Specification of heart tissue and fusion of heart rudiments

In amniote vertebrates, the embryo is a flattened disc, and the lateral plate mesoderm does not completely encircle the yolk sac. The presumptive heart cells originate in the early primitive streak, just posterior to Hensen's node and extending about halfway down its length. These cells migrate through the streak and form two groups of mesodermal cells lateral to (and at the same level as) Hensen's node (Figure 15.2; Garcia-Martinez and Schoenwolf 1993). These groups of cells are called the cardiogenic mesoderm. The cells forming the atrial and ventricular musculature, the cushion cells of the valves, the Purkinje conducting fibers, and the endothelial lining of the heart are all generated from these two clusters (Mikawa 1999).

Figure 15.2

Heart-forming cells of the chick embryo. (A) Model for the specification of cardiogenic mesoderm. The routes of mesodermal migration at various regions of the primitive streak are represented by arrows. Signals that induce cardiac myogenesis are represented (more...)

When the chick embryo is only 18–20 hours old, the presumptive heart cells move anteriorly between the ectoderm and endoderm toward the middle of the embryo, remaining in close contact with the endodermal surface (Linask and Lash 1986). When these cells reach the lateral walls of the anterior gut tube, migration ceases. The directionality for this migration appears to be provided by the foregut endoderm. If the cardiac region endoderm is rotated with respect to the rest of the embryo, migration of the cardiogenic mesoderm cells is reversed. It is thought that the endodermal component responsible for this movement is an anterior-to-posterior concentration gradient of fibronectin. Antibodies against fibronectin stop the migration, while antibodies against other extracellular matrix components do not (Linask and Lash 1988a,b).

The endoderm and primitive streak also specify the some of the cardiogenic cells to become heart muscles. Cerberus and an unknown factor, possibly BMP2 in the anterior endoderm, induce the synthesis of the Nkx2-5 transcription factor in the migrating mesodermal cells that will become the heart^* (Komuro and Izumo 1993; Lints et al. 1993; Sugi and Lough 1994; Schultheiss et al. 1995; Andrée et al. 1998). Nkx2-5 is a critical protein in instructing the mesoderm to become heart tissue, and it activates the synthesis of other transcription factors (especially members of the GATA and MEF2 families). Working together, these transcription factors activate the expression of genes encoding cardiac muscle-specific proteins (such as cardiac actin, atrial naturetic factor, and the alpha myosin heavy chains). Specification of the heart cells occurs gradually, with the ventricular cells becoming specified prior to the atrial cells (Markwald et al. 1998).

Cell differentiation occurs independently in the two heart-forming primordia that are migrating toward each other (Figure 15.3). As they migrate, the cells begin to express N-cadherin on their apices and join into an epithelium. A small population of these cells then downregulates N-cadherin and delaminates from the epithelium to form the endocardium, the lining of the heart that is continuous with the blood vessels.^† The epithelial cells form the myocardium (Manasek 1968; Linask and Lash 1993; Linask et al. 1997). The myocardium will form the heart muscles that will pump for the lifetime of the organism. The endocardial cells produce many of the heart valves, secrete the proteins that regulate myocardial growth, and regulate the placement of nervous tissue in the heart.

Figure 15.3

Formation of the chick heart from the splanchnic lateral plate mesoderm. The endocardium forms the inner lining of the heart, the myocardium forms the heart muscles, and the epicardium will eventually cover the heart. Transverse sections through the heart-forming (more...)

As neurulation proceeds, the foregut is formed by the inward folding of the splanchnic mesoderm (Figures 15.3 and 15.4). This movement brings the two cardiac tubes together, eventually uniting the myocardium into a single tube. The bilateral origin of the heart can be demonstrated by surgically preventing the merger of the lateral plate mesoderm (Gräper 1907; DeHaan 1959). This results in a condition called cardia bifida, in which a separate heart forms on each side of the body (Figure 15.4E). The two endocardia lie within this common tube for a short while, but these will also fuse. At this time, the originally paired coelomic chambers unite to form the body cavity in which the heart resides.

Figure 15.4

Fusion of the right and left heart rudiments to form a single cardiac tube. The cells fated to form the heart myocardium are shown by staining for the Xin message, whose protein product will be essential for the looping of the heart tube. (A) Stage 9 (more...)

This fusion occurs at about 29 hours in chick development and at 3 weeks in human gestation (see Figure 15.3C,D). The unfused posterior portions of the endocardium become the openings of the vitelline veins into the heart. These veins will carry nutrients from the yolk sac into the sinus venosus. The blood then passes through a valvelike flap into the atrial region of the heart. Contractions of the truncus arteriosus speed the blood into the aorta.

Pulsations of the heart begin while the paired primordia are still fusing. The pacemaker of this contraction is the sinus venosus. Contractions begin here, and a wave of muscle contraction is propagated up the tubular heart. In this way, the heart can pump blood even before its intricate system of valves has been completed. Heart muscle cells have their own inherent ability to contract, and isolated heart cells from 7-day rat or chick embryos will continue to beat in petri dishes (Harary and Farley 1963; DeHaan 1967). In the embryo, these contractions become regulated by electrical stimuli from the medulla oblongata via the vagus nerve, and by 4 days, the electrocardiogram of a chick embryo approximates that of an adult.

Looping and formation of heart chambers

In 3-day chick embryos and 5-week human embryos, the heart is a two-chambered tube, with one atrium and one ventricle (Figure 15.5). In the chick embryo, the unaided eye can see the remarkable cycle of blood entering the lower chamber and being pumped out through the aorta. The looping of the heart converts the original anterior-posterior polarity of the heart tube into the right-left polarity seen in the adult (Figures 15.5 and 15.6). Thus, the portion of the heart tube destined to become the right ventricle lies anterior to the portion that will become the left ventricle. This looping is dependent upon the left-right patterning proteins (Nodal, Lefty-2) discussed in Chapter11. Within the heart primordium, Nkx2–5 regulates the Hand1 and Hand2 transcription factors. Although the Hand proteins appear to be synthesized throughout the early heart tube, Hand1 becomes restricted to the future left ventricle, and Hand2 to the right, as looping commences. Without these proteins, looping fails to occur normally and the ventricles fail to form properly (Srivastava et al. 1995; Biben and Harvey 1997). The Pitx-2 transcription factor, activated solely in the left side of the lateral plate mesoderm, is also critical for proper heart looping, and it may regulate the expression of proteins such as the extracellular matrix protein flectin to regulate the physical tension of the heart tissues on the different sides (Figure 15.7; Tsuda et al. 1996). Transcription factors Nkx2-5 and MEF2C also activate the Xin gene, whose protein product, Xin (Chinese for “heart”), may mediate the cytoskeletal changes essential for heart looping (Wang et al. 1999).

Figure 15.5

Specification of the atrium and ventricles occurs even before heart looping. The atrium and ventricles of the mouse embryo have separate types of myosin proteins, which allows them to be differentially stained. In these photographs, the atrium is stained (more...)

Figure 15.6

Cascade of heart development. A correlation is made between the morphological stage and the transcription factors present in the nucleus of the heart precursor cells. The cardioblasts are the committed heart precursor cells containing Nkx2-5 and GATA (more...)

Figure 15.7

Asymmetric expression of the flectin protein in the developing chick heart. This extracellular protein accumulates predominantly on the left side of the heart in the stage 10 chick embryo. This photograph is taken from the ventral side (looking “up” (more...)

The separation of atrium from ventricle is specified by the several transcription factors that become restricted to either the anterior or the posterior portion of the heart tube (see Figure 15.6; Bao et al. 1999; Bruneau et al. 1999; Wang et al. 1999). The partitioning of this tube into a distinctive atrium and ventricle is accomplished when cells from the myocardium produce a factor (probably transforming growth factor β3) that causes cells from the adjacent endocardium to detach and enter the hyaluronate-rich “cardiac jelly” between the two layers (Markwald et al. 1977; Potts et al. 1991). In humans, these cells cause the formation of an endocardial cushion that divides the tube into right and left atrioventricular channels (Figure 15.8). Meanwhile, the primitive atrium is partitioned by two septa that grow ventrally toward the endocardial cushion. The septa, however, have holes in them, so blood can still cross from one side into the other. This crossing of blood is needed for the survival of the fetus before circulation to functional lungs has been established. Upon the first breath, however, these holes close, and the left and right circulatory loops become established (see Sidelights & Speculations). With the formation of the septa (which usually occurs in the seventh week of human development), the heart is a four-chambered structure with the pulmonary artery connected to the right ventricle and the aorta connected to the left.

Figure 15.8

Formation of the chambers of the heart. (A) Diagrammatic cross section of the human heart at 4.5 weeks. The atrial and ventricular septa are growing toward the endocardial cushion. (B) Cross section of the human heart during the third month of gestation. (more...)

Constraints on how blood vessels may be constructed

The first constraint on vascular development is physiological. Unlike new machines, which do not need to function until they have left the assembly line, new organisms have to function even as they develop. The embryonic cells must obtain nourishment before there is an intestine, use oxygen before there are lungs, and excrete wastes before there are kidneys. All these functions are mediated through the embryonic circulatory system. Therefore, the circulatory physiology of the developing embryo must differ from that of the adult organism. Food is absorbed not through the intestine, but from either the yolk or the placenta, and respiration is conducted not through the gills or lungs, but through the chorionic or allantoic membranes. The major embryonic blood vessels must be constructed to serve these extraembryonic structures.

The second constraint is evolutionary. The mammalian embryo extends blood vessels to the yolk sac even though there is no yolk inside. Moreover, the blood leaving the heart via the truncus arteriosus passes through vessels that loop over the foregut to reach the dorsal aorta. Six pairs of aortic arches loop over the pharynx (Figure 15.13). In primitive fishes, these arches persist and enable the gills to oxygenate the blood. In the adult bird or mammal, in which lungs oxygenate the blood, such a system makes little sense, but all six pairs of aortic arches are formed in mammalian and avian embryos before the system eventually becomes simplified into a single aortic arch. Thus, even though our physiology does not require such a structure, our embryonic condition reflects our evolutionary history.

Figure 15.13

The aortic arches of the human embryo. (A) Originally, the truncus arteriosus pumps blood into the aorta, which branches on either side of the foregut. The six aortic arches take blood from thetruncus arteriosus and allow it to flow into the dorsal aorta. (more...)

The third set of constraints is physical. According to the laws of fluid movement, the most effective transport of fluids is performed by large tubes. As the radius of a blood vessel gets smaller, resistance to flow increases as r^-4 (Poiseuille's law). A blood vessel that is half as wide as another has a resistance to flow 16 times greater. However, diffusion of nutrients can take place only when blood flows slowly and has access to cell membranes. So here is a paradox: the constraints of diffusion mandate that vessels be small, while the laws of hydraulics mandate that vessels be large. Living organisms have solved this paradox by evolving circulatory systems with a hierarchy of vessel sizes (LaBarbera 1990). This hierarchy is formed very early in development (and is already well established in the 3-day chick embryo). In dogs, blood in the large vessels (aorta and vena cava) flows over 100 times faster than it does in the capillaries. With a system of large vessels specialized for transport and small vessels specialized for diffusion (where the blood spends most of its time), nutrients and oxygen can reach the individual cells of the growing organism.

But this is not the entire story. If fluid under constant pressure moves directly from a large-diameter tube into a small-diameter tube (as in a hose nozzle), the fluid velocity increases. The evolutionary solution to this problem was the emergence of many smaller vessels branching out from a larger one, making the collective cross-sectional area of all the smaller vessels greater than that of the larger vessel. Circulatory systems show a relationship (known as Murray's law) in which the cube of the radius of the parent vessel approximates the sum of the cubes of the radii of the smaller vessels. The construction of any circulation system must negotiate among all of these physical, physiological, and evolutionary constraints.

Vasculogenesis: formation of blood vessels from blood islands

Blood vessel formation is intimately connected to blood cell formation. Indeed, blood vessels and blood cells are believed to share a common precursor, the hemangioblast^‡ (Figure 15.14). Not only do blood vessels and blood cells share common sites of origin, but mutations of certain transcription factors in mice and zebrafish will delete both blood cells and blood vessels. In addition, the earliest blood cells and the earliest capillary cells share many of the same rare proteins on their cell surfaces (Wood et al. 1997; Choi et al. 1998; Liao and Zon 1999).

Figure 15.14

Origin and fate of the hemangioblast cells. Hemangioblasts are derived from mesodermal cells that are exposed to relatively high concentrations of certain bone morphogenetic proteins during early development. They give rise to the angioblasts—the (more...)

Blood vessels are constructed by two processes: vasculogenesis and angiogenesis (Figure 15.15). During vasculogenesis, blood vessels are created de novo from the lateral plate mesoderm. In the first phase of vasculogenesis, groups of splanchnic mesoderm cells are specified to become hemangioblasts, the precursors of both the blood cells and the blood vessels (Shalaby et al. 1997). These cells condense into aggregations that are often called blood islands. The inner cells of these blood islands become hematopoietic stem cells (the precursors of all the blood cells), while the outer cells become angioblasts, the precursors of the blood vessels. In the second phase of vasculogenesis, the angioblasts multiply and differentiate into endothelial cells, which form the lining of the blood vessels. In the third phase, the endothelial cells form tubes and connect to form the primary capillary plexus, a network of capillaries. In the second process, angiogenesis, this primary network will be remodeled and pruned into a distinct capillary bed, arteries, and veins (Risau 1997; Hanahan 1997). It is important to realize that the capillary networks of each organ arise within the organ itself, and are not extensions from larger vessels (Auerbach et al. 1985; Pardanaud et al. 1989).

Figure 15.15

Vasculogenesis and angiogenesis. Vasculogenesis involves the formation of hemangioblastic blood islands and the construction of capillary networks from them. Angiogenesis involves the formation of new blood vessels by remodeling and building on older (more...)

The aggregation of hemangioblasts in extraembryonic regions is a critical step in amniote development, for the blood islands that line the yolk sac produce the vitelline (omphalomesenteric) veins that bring nutrients to the embryo and transport gases to and from the sites of respiratory exchange (Figure 15.16). These cells are first seen in the area opaca of chick embryogenesis, when the primitive streak is at its fullest extent (Pardanaud et al. 1987). These cords of hemangioblasts soon become hollow. The outer cells become the flat endothelial cell lining of the vessel. The central cells of the blood islands differentiate into the embryonic blood cells. As the blood islands grow, they eventually merge to form the capillary network draining into the two vitelline veins, which bring food and blood cells to the newly formed heart.

Figure 15.16

Vasculogenesis. Blood vessel formation is first seen in the wall of the yolk sac, where (A) undifferentiated mesenchyme condenses to form (B) angiogenetic cell clusters. (C) The centers of these clusters form the blood cells, and the outsides of the clusters (more...)

Three growth factors may be responsible for initiating vasculogenesis (see Figure 15.15). One of these, basic fibroblast growth factor (FGF2) is required for the generation of hemangioblasts from the splanchnic mesoderm. When cells from quail blastodiscs are dissociated in culture, they do not form blood islands or endothelial cells. However, when these cells are cultured in FGF2, blood islands emerge and form endothelial cells (Flamme and Risau 1992). FGF2 is synthesized in the chick embryonic chorioallantoic membrane and is responsible for the vascularization of this tissue (Ribatti et al. 1995). The second protein involved in vasculogenesis is vascular endothelial growth factor (VEGF). VEGF appears to enable the differentiation of the angioblasts and their multiplication to form endothelial tubes. VEGF is secreted by the mesenchymal cells near the blood islands, and the hemangioblasts and angioblasts have receptors for VEGF (Millauer et al. 1993). If mouse embryos lack the genes encoding either VEGF or the major receptor for VEGF (the Flk1 receptor tyrosine kinase), yolk sac blood islands fail to appear, and vasculogenesis fails to take place (Figure 15.16E; Ferrara et al. 1996). Mice lacking genes for the second receptor for VEGF (the Flt1 receptor tyrosine kinase) have differentiated endothelial cells and blood islands, but these cells are not organized into blood vessels (Fong et al. 1995; Shalaby et al. 1995). A third protein, angiopoietin-1 (Ang1), mediates the interaction between the endothelial cells and the pericytes—smooth musclelike cells they recruit to cover them. Mutations of either angiopoietin-1 or its receptor lead to malformed blood vessels, deficient in the smooth muscles that usually surround them (Davis et al. 1996; Suri et al. 1996; Vikkula et al. 1996).

Angiogenesis: sprouting of blood vessels and remodeling of vascular beds

After an initial phase of vasculogenesis, the primary capillary networks are remodeled. At this time, veins and arteries are made. This process is called angiogenesis (see Figure 15.15). First, VEGF acting alone on the newly formed capillaries causes a loosening of cell contacts and a degradation of the extracellular matrix at certain points. The exposed endothelial cells proliferate and sprout from these regions, eventually forming a new vessel. New vessels can also be formed in the primary capillary bed by splitting an existing vessel in two. The loosening of the cell-cell contacts may also allow the fusion of capillaries to form wider vessels—the arteries and veins. Eventually, the mature capillary network forms and is stabilized by TGF-β (which strengthens the extracellular matrix) and platelet-derived growth factor (PDGF, which is necessary for the recruitment of the pericyte cells that contribute to the mechanical flexibility of the capillary wall) (Lindahl et al. 1997).

A key to our understanding of the mechanism by which veins and arteries form was the discovery that the primary capillary plexus in mice actually contains two types of endothelial cells. The precursors of the arteries contain EphrinB2 in their cell membranes. The precursors of the veins contain one of the receptors for this molecule, EphB4 tyrosine kinase, in their cell membranes (Figure 15.17; Wang et al. 1998). If EphrinB2 is knocked out in mice, vasculogenesis occurs, but angiogenesis does not. It is thought that EphB4 interacts with its ligand, EphrinB2, during angiogenesis in two ways. First, at the borders of the venous and arterial capillaries, it ensures that arterial capillaries connect only to venous ones. Second, in non-border areas, it might ensure that the fusion of capillaries to make larger vessels occurs only between the same type of vessel.^§

Figure 15.17

Model of the roles of ephrin and Eph receptors during angiogenesis. (A) Primary capillary plexus produced by vasculogenesis. The arterial and venous endothelial cells have sorted themselves out by the presence of EphrinB2 or EphB4 in their respective (more...)

In some instances, angiogenesis may be the major way of making blood vessels. In the forelimb bud, for instance, the capillary network is probably derived by the sprouting of cells from the aorta (Evans 1909; Feinberg 1991). Within this capillary network, a central artery (which becomes the subclavian artery) forms as the major feeding vessel. Blood returns to the body through marginal veins that form from the anterior and posterior capillaries (Figure 15.18). The organ-forming regions of the body are thought to secrete angiogenesis factors that promote sprouting by enabling the mitosis and migration of endothelial cells into those areas. VEGF (mentioned earlier as a vasculogenesis factor) also promotes the migration of endothelial cells into the organs from preexisting blood vessels on the organs’ surfaces.

Figure 15.18

Vascularization of the chick forelimb. (A) Development of the vascular system during the early development of the chick wing bud. The periphery of the bud is avascular, and more avascular regions will form at the regions where chondrocytes condense to (more...)

Several organs make their own angiogenesis factors. The placenta is one organ whose function depends on redirecting existing blood vessels into it. When the placenta is first being formed, it induces angiogenesis by secreting proliferin (PLF), a factor that resembles growth hormone. When the placental blood vessels have become established (after day 12 in the mouse), the placenta secretes proliferin-related protein (PRP), a peptide that acts as an inhibitor of angiogenesis (Jackson et al. 1994). Ovarian follicle cells and placental cells also secrete leptin, a hormone that is involved in appetite suppression in the adult. However, it can also act locally to induce angiogenesis and cause endothelial cells to organize into tubes (Figure 15.19; Antczak et al. 1997; Sierra-Honigmann et al. 1998).

Figure 15.19

Leptin-induced angiogenesis. (A-C) Corneal response assay. Pellets (indicated by dashed circles) containing either saline (A), leptin (B), or VEGF (C) were implanted into rat corneas. The cornea normally lacks blood vessels, but is close to a highly vascularized (more...)

The developing bone is another organ that redirects blood vessels into it while it is forming. As mentioned in Chapter 14, cartilage is usually an avascular tissue, except when capillaries invade the growth plate to convert the cartilage into bone. Hypertrophic cartilage (but not mature or dividing cartilage) secretes a 120-kDa angiogenesis factor (Alini et al. 1996). It is interesting that this factor is made only when the early hypertrophic chondrocytes have been exposed to vitamin D. This finding suggests an explanation for the bone deformities seen in patients with rickets.

Angiogenesis is critical in the growth of any tissue, including tumors. Tumors are “successful” only when they are able to direct blood vessels into them. Therefore, tumors secrete angiogenesis factors. The ability to inhibit such factors may have important medical applications as a way to prevent tumor growth and metastasis (Fidler and Ellis 1994).

The stem cell concept

Many adult tissues are formed such that once the cells are created, they are not expected to be replaced. Most neurons and bones, for instance, cannot be replaced if they are damaged or lost. There are several populations of cells, however, that are constantly dying and being replaced. Each day, we lose and replace about 1.5 grams of skin cells and about 10¹¹ red blood cells. The skin cells are sloughed off, and the red blood cells are killed in the spleen. Where are their replacements coming from? They come from populations of stem cells. A stem cell is a cell that is capable of extensive proliferation, creating more stem cells (self-renewal) as well as more differentiated cellular progeny (Figure 15.20). Stem cells are, in effect, a population of embryonic cells, continuously producing cells that can undergo further development within an adult organism. Thus, the adult vertebrate body retains populations of stem cells, and these stem cell populations can produce both more stem cells and a population of cells that can undergo further development (Potten and Loeffler 1990). Our blood cells, intestinal crypt cells, epidermis, and (in males) spermatocytes are populations in a steady-state equilibrium in which cell production balances cell loss (Hay 1966). In most cases, stem cells can produce either more stem cells or more differentiated cells when body equilibrium is stressed by injury or environment. (This is seen by the production of enormous numbers of red blood cells when the body suffers from anoxia.)

Figure 15.20

The concept of stem cells. A stem cell divides to produce another stem cell and a committed cell. The committed cell can divide again, but its progeny will form a very restricted set of differentiated cell types. A stem cell can also produce two stem (more...)

Stem cells have been identified in all the tissues mentioned above, but they are most readily studied in blood cell development. Blood cell formation in the adult occurs in the bone marrow and spleen; it also occurs in the fetal liver. The path of development that a stem cell descendant enters depends on the molecular milieu in which it finds itself. This became apparent when experimental evidence showed that red blood cells (erythrocytes), white blood cells (granulocytes, neutrophils, and platelets), and lymphocytes shared a common precursor—the pluripotential hematopoietic stem cell.

Pluripotential stem cells and hematopoietic microenvironments

The pluripotential hematopoietic stem cell

The hemangioblast cells of the lateral plate mesoderm can give rise to both the angioblasts of the vascular system and the pluripotential hematopoietic stem cells of the blood and lymphoid systems. The pluripotential hematopoietic stem cell is one of our body's most impressive cells. From it will emerge erythrocytes, neutrophils, basophils, eosinophils, platelets, mast cells, monocytes, tissue macrophages, osteoclasts, and the T and B lymphocytes (Figure 15.21). The pluripotential hematopoietic stem cell (also known as the colony-forming unit of the myeloid and lymphoid cells, or CFU-M,L, and the CD34 cell) was discovered when irradiated bone marrow (which contains blood-forming cells) was injected into mice that had a hereditary deficiency of blood-forming cells. Abramson and her colleagues (1977) showed that the same radiation-induced chromosomal abnormalities were present in both the nucleated blood cells and the circulating lymphocytes of these mice. This finding was confirmed by studies in which marrow cells were injected with certain viruses that become incorporated into cellular DNA at various random places. The same virally derived genes were seen in the same region of the genome in both lymphocytes and blood cells (Keller et al. 1985; Lemischka et al. 1986). In 1995, Berardi and colleagues isolated a fraction of cells that may be the human CFU-M,L. By killing all the cells that divided when exposed to proteins that would activate more committed stem cells, they were left with about one nucleated cell for every 10,000 originally found in the bone marrow. These cells could generate both the blood and lymphoid lineages.

Figure 15.21

A model for the origin of mammalian blood and lymphoid cells. (Other models are consistent with the data, and this one summarizes features from several models.) Ag, antigen; EPO, erythropoietin; G-CSF, granulocyte colony-stimulating factor; GM-CSF, granulocyte-macrophage (more...)

The CFU-S

If the pluripotential hematopoietic stem cell can give rise to both the lymphocytes and the blood cells, the next stem cell in the sequence is somewhat more committed, being able to give rise to all the blood cells, but not to the lymphocytes. The existence of a such a multipotent hematopoietic stem cell was demonstrated by Till and McCulloch (1961), who injected bone marrow cells into lethally irradiated mice of the same genetic strain as the marrow donors. (Irradiation kills the hematopoietic cells of the host, so that any new blood cells formed must come from the donor mouse.) Some of these donor cells produced discrete nodules, or colonies, on the spleens of the host animals (Figure 15.22). Microscopic studies showed these colonies to be composed of erythrocyte, granulocyte, and platelet precursors. Thus, a single cell from the bone marrow was capable of forming many different blood cell types. The cell type responsible was called the CFU-S, the colony-forming unit of the spleen. This type of cell has also been called the CFU-GEMM, the colony-forming unit for granulocytes, erythrocytes, macrophages, and megakaryocytes. Further studies used chromosomal markers to prove that the different types of cells within a single colony were formed from the same CFU-S. In these studies, bone marrow cells were irradiated so that very few survived. Many of those that did survive developed chromosomal abnormalities that could be detected microscopically. When such irradiated CFU-S cells were injected into a mouse whose own blood-forming stem cells had been destroyed, each cell of a spleen colony, be it granulocyte or erythrocyte precursor, had the same chromosomal anomaly (Becker et al. 1963).

Figure 15.22

Isolated blood-forming colonies. When bone marrow containing hematopoietic stem cells is injected into an irradiated mouse, discrete colonies of blood cells derived from a single precursor cell are seen on the surface of the spleen of that mouse. The (more...)

An important part of the stem cell concept is the requirement that the stem cell be able to form more stem cells in addition to its differentiated cell types. This has indeed been found to be the case for hematopoietic stem cells. When spleen colonies derived from a single CFU-S are resuspended and injected into other mice, many spleen colonies emerge (Juŕrsÿsková and Tkadleÿcek 1965; Humphries et al. 1979). Thus, we see that a single marrow cell can form numerous different cell types and can also undergo self-renewal; in other words, the CFU-S is a multipotent hematopoietic stem cell.

The CFU-M,L and the CFU-S are both supported by stem cell factor (SCF), a paracrine protein that promotes cell division in numerous stem cell populations. As mentioned in Chapter 6, SCF also promotes the division of melanoblasts (which will form pigment cells) and germ stem cells (which will form the gametes). Thus, mice lacking SCF or its receptor (the c-Kit protein) are sterile (no germ cells), white (no pigment cells), anemic (no red blood cells), and immunodeficient (no lymphocytes).

Sites of hematopoiesis

Vertebrate blood development occurs in two phases: a transient embryonic (“primitive”) phase of hematopoiesis is followed by the definitive (“adult”) phase. These phases differ in their sites of blood cell production, the timing of hematopoiesis, the morphology of the cells produced, and even the type of globin genes used in the red blood cells. The embryonic phase of hematopoiesis is probably used to initiate the circulation that provides the embryo with its initial blood cells and with its capillary network to the yolk. The definitive phase of hematopoiesis is used to generate more cell types and to provide the stem cells that will last for the lifetime of the individual.

Embryonic hematopoiesis is associated with the blood islands in the ventral mesoderm near the yolk. In the mouse, embryonic erythropoiesis is seen in the blood islands in the mesoderm surrounding the yolk sac. In chick embryos, the first blood cells are seen in those blood islands forming in the posterior marginal zone near the site of hypoblast initiation (Wilt 1974; Azar and Eyal-Giladi 1979). In Xenopus, the ventral mesoderm forms a large blood island that is the first site of hematopoiesis. Zebrafish are the exceptions to this pattern, as their first blood cells form in the paraxial mesoderm. However, this region of paraxial mesoderm contains the same hematopoietic transcription factors as the ventral mesoderm in Xenopus, mice, and fish (Detrich et al. 1995). Since vertebrate ventral mesoderm is associated with high concentrations of BMPs, it is not surprising that ectopic BMP 2 and 4 can induce blood and blood vessel formation in Xenopus, and that interference with BMP signaling prevents blood formation (Maeno et al. 1994; Hemmati-Brivanlou and Thomsen 1995). Moreover, in the zebrafish, the swirl mutation, which prevents BMP2 signaling, also abolishes ventral mesoderm and blood cell production (Mullins et al. 1996).

The embryonic hematopoietic cell population, however, is transitory. The hematopoietic stem cells that last the lifetime of the organism are derived from the mesodermal area surrounding the aorta. This was shown in the chick by a series of elegant experiments by Dieterlen-Lièvre, who grafted the blastoderm of chickens onto the yolk of Japanese quail (Figure 15.23). Chick cells are readily distinguishable from quail cells because the quail cell nucleus stains much more darkly (owing to its dense nucleoli), thus providing a permanent marker for distinguishing the two cell types (see Figure 1.10). Using these “yolk sac chimeras,” Dieterlen-Lièvre and Martin (1981) showed that the yolk sac stem cells do not contribute cells to the adult animal. Instead, the definitive stem cells are formed within nodes of mesoderm that line the mesentery and the major blood vessels. In the 4-day chick embryo, the aortic wall appears to be the most important source of new blood cells, and it has been found to contain numerous hematopoietic stem cells (Cormier and Dieterlen-Lièvre 1988).

Figure 15.23

Blood cell mapping using chick-quail chimeras. (A) Photograph of a “yolk sac chimera,” created by transplanting the blastoderm of a quail onto the yolk sac of a chick. (B) Photograph of chick and quail cells in the thymus of a chimeric (more...)

Similarly, studies in fishes, mammals, and frogs indicate that the definitive hematopoietic cells are formed near the aorta in a domain called the aorta-gonad-mesonephros (AGM) region. The first blood islands in the mouse embryo appear in the mesoderm around the yolk sac, but by day 11, pluripotential hematopoietic stem cells and CFU-S cells can be found in the AGM (Kubai and Auerbach 1983; Godlin et al. 1993; Medvinsky et al. 1993). These are the hematopoietic stem cells that will colonize the liver and constitute the fetal and adult circulatory system (Medvinsky and Dzierzak 1996). Müller and colleagues (1994) have proposed that two waves of cells colonize the fetal liver. According to this hypothesis, the first population of these cells comes from the yolk sac and comprises predominantly pluripotential stem cells. The majority population, however, comes from the AGM, and comprises both CFU-S and CFU-M,L cells (Figure 15.24). This hypothesis was supported by the finding that mice deficient in the transcription factor AML1 undergo normal yolk sac hematopoiesis, but no definitive (AGM) hematopoiesis (Okuda et al. 1996). These mutant mice die at embryonic day 12.5. Their livers contain a small number of primitive nucleated red blood cells, whereas control livers are full of blood cells derived from the AGM. The AML protein is critical for activating the genes involved in definitive hematopoiesis. At around the time of birth, stem cells from the liver populate the bone marrow, which then becomes the major site of blood formation throughout adult life.

Figure 15.24

Colonization of the mouse liver by two waves of hematopoietic stem cells. The two main sources of hematopoietic progenitor cells are the yolk sac and the AGM region. (A) At day 9, the yolk sac contributes an early line of CFU-C cells that probably does (more...)

The pathways of hematopoiesis are difficult to unravel, but recently a new approach has gained momentum. Since zebrafish can be easily screened for mutations of developmentally important genes, over 26 different mutations of hematopoiesis have been found in this species (Liao and Zon 1999). Some of these, such as the swirl mutation, inhibit the production of ventral mesoderm. Mutations of the moonshine and cloche genes prevent hemangioblast development; mutations of frascati and thunderbird act at the level of the pluripotential hematopoietic stem cells, and several other mutations affect the pathways leading to the various differentiated blood cell types. By studying gene expression patterns in these mutants, the stage at which the mutant gene operates can be discerned (Figure 15.25; Thompson et al. 1998). For instance, the cloche and spadetail mutants have defects in both primitive and definitive hematopoiesis. The cloche mutant, however, also has defects in vascularization, indicating that cloche works at the level of the hemangioblast, while the spadetail gene works later, probably at the level of the pluripotential hematopoietic stem cell.

Figure 15.25

Expression of the flt4 and GATA1 genes (dark areas) in wild-type and mutant zebrafish embryos. GATA1 is a transcription factor essential for blood cell development, and it is absent in both the cloche and spadetail mutants. The flt4 gene is essential (more...)

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Redirecting Blood Flow in the Newborn Mammal.