be disturbing the results.

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Fig. 7 Models of intraembryonic hematopoietic origin. (A-D) Hemogenic endothelium: HSC clusters emerge from the endothelium, bulging towards the lumen and also ingressing in the subjacent mesenchyme. (E-F) PSp mesoderm hemangioblast: both endothelial and HCs emerge form a mesodermic hemangioblast. (G-H) Undifferentiated mesoderm:

HCs are generated in mesodermic foci and migrate into the arterial lumen.

1.2 Gene regulatory networks in embryonic hematopoiesis

HSC production from embryonic mesoderm is controlled by both intrinsic and extrinsic cues. Intrinsic cues consist mainly of transcriptional regulatory networks, sometimes called genetic regulatory networks (GRNs). GRNs consist of the interactions between TFs and extracellular signals that promote the specification and properties of a given cell type or tissue, through the tight control of the spatial and temporal aspects of gene expression (Loose et al., 2007). Although not fully understood, the GRNs for the ontogeny of HSCs are evolutionarily well conserved among vertebrates (Hsia et al., 2005, Pimanda et al., 2007, Liu et al., 2008). Extrinsic cues are represented mainly by endodermal inductive signals and general ventralizing embryonic signals.

1.2.1 Transcription factor signature of nascent HSC population The main players in these GRNs are Scl (Tal-1), Gata1, Gata2, Lmo2, Fli1, c-myb, PU.1 (Sfpi1) and Runx1 (Aml1). Those are genes known to be implicated in the induction of leukemias and other tumors and whose deletion impairs primitive hematopoiesis, definitive hematopoiesis or mainly both (table 1).

1.2.2 Extrinsic signals involved in HSC specification

In Danio rerio and Xenopus, establishment of the ventral mesoderm results in the start of the primitive hematopoietic wave. Anamniotes AGM counterpart will arise from the LPM.

Patterning of LPM depends on transforming-growth factor beta (TGF) and bone morphogenic proteins (BMPs) signalling pathways (de Robertis et al., 2000, He et al., 2005), for instance, BMP4 has been implicated in Fli1 expression in the definitive lineage in Xenopus. In addition, recent data suggest that FGF signalling pathway may act to regulate the

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expression of Scl and Gata1 in Xenopus and the chick, respectively (rev. in Loose et al., 2007).

In mouse, commitment of mesodermal progenitors to the hematopoietic and endothelial lineages begins within or shortly after these cells emerge from the primitive streak (rev. in Baron et al., 2005). Different mesodermal derivatives form within distinct posterior- anterior regions of the streak. Little is known about the cellular and molecular mechanisms by which nascent mesoderm is allocated to its various lineages: signals from the visceral endoderm regulate the formation and positioning of the streak, and the specification and differentiation of early hematopoietic and endothelial progenitors. Hedgehog (Ihh-indian hedgehog), BMPs and Wnt signalling pathways seem implicated in specifying posterior streak epiblast to the hematopoietic and endothelial fates (rev. in Baron et al., 2005). VEGF-A is also secreted by visceral endoderm and may function as a chemoattractant for nascent mesoderm cells migrating out of the primitive streak (Damert et al., 2002). Moreover, BMPs and Notch have been recognized upstream activators of Runx1 (Pimanda et al., 2007, Robert- Moreno et al., 2005, Nakagawa et al., 2006).

With respect to the specific inductive events that commit mesoderm to the hematopoietic fate in 2 very different tissue microenvironments (YS and PSp), it is known that the Brachyury⁺ cells that contribute to the hematopoietic lineages initiate expression of the receptor tyrosine kinase Flk-1. Subsequent Scl expression in this population marks the establishment of the hematopoietic and endothelial lineages. Later, Gata-1 expression marks the onset of hematopoietic commitment, from which primitive hematopoiesis and the initial wave of definitive erythropoiesis arises. Establishing a positive regulatory loop between Flk-1 and Scl might be more important than the order in which these two genes are expressed (rev.

in Loose et al., 2007).

1.3. Homeodomain transcription factors in normal and leukemic hematopoiesis

Homeodomain TFs take part in both regulation of normal hema-topoiesis and generation of hematological diseases such as cytopenias and leukemias. Recently, members of this family were found to be involved in definitive HSC generation.

Homeodomain genes constitute a big family of TFs whose common feature is a conserved sequence of 183 nucleotides, the homeobox, which encodes a 61-aminoacid (aa) helix-turn-helix type of DNA-binding motif, the homeodomain (HD) (Gehring et al., 1986, Scott et al., 1989), in which the third helix is the recognition one. They were first described in arthropods, when a mutation in Drosophila induced the so-called homeotic transformation of fly halteres into wings, generating a fly with four wings, (Bridges et al., 1923, Lewis, 1978).

Different homeobox gene families have evolved that encode HDs of different types or classes.

Among these HDs, the Drosophila Antennapeaedia (Antp) defines one consensus sequence referred to as class I HDs. Class I homeobox genes (in mammals, Hox genes) are essential in the genetic control of the body plan development of all animals (fig. 8). Perturbations in the

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anterior expression boundaries of Hox genes result in changes in cell fate, and this has led to the ‘Hox code’ hypothesis, in which specific combinations of Hox genes are believed to specify tissue identities along the AP axis (Krumlauf, 1994, Lumsden et al., 1996, Capecchi et al., 1997).

Hox proteins bind target DNA sites in vitro with little obvious selectivity (Hoey et al., 1988, Ekker et al., 1994, Mann et al., 1995), actually, their DNA-binding specificity is modified through interactions with other DNA-binding proteins, which act as cofactors (Mann et al. 1995, 1996, 1998).

Fig. 8 Hox genes. Comparison of the Hox complex in an insect and in mammals: In vertebrates, there are 39 Hox genes that are grouped in four clusters (Hox A, -B, -C and –D) mapping to human chromosomes 7, 17, 12 and 2, respectively (chromosomes 6, 11, 15 and 2 in mice), and presumably evolved by quadruplication of an ancestral gene cluster. According to the primary sequence of the homeobox, 13 paralogous goups are defined. The order of the homeotic genes on the chromosome is colinear with the patters of expression in the body, with the most proximal gene having the most anterior limit of expression. This colinearity is a property associated with the ability of these genes to regulate regional identity.

(Alberts B et al., 2002)

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Table I Main members of the GRNs for the ontogeny of HSCs.

GENE FAMILY EXPRESSION PATTERN MUTANT PHENOTYPE INTERACTION PARTNERS REGULATORS AND TARGET

GENES REFERENCES

ScL/Tal1 Basic helix- loop-helix

- Mesodermal expression of Scl restricts cells to endothelial or hematopoietic fates.

- In the adult, functions in

erythropoiesis and megakaryopoiesis.

- Not required for the maintenance of established LTR-HSCs in the adult.

- 9.5-10.5dpc lethality in mouse.

- Severe defects in primitive and definitive blood formation, and in vasculogenesis.

- Not recapitulated in Fli1, Elf1 or Gata2 k.o.

- Mutant ES cells unable to contribute to any hematopoietic lineages without affecting angioblast specification.

- Lmo2 and Gata2 in a multimeric complex.

- Members of the E-protein family (E2A) in dimers to bind DNA.

- DNA binding-domain dispensable for hematopoietic and vascular development.

- Cross -regulatory interactions among Scl, Lmo2 and Hhex.

- c-kitis a downstream target of Scl in vitro.

- GATA motif in its promoter.

- Runx genes are direct targets of Scl/Tal1 in the yolk sac and fetal liver.

Aplan, 1990;

Robb, 1995;

Shivdasani, 1995;

Green, 1996;

Gering, 1998, 2003; Krosl,1998;

Porcher, 1999;

Liao, 2000;

Schlaeger, 2005;

Brunet de la Grange, 2006;

Gottgens, 2002;

Patterson, 2007;

Landry, 2008

Lmo2/

Rbtn2

LIM zinc- finger

- Early role in specification of hemangioblast.

- Commitment into the Ery/Mk lineages.

- Regulates angiogenesis but not vasculogenesis.

- 10.5dpc lethality in mouse.

Severe defects in primitive and definitive blood formation, and in vasculogenesis.

- Similar to Scl and Gata1 mutants.

- Bridging molecule in a Scl, Ldb1, E2A, Gata1/2 complex: Gata2 in HSCs, Gata1 in Ery/Mk lineage decision.

- No autoregulation or feedback control.

Wadman, 1997;

Vyas 1999;

Warren, 1994:

Gering, 2003

Gata1

WGATAR binding zinc-finger

- Erythroid and mast cells differentiation and megakaryocyte development.

- 11.5dpc lethality in mouse.

- 9.5dpc: extreme pallor: embryonic erythroid cells arrested at an early proerythroblast-like stage.

- Scl/Tal1, Lmo2, Ldb1, E2A complex at positively acting Gata-1- bound elements.

- At sites where Gata-1 functions as a repressor, the Scl complex is depleted.

- Positively regulates c-Myb in the absence of Fog1.

- Notch1 inhibits the development of Ery/Mk cells by suppressing Gata1 activity through Hes1

Pevny, 1991, 1995; Fujiwara, 1996; Migliaccio, 2005; Ishiko, 2005; Tripic, 2009

Gata2

- Expression in PSp and AGM region.

- Expression in hematopoietic and endothelial lineages.

- Control of proliferation rate and growth factor responses of early HPCs.

- Role in HSC proliferation in adult BM.

- Differentiation of the Ery lineage.

- 11.5 dpc embryonic lethality in mouse.

- Anemia, defects in HSC expansion and in angiogenic factors secretion by the placenta.

- Gata1^-/-;Gata2^-/- double mutant embryos show almost complete failure in YS erythropoiesis.

- Forced activity inhibits differentiation and enhances proliferation of HPCs.

- Gata2, Fli1 and Elf1 are key components of an enhanceosome responsible for activating Scl transcription.

- Maybe c-Myb, as Gata2 and Gata1 bind to the same sites.

- Gata2, Fli1, and Scl form a recursively wired gene-regulatory circuit during early

hematopoietic development.

Orkin, 1992; Tsai, 1994; Minegishi, 1999;Gottgens, 2002; Ling, 2004;

Fujiwara, 2004;

Pimanda, 2007

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GENE FAMILY EXPRESSION PATTERN MUTANT PHENOTYPE INTERACTION PARTNERS REGULATORS AND TARGET GENES

REFERENCES

Gata3

- Pluripotent HSCs and possibly hemangioblast.

- Subaortic patches.

- 11-12 dpc embryonic lethality in mouse.

- Massive internal bleeding and markedly suppressed fetal liver hematopoiesis.

- Normal YS hematopoiesis and fetal liver contain erythroid precursors and normal megakaryocytes.

- Tal1 and LIM-only proteins are Gata3 cofactors in T-cell ALL..

Pandolfi, 1995;

Ono, 1998;

Manaia, 2000

Fli1 ETS - One of the earliest markers of hemangioblast formation.

- 11.5 dpc embryonic lethality in mouse.

- Impaired hematopoiesis and haemorrhaging.

- In Xenopus, substantial reduction or absence of hemangioblast.

- Overexpression in mouse leads to erythroleukemia, suggesting stimulation of proliferation or inhibition of differentiation of HPCs.

- A constitutively active form of Fli1 is sufficient to induce expression of Scl/Tal1, Lmo2, Gata2, Etsrp, and Flk1.

Ben-David, 1991;

Hart, 2000;

Spyropoulos, 2000;

Liu, 2008

Runx1/

Aml1

Core binding factor

- Mesoderm masses and endoderm of the prospective YS BI at the neural plate stage.

- Mesoderm of the distal allantois and chorion at head fold stage.

- ECs in the distal allantois, DA and vitelline and umbilical arteries starting the 4-6 sp stage.

- At 9.5-10.5 dpc, both endothelial and mesenchymal cells in the DA and placental labyrinth.

- 11.5-12.5 dpc lethality

- Haemorrhaging, impaired definitive erythropoiesis and myelopoiesis, lack of intra-aortic hematopoietic clusters - Runx1 functions in vasculature formation, primitive blood maturation, and definitive hematopoietic

development seeming a molecular switch that regulates hemangioblast differentiation towards HSC commitment.

- Component of the alfa subunit of a heterodimeric core-binding factor transcriptional complex

- BMP and Notch positively regulate the expression of Runx1.

- Autoregulatory loop in the Runx1 promoter.

Levanon, 1994;

Chen, 2009;

North, 1999;

Okuda, 1996;

Wang, 1996;

Kalev-Zylinska, 2002; Burns, 2002; Ichikawa, 2004; Robert- Moreno, 2005;

Pimanda, 2007;

Nakagawa, 2006

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GENE FAMILY EXPRESSION PATTERN MUTANT PHENOTYPE INTERACTION PARTNERS REGULATORS AND TARGET

GENES

REFERENCES

cMyb Zinc-finger

- Marks the initial developing population of HSCs found in the ventral wall of the DA.

- High in proliferating multipotent progenitor cells and declines as these cells differentiate, with no expression detectable in mature erythroid cells.

- Indispensable for primitive erythropoiesis and megakaryopoiesis.

- 15dpc lethality in mouse

- Severe anemia due to impaired adult erythropoiesis without perturbation on primitive erythropoiesis.

- With p300 controls the proliferation and differentiation of HSCs.

- Indispensable target for homeobox-genes induced hematopoietic cell transformation.

Beug, 1982;

Vandenbunder, 1989;

Mukouyama, 1999, 2000;

Lecuyer, 2002;

Mucenski, 1991;

Sandberg, 2005;

Hess, 2006;

Tober, 2008

PU.1/

Sfpi1 ETS

- Widely expressed in pluripotent HSCs and in precursors of the lymphoid and myeloid lineages.

- Maintenance of the HSC pool in BM.

- Directs the entrance of HSCs into the lymphoid-myeloid pathways.

- 18.5dpc- birth lethality in mouse - Loss of B cells.

- May regulate the maintenance or function of LTR-HSCs or primitive HPCs in the fetal liver.

- Inhibits the erythroid program by binding to Gata1 on its target genes.

- Downstream target of Runx1.

Moreau-gachelin, 1988, 1996; Scott, 1994; McKercher, 1996; Kim, 2004;

Iwasaki, 2005;

Nutt, 2005;

Stopka, 2005

Hhex HD - First identified as a hematopoietically expressed homeobox gene

- A hhex deficiency allele shows that it is sufficient but not essential for blood and endothelial development, suggesting that another gene, such as scl, may compensate for loss of hhex.

- Ectopic expression leads to premature expression of early blood and

endothelial differentiation.

Crompton,1992;

Bedford, 1993;

Yatskievych, 1999

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1.3.1 TALE homeodomain transcription factors

This family of TFs contain a divergent HD (Bertolino et al., 1995) containing a 3 amino-acid loop extension (TALE) between -helix 1 and -helix 2 (fig. 9). In vertebrates, four TALE groups have been identified so far:

- the Meinox family: Meis1, 2 and 3 and Prep 1 and 2 (from PBX REgulatory Protein), - the PBC family: Pbx 1, 2, 3 and 4 proteins (from Pre-B cell leukemia),

- TGIF (5'TG3' interacting factor), and - IRO (Iroquois cluster genes).

The Meinox and PBC TALE homeodomain proteins cooperate as cofactors with Hox proteins (fig. 10). By physically interacting with Hox proteins, they increase DNA-binding affinity and specificity of the latter and modify their transregulatory properties (rev. in Moens et al., 2006). Mostly, Hox proteins from paralogous groups 1-10 (3’) interact physically with PBC proteins, while those from paralogous groups 9-13 (5’) interact with Meis/Prep proteins (Shen et al., 1997). Meis-Pbx dimerization as well as that of their insect orthologs (Hth and Exd), is required for nuclear localization of PBC class proteins, and Meis-Pbx interaction is thus considered to be essential for their function (Berthelsen et al., 1999, Abu-shaar et al., 1999, Pai et al., 1998, Rieckhof et al., 1997).

Fig. 9 Structure of TALE HD TFs family. (A) TALE HD structure. TALE proteins share a three-amino-acid loop extension (*) proline (P)– tyrosine (Y)– proline (P) in positions 24– 26 between helix 1 and helix 2 of their HD (Burglin et al., 1997, 1998). (B) Prototype members of Meinox, PBC, Iro and TGIF classes. Meis/Prep member share an N-terminal conserved domain, termed MH in Meis and HR in Prep proteins (Burglin et al., 1997, Mann et al., 1998). The PBC class bears an N-terminal domain termed PBC (which can be subdivided into domains PBC-A and PBC-B). In addition, some isoforms of PBC proteins include a transactivation domain (T). Iro proteins differ from the rest of TALE members by a C-terminal specific Iro-domain. Finally, the TGIF (5'TG3' interacting factor) subclass contains several repressor domains (R). (C) Schematic representation of the Meis1 and Meis2 isoforms.

Meis1 encodes two splicing variants, Meis1a and Meis1b that differ in their C-termini, as Meis1b contains an additional stretch of 75 aa (dark orange). Meis2 also encodes isoforms that differ in their length and, in addition, can contain a 7 aa insertion (cyan) downstream of the homeobox. Meis2e is a truncated isoform containing a stop codon upstream the homeobox. Recently, various HD-less isoforms encoded by Prep2, Meis1 (and its fly ortholog Hth) were described (Haller et al., 2004, Noro et al., 2006).

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1.3.2 Hox, Meis and Pbx HD transcription factors in normal and leukemic hematopoiesis

Hox gene expression in HCs was first demonstrated using immortalised cell lines of human and murine origin (Lonai et al., 1987, Kongsuwan et al., 1988, Shen et al., 1989, Lowney et al., 1991, Magli et al., 1991, Mathews et al., 1991, Vieille-Grosjean et al., 1992).

These studies suggested that Hox genes were expressed in a lineage-restricted manner, with Hoxa expression being associated with myeloid cells, Hoxb expression with erythroid cells and Hoxc expression with lymphoid cells. Hoxd genes seemed not expressed in the hematopoietic compartment. Later, gain-of-function models showed that Hox genes are powerful regulators of normal and aberrant hematopoietic system development (Kroon et al., 1998, Sauvageau et al., 1995, 1997, Thorsteinsdottir et al., 1997). The current view is that, although multiple proteins may have similar or overlapping effects, each protein has a specific function and regulatory combinations play a critical role in controlling HC processes, for instance hematopoietic lineage decisions (rev. in Lawrence et al., 1996, Chiba et al., 1998, van Oostveen et al., 1999, Owens et al., 2002, Abramovich et al., 2005, Argiropoulos et al., 2007).

Meis1 was described as a viral insertion site implicated in leukemias of myeloid origin in the BXH-2 mouse strain (Moskow et al., 1995) (fig. 11), while Pbx1 was initially discovered as part of a fusion protein resulting from the t(1,19) chromosomal translocation in human pre-B cell leukemias. This fusion protein with transforming ability involves the HD and the Hox protein interaction motif of Pbx and the activation domain of E2A (Kamps et al., 1990, Monica et al., 1994, Chang et al., 1997).

The majority of Hox genes of the a, b and c clusters are preferentially expressed in HSC-enriched subpopulations and in immature progenitor compartments and they are down- regulated during differentiation and maturation, as revealed by gene expression analysis of mouse and human BM samples and of murine FL samples (Giampaolo et al., 1994, Moretti et al., 1994, Sauvageau et al., 1994, Kawagoe et al., 1999, Pineault et al., 2002). Thus, Hox genes might potentially regulate HSC self-renewal and/or differentiation both directly or indirectly.

In agreement with that expression pattern, progenitors and HCs that appear in differentiating embryoid bodies up-regulate multiple Hox genes along with Meis1 and Pbx1.

Consistent with this model, Pbx1 and Meis1 are also expressed at their highest levels in the most primitive subpopulations in both BM and FL, as observed with Hox genes. In contrast to Pbx1, the expression pattern of Meis1 closely matches that found for Hox genes in BM, FL, and differentiating ES cells. Thus, Hox gene functions in hematopoiesis might be modulated at different stages by concurrent expression with Meis1 or Pbx1 or with both (Pineault et al., 2002).